Determining composition of volatiles in Couroupita guianensis Aubl. through headspace-solid phase micro-extraction (HS-SPME) Arpita Mandal Khan1, K.S. Shivashankara and T.K. Roy ICAR-Indian Institute of Horticultural Research Hesaraghatta Lake post, Bengaluru – 560089, India E-mail: meet.arpitakhan@gmail.com ABSTRACT Composition of volatile components in Couroupita guianensis Aubl. flowers was analyzed using headspace-solid phase micro-extraction (HS-SPME), followed by capillary gas chromatography and mass spectrometry (GC-MS) separation and identification. In all, 75 compounds were identified accounting for 96.32% of the total volatiles present. The major groups of compounds present were oxygenated terpenoids (35.66%), alcohols (26.92%), esters (17.36%), mono-and sesqui-terpenoids (8.64%), aldehydes and ketones (4.71%), hydrocarbons (1.68%), phenols (0.18%), acids (0.754%) and heterocyclic compounds (0.42%) constituted a small proportion of the volatile profile. The most abundant individual constituent was eugenol (18.95%) followed by nerol (13.49%), (E,E) farnesol (12.88%), (E,E)-farnesyl acetate (6.68%), trans ocimene (6.02%), nootkatone (4.64%), geraniol (2.94%), 2-isopropenyl-5- methyl-4-hexenyl acetate (2.69%), cedr-8-en-13-ol (2.58%), (E,Z)-farnesyl acetate (2.40%) and methyl (11E)-11- hexadecenoate (2.041%). Analytical comparison of composition of volatiles in the flowers, obtained by different methods of extraction, viz., solvent extraction, micro-simultaneous extraction and headspace-solid phase micro- extraction, revealed specific variations in relative concentrations of the constituent chemicals. Linalool was the major chemical (21.5% and 14.9%) in solvent extract and micro-simultaneous extract, respectively, but appeared in negligible quantity (0.16%) in head-space analysis. Key words: Couroupita guianensis, volatiles, headspace-solid phase micro-extraction (HS-SPME), capillary gas chromatography and mass spectrometry (GC/MS) INTRODUCTION Couroupita guianensis Aubl, or the Cannonball tree, has always been a botanical curiosity due to the unique shape of its flowers and fruits. The plant, belonging to the family Lecythidaceae, is native to the tropics of the northern part of South America and to the West Indies (Heywood and Chant, 1982). In India, the tree is grown in the vicinity of Shiva temples, as, Hindus revere it as sacred, it being known as ‘Shivalingam’ in Hindi. It is a fast growing, evergreen tree attaining a height of up to 30m. The fragrant, orange- red flowers are borne on long, thick, tangled extrusions from the trunk. Fruits are spherical, brown and large as a cannon ball. Besides its ornamental value, the tree has several medicinal properties. Infusion from the flowers is used for treating colds and stomach ache (Anon., 1950) and the bark is used for treating hypertension, tumors and inflammations (Stanz et al, 2009). In Brazil, its leaves are widely used as an analgesic (Mariana et al, 2010) and for treating skin diseases (Satyavati et al, 1976). The flowers emit a strong, 1National Research Centre for Orchids, P.O. Pakyong, Sikkim-737106, India sweet, spicy fragrance. Previous efforts on chemical examination revealed presence of linalool, eugenol, nerol, tryptanthrin, farnesol, indigo, indirubin, isatin, linoleic acid, α, β- amirins, carotenoids, sterols and some acidic and phenolic compounds (Sen et al, 1974; Bergman et al, 1985; Wong and Tie, 1995; Rane et al, 2001; Rajamanickam et al, 2009). Wong and Tie (1995) identified 41 compounds responsible for fragrance in Couroupita flowers using solvent extraction, of which eugenol, linalool, (E,E)-farnesol and nerol were the major ones. Similar results were obtained by Andrade et al (2000) from fresh flowers using the micro- simultaneous extraction method. Variation in relative concentrations of the major fragrance components occurs due to a difference in the method of extraction employed. The objective of the present study was to identify aroma compounds that most likely represent the fragrance of Couroupita guianensis flowers, using the headspace-solid phase micro-extraction (HS-SPME) technique. HS–SPME is now a well-established and very popular technique for head-space (HS) sampling in several fields, including study J. Hortl. Sci. Vol. 9(2):161-165, 2014 162 of composition of HS volatiles in medicinal and aromatic plants, flowers and fruits where it has assumed an ever- increasing importance. HS-SPME is an easy and non- destructive method of extraction of volatiles, therefore, a more accurate method than others. Solvent extraction and simultaneous micro-extraction method could modify the compounds due to the destructive way of sample preparation besides the high temperatures used for extraction. Studies on head-space extraction and analysis of flower volatiles (Flamini et al, 2003; Deng et al, 2004 and Belliardo et al., 2006) report direct sampling using SPME to avoid interferences from non-volatile matrix components (Pawliszyn, 1997). MATERIAL AND METHODS Plant material Fresh, fully opened Couroupita guianensis flowers were collected in the morning during the month of May, 2013 from full-grown plants located near the garden of ICAR-Indian Institute of Horticultural Research, Bengaluru. Volatile fragrance constituents were extracted by headspace- solid phase micro-extraction (HS-SPME) technique and analyzed using GC–MS/MS. SPME extraction of volatiles A manual SPME holder and three commercial SPME fibers (procured from Supelco Inc. Bellefonte, PA, USA) were used in the study. SPME fibers were conditioned in a GC injector port as recommended by the manufacturer, at a temperature of 250°C for 3hrs before use in volatile extraction. SPME fiber types DVB/CAR/PDMS (Divinylbenzene/Carboxen/polydimethylsiloxane), 50/30 μm, highly crossed-linked (Supelco Inc., Bellefonte, PA, USA) were used for extraction of head-space volatile compounds from flowers. Extraction process used for head-space volatiles was as per Flamini et al (2003) and Deng et al (2004). Soon after plucking, six Couroupita flowers were transferred to each of the two 250ml conical flasks (with screw caps and silicon rubber septum) and capped immediately. The samples were kept at room temperature (25 ±1°C) for 10-15 minutes to accelerate transfer of analytes for reaching equilibration in the head-space. After the equilibration-time was up, sampling was done by inserting pre-conditioned SPME fiber into the head-space of the flask for 1 hour at room temperature (25 ±1°C). GC analysis After extraction of head-space volatiles, the SPME device was inserted into the injector port for gas chromatographic analysis, and was held in the inlet for 10 minutes for desorption. GC-FID analysis was done using Varian-3800 Gas Chromatograph, equipped with FID detector. Nitrogen (1ml/min) was used as a carrier gas. The components were separated on VF-5, capillary column from Varian, USA, 30m x 0.25mm i.d., 0.25μm film thickness. The injector temperature was set at 260°C and all injections were made in split mode (1:5). The detector temperature was maintained at 270°C and the temperature programme used for the column was as follows: 50°C for 5 min, followed by an increment of 4°C/min till 170°C, held for 2 min; subsequently, increased by 5°C/min till it reached 250°C and, then, a constant temperature of 250°C was maintained for 7 minutes. The total run-time was 60 minutes. GC/MS analysis GC/MS analysis was carried out in the system consisting of a Varian-3800 Gas Chromatograph coupled to a Varian-4000 Ion-Trap mass spectra detector. The ion trap, transfer line and ion source temperatures were maintained at 190°C, 240°C and 200°C, respectively. A fused-silica capillary column VF-5ms from Varian, USA, with 30m x 0.25mm id, 0.25mm film thickness was used for the analysis. Helium was used as carrier gas with flow rate of 1ml/min. The mass spectrometer was operated in the external electron ionization mode of 70eV, with full mass scan-range 45–450amu. Temperature programmes used for the column were the same as described for GC-FID analysis. Total volatile production was estimated by a sum of all GC-FID peak areas in the chromatogram and individual compounds were quantified as relative per cent area. Individual volatile compounds were identified by comparing their retention index (RI) which was determined using homologous series of n-alkanes (C5 to C32, procured from Sigma-Aldrich) as Standard (Kovats, 1965) and comparing mass spectra with the available two spectral libraries, using Wiley and NIST-2007. RESULTS AND DISCUSSION GC and GC-MS separation and identification of volatile components of Couroupita flowers extracted by headspace-solid phase micro-extraction (HS-SPME) resulted in identification of 75 compounds (Table 1). The total percentage of compounds identified was 96.32%, in Arpita Mandal Khan et al J. Hortl. Sci. Vol. 9(2):161-165, 2014 163 Table 1. Volatile components of Couroupita guianensis flowers estimated using headspace-solid phase micro-extraction (HS- SPME) method Name of the compound/group Retention Area Index (%) Hydrocarbons 1. 1,3,5,5-Tetramethyl-1,3-cyclohexadiene 1028 0.058 2. 2-Methyl-2-bornene 1045 0.132 3. Eicosane 2011 0.569 4. Heneicosane 2103 0.707 5. Triecosane 2298 0.217 Total 1.684 Monoterpenoids 6. β-Pinene 974 0.143 7. 3-Carene 1010 0.112 8. γ-Terpinene 1018 0.112 9. β-Phellandrene 1027 0.215 10. Limonene 1033 0.122 11. cis-Ocimene 1039 0.781 12. trans-Ocimene 1052 6.021 13. α-Terpinene 1057 0.096 14. Terpinolene 1075 0.112 15. Mentha-1,3,8-triene 1111 0.098 16. allo-Ocimene 1127 0.086 Total 7.899 Sesquiterpenoids 17. α-Bergamotene 1445 0.095 18. β-Caryophyllene 1455 0.108 19. Germacrene D 1468 0.102 20. (Z,E)-α-Farnesene 1491 0.138 21. (E,E)α-Farnesene 1504 0.195 22. Bicyclogermacrene 1528 0.098 Total 0.736 Oxygeneted terpenoids 23. Linalool 1095 0.164 24. 6-Camphenol 1118 0.112 25. cis-Verbenol 1131 0.665 26. 2-Pinen-4-ol 1146 0.095 27. cis-Limonene oxide 1148 0.103 28. Z-Thujanol 1165 0.055 29. (-)-Borneol 1173 0.121 30. Myrtenol 1192 0.132 31. Nerol 1223 13.489 32. Isogeraniol 1232 1.128 33. Geraniol 1258 2.942 34. Geranial 1268 1.178 35. Nerolidol 1568 0.153 36. Caryophyllene oxide 1585 0.112 37. (2Z,6E)-Farnesol 1682 0.266 38. (Z,Z)-Farnesol 1715 0.924 39. (E,E)-Farnesol 1725 12.881 40. (E,Z)-Farnesol 1742 1.072 41. Longifolenaldehyde 1876 0.065 Total 35.657 Phenolics 42. Carvacrol 1304 0.096 43. 2,3,5,6-Tetramethylphenol 1321 0.088 Total 0.184 Table 1. Contd. Name of the compound/group Retention Area Index (%) Alcohols 44. (E)-6-Nonen-1-ol 1124 0.095 45. (5-Isopropyl-2-methyl-1-cyclopenten-1-yl) 1199 0.064 methanol 46. α-Methyl-benzeneethanol 1208 1.490 47. 2-(2,2,4-Trimethyl-3-cyclopenten-1-yl) 1233 0.385 ethanol 48. Eugenol 1358 18.952 49. Methyleugenol 1392 0.112 50. Dihydro-β-ionol 1405 0.054 51. (E)-Isoeugenol 1463 0.059 52. Cedrenol 1603 0.079 53. Cedr-8-en-13-ol 1672 2.576 54. Z-9-Pentadecenol 1749 1.077 55. Z-11-Pentadecenol 1772 0.403 56. (6E,10E)-3,7,11,15-Tetramethyl-1,6,10, 2049 1.577 14-hexadecatetraen-3-ol Total 26.923 Acids 57. Myristic acid 1765 0.652 58. Pentadecanoic acid 1821 0.102 Total 0.754 Aldehydes and Ketones 59. Isopulegone 1155 0.068 60. Nootkatone 1845 4.637 Total 4.705 Esters 61. Methyl salicylate 1193 0.209 62. Z-Methyl geranate 1298 0.470 63. Citronellyl acetate 1348 0.122 64. 2-Isopropenyl-5-methyl-4-hexenyl acetate 1375 2.693 65. (Z,Z)-Farnesyl acetate 1810 0.507 66. (E,E)-Farnesyl acetate 1818 6.682 67. (E,Z)-Farnesyl acetate 1838 2.396 68. Methyl (11E)-11-hexadecenoate 1883 2.041 69. Methyl (9Z)-9-hexadecenoate 1892 1.221 70. (3Z)-3-Hexenyl benzoate 1568 0.121 71. Hexyl benzoate 1577 0.132 72. Ethyl (9E)-9-hexadecenoate 1969 0.762 Total 17.356 Heterocyclic compounds 73. 2-Methylfuran 603 0.210 74. Indole 1289 0.156 75. (E)-3-(4,8-dimethyl-3,7-nonadienyl)-furan 1552 0.053 Total 0.419 which the major groups of compounds were: oxygenated terpenoids (35.66%), alcohols (26.92%), esters (17.36%), mono-and sesqui-terpenoids (8.64%) and aldehydes and ketones (4.71%) (Fig. 1). Hydrocarbons (1.68%), phenols (0.18%), acids (0.754%) and heterocyclic compounds (0.42%) constituted a small proportion of the volatile profile. Volatiles in Couroupita guianensis flower fragrance J. Hortl. Sci. Vol. 9(2):161-165, 2014 164 The most abundant individual constituent was eugenol (18.95%), followed by nerol (13.49%), (E,E)-farnesol (12.88%), (E,E)-farnesyl acetate (6.68%), trans-ocimene (6.02%), nootkatone (4.64%), geraniol (2.94%), 2-isopropenyl-5-methyl-4-hexenyl acetate (2.69%), cerd- 8-en-13-ol (2.58%), (E, Z)-farnesyl acetate (2.40%) and methyl (11E)-11-hexadecenoate (2.041%). Comparison of volatile composition of Couroupita guianensis flowers obtained in the present study with earlier published methods of solvent extraction (Wong and Tie, 1995) and micro-simultaneous extraction (Andrade et al, 2000) revealed some variations in relative concentrations of the constituent chemicals (Fig. 2). Earlier studies reported linalool as a major constituent imparting aroma to orange- flower (21.5% and 14.9%), respectively in the volatiles profile. However, it appeared in negligible quantity (0.16%) in head-space analysis, where citrus aroma is attributed to higher percentage of nerol (13.49%). Eugenol, which is responsible for strong spicy nutmeg or clove-type odor of the flower, registered high percentage (18.9%) in both micro-simultaneous extraction and HS-SPME method, but comparatively lower than in the solvent extraction method (41.6%). Head space analysis also recorded higher percentage of (E,E)-farnesol (12.88%) and (E,E)-farnesyl acetate (6.68%) among the volatiles. These compounds add an oily floral note to fragrance-profile. Presence of ocimine, similarly, was observed only in HS-SPME method, and was reported to be negligible when estimated by the other methods. The variation in relative concentrations of major fragrance components observed in earlier studies could be due to different methods of sample preparation. 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