NPC Natural Product Communications Chemical Variation in the Essential Oil Composition of Hyptis suaveolens (L.) Poit. (Lamiaceae) 2008 Vol. 3 No. 7 1137 - 1140 Paolo Grassi1, Marvin José Nuñez2, Tomás Sigfrido Urías Reyes3 and Chlodwig Franz1 1 Institute for Applied Botany, University of Veterinary Medicine, Veterinärplatz 1, A-1210 Vienna, Austria 2 Facultad de Química y Farmacia, Universidad de El Salvador, Final 25 Avenida Norte, San Salvador, El Salvador, C.A. 3 Facultad de Química y Farmacia-Biología, Universidad Salvadoreña Alberto Masferrer - USAM, 19a Avenida Norte entre 3a Calle Poniente y Alameda Juan Pablo II, San Salvador, El Salvador, C.A. paolo.grassi@vu-wien.ac.at Received: April 13th, 2008; Accepted: June 9th, 2008 Hyptis suaveolens (Lamiaceae) is an annual species which is used for aromatic and medicinal purposes. The species is polymorphic in its essential oil composition, with three distinct chemotypes in El Salvador of which the fenchone-fenchole type will be the focus of this communication. The fenchone-fenchol chemotype showed strong relative changes, especially within the monoterpenes. The main compounds of the essential oil were sabinene, 1,8-cineole, γ-terpinene, fenchone, fenchol and the sesquiterpene β-caryophyllene. Due to strong inter-individual differences, these changes are explained based on several individuals separately. Fenchol was found to decrease drastically when leaves enter the last development stage of yellowing, whereas the proportion of the ketone, fenchone, increased. Furthermore, the proportion of sesquiterpenes decreased in older tissues compared to the monoterpenes. These changes were surprising, because it was expected that yellowish leaves would lose compounds with higher vapor pressures, like sabinene and 1,8-cineole. Keywords: Hyptis suaveolens, Lamiaceae, monoterpenes, fenchone, fenchol. Hyptis suaveolens (L.) Poit., known in El Salvador as Chichinguaste, grows mainly in hot subtropical savannas from 0 – 400 m above sea level. It is clearly differentiated morphologically and geographically by another Chichinguaste (H, mutabilis), which grows at higher altitudes (400 – 1000 m) [1]. H. suaveolens is a weedy species that was used and cultivated in ancient pre-Columbian times for its seeds, called “chia” or “chan” (together with Salvia hispanica and S. polystachya) [2]. In recent times, this species started its renaissance, especially due to its strongly aromatic and unique scent. The use of its extracts and essential oils for hair care products, as shampoos, are the main purpose of Chichinguaste in El Salvador. The medicinal use of this species is rather restricted. It is used as a wound remedy and/or as a skin disinfectant, as a carminative, and the seeds for the treatment of gastrointestinal disorders [3]. Similar ethnobotanical uses are known from other countries of Central America [4]. Three chemotypes were reported from El Salvador: 1. the 1,8-cineole chemotype, with more than 30% 1,8-cineole and less than 1% fenchone, fenchol and α-terpinolene, 2. the α-terpinolene chemotype, with 20% sabinene, 10% α-terpinolene and less than 1% fenchone and fenchol, and 3. the fenchone-fenchol chemotype, with more than 10% fenchone and more than 5% fenchol and with less than 1% α-terpinolene [5]. This work includes the description of the chemical changes within individual plants of the fenchone-fenchol chemotype. Essential oil distillations and extractions were performed using different parts of the plants, and leaves of different sizes from single lateral shoots, respectively. H. suaveolens is a fast growing weedy species, reaching heights of almost 3 m within one season. Especially in the rainy season, from July to October, until blooming the plants grow 2 cm per day. A characteristic plant in bloom was collected and 0,9 0,8 0,7 0,6 0,5 0,4 0,752 0,611 0,578 0,288 0,338 0,3 0,139 0,2 0,1 0,0 0-50 cm 50-100 cm 50-100 cm 50-100 cm leaves calyx 100-150 cm 150-267 cm 0,7 ratio fenchol / fenchone separated into four sections of the main shoot, including all its lateral shoots. The plant material was distilled separately by hydrodistillation. The second section (50 cm – 100 cm above the ground) was the section with the highest amount of distillable material (about 100 g dry weight) and was, therefore, separated into inflorescences and leaves. The leaves were formed explicitly before the inflorescences and represent older tissue. The essential oil yield was shown to strongly depend on the age of the plant material and the type of organ. Figure 1 shows the increase of the essential oil yield from 0.29% (wt/wt) in the lowest section (0 – 50 cm) to 0.75% in the upper section (150 – 267 cm). This increase is the result of a higher proportion of inflorescences in the upper parts. Compared to the leaves, the inflorescences represent younger tissue with higher essential oil yields. Grassi et al. essential oil yield [%] 1138 Natural Product Communications Vol. 3 (7) 2008 0,457 0,5 0,4 0,587 0,538 0,6 0,352 0,319 0,3 0,2 0,137 0,1 0,0 0-50 cm 50-100 cm 50-100 cm 50-100 cm leaves calyx 100-150 cm 150-267 cm Figure 1: Change of essential oil yield (top) and the fenchol / fenchone ratio (bottom) in different plant segments and between inflorescences and leaves within one segment. Table 1: Essential oil composition of different sections of a plant of the fenchone-fenchol chemotype in area-%. Compound α-Thujene α-Pinene Camphene Sabinene β-Pinene Myrcene α-Phellandrene α-Terpinene p-Cymene Limonene 1,8-Cineole γ-Terpinene cis-Sabinene hydrate Fenchone Fenchol (endo) Camphor Borneol Terpinen-4-ol α-Terpineol α-Copaene β-Bourbonene β-Elemene β-Caryophyllene α-Bergamotene α-Humulene Germacrene D α-Selinene Bicyclogermacrene Spathulenol Caryophyllene oxide Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Sesquiterpenes Others RI 931 937 952 977 979 994 1006 1020 1029 1033 1035 1064 1074 1091 1119 1151 1173 1183 1197 1381 1389 1396 1425 1440 1460 1486 1499 1501 1586 1590 section 0 – 50 cm section 50 – 100 cm [area-%] 1.1 2.7 0.6 11.9 4.24 0.6 0.6 1.0 4.4 3.1 12.5 8.1 < 0.1 20.1 7.1 0.5 0.8 0.6 0.7 1.3 0.2 0.5 4.9 1.7 0.4 0.7 0.5 1.7 0.2 0.2 38.4 42.2 80.6 11.9 0.4 12.3 7.0 [area-%] 1.0 2.7 0.7 10.4 4.0 0.6 0.6 1.0 4.0 3.2 12.9 8.0 0.2 19.8 6.3 0.6 0.8 0.7 1.0 1.4 0.1 0.5 4.7 2.2 0.5 0.7 0.5 1.8 0.3 0.2 36.3 42.3 78.7 12.4 0.5 12.8 8.5 section 50 – 100 cm leaves [area-%] 0.8 2.7 0.9 7.0 3.7 1.0 0.6 0.9 4.7 4.2 12.9 7.3 0.2 23.4 3.2 0.9 0.9 1.1 1.7 1.0 0.3 0.3 4.2 1.7 0.4 0.4 0.6 1.3 0.2 0.2 34.0 44.4 78.3 10.3 0.4 10.6 11.0 section 50 – 100 cm inflor. / calyx [area-%] 1.1 2.6 0.5 13.2 4.2 0.3 0.5 1.0 3.3 2.3 12.7 8.6 0.2 16.4 8.8 0.4 0.7 0.4 0.4 1.6 < 0.1 0.6 5.0 2.6 0.5 1.0 0.4 2.2 0.3 0.2 37.8 40.1 77.9 14.0 0.5 14.5 7.5 section 100 – 150 cm [area-%] 1.1 2.6 0.5 13.0 4.1 0.4 0.6 1.0 3.8 2.5 14.4 8.5 0.2 16.9 7.7 0.5 0.6 0.5 0.6 1.7 < 0.1 0.4 4.5 2.6 0.4 0.9 0.4 2.1 0.3 1.2 38.2 41.5 79.6 13.2 1.5 14.7 5.7 section 150 – 267 cm [area-%] 1.1 2.7 0.5 14.4 4.5 < 0.1 0.5 0.8 4.6 2.3 14.7 7.8 < 0.1 14.0 8.2 0.4 0.5 0.4 0.4 1.9 < 0.1 0.6 4.7 2.9 0.5 1.1 0.4 2.2 0.4 0.2 39.3 38.5 77.8 14.2 0.6 14.8 7.3 Natural Product Communications Vol. 3 (7) 2008 1139 In total, 30 essential oil compounds were identified in the essential oil, including 19 monoterpenes and 11 sesquiterpenes. The main compounds of the fenchol-fenchone chemotype of H. suaveolens were the monoterpenes fenchone, fenchol (endo), 1,8-cineole, sabinene and γ-terpinene, as well as the sesquiterpene β-caryophyllene (Table 1). To verify this suggestion, three lateral shoots of three individual plants were investigated in detail. Different parts of the inflorescences and single leaves of different sizes were analyzed by solvent extraction and gas chromatography. The results showed that the changes between fenchol and fenchone were not directly negatively correlated. On the contrary, these analyses rather showed a strong negative correlation of sabinene and fenchol, which was not observed in the case of the distilled essential oils. The proportion of fenchone increased continuously with increases in the size of the leaf, similar to 1,8-cineole (Figure 2). Fenchol, however, became drastically reduced, mainly in the last leaf developmental stages, where the leaves became more rigid and start to yellow. This change cannot be explained by low fenchol production in the early season. The second diagram of Figure 2 shows the tremendous fenchol reduction from almost 30% to 30 area-% 25 20 15 10 5 leaf < 4 cm leaf < 8 cm yellow leaf leaf < 2 cm leaf < 4 cm leaf < 8 cm leaf < 12cm yellow leaf leaf < 2 cm leaf < 4 cm leaf < 8 cm leaf < 12cm yellow leaf leaf < 2 cm leaf = 1 cm leaf = 1 cm calyx leaf = 1 cm leaf < 1 cm leaf < 1 cm flower bud leaf < 1 cm calyx calyx inflorescence inflorescence 0 35 30 area-% 25 20 15 10 5 0 35 30 25 area-% It was observed that the proportion of monoterpenes, especially that of the oxygenated monoterpenes, was reduced in younger tissues of the upper sections. The main reason for these changes was the accumulation of fenchone, which represented the dominant terpene in the lower sections of the plant. The second section (50 – 100 cm), with the two separately obtained essential oils for inflorescences and leaves, clarified the actual situation. The leaves of this section, representing the older tissue, accumulated up to 23% of fenchone, whereas the inflorescences yielded only 16%. On the other hand the corresponding alcohol, fenchol, behaved contrarily. Old tissue, like the leaves of the second segment, accumulated only about 3% and the young tissue up to almost 9%. A ratio between fenchol and fenchone was calculated, showing that the chemical change between these two compounds coincided with the change in essential oil content. The lower the essential oil content, the lower was the fencholfenchone ratio (Figure 1). These data suggested that a conversion of fenchol to fenchone could take place during leaf development, similar to the conversion of menthone to menthol in the genus Mentha [6]. 35 inflorescence Essential oil composition of Hyptis suaveolens 20 15 10 5 0 Figure 2: Changes of the main essential oil compounds fenchone (□), fenchol (▲), sabinene (○), 1,8-cineole (×) and the sum of sesquiterpene hydrocarbons (−) in three individuals of Hyptis suaveolens. about 7%. In younger leaf stages (up to 4 cm of blade length), the proportion of fenchol increased, together with the other oxygenated monoterpenes. These data do not support a direct conversion from fenchol to fenchone. From other Labiatae species it is known that older leaves loose the essential oil simply by its evaporation from disrupted essential oil glands. However, in this case it would be more likely that compounds with higher vapor pressures [7], such as the monoterpene hydrocarbon, sabinene evaporate before the hydroxylated monoterpene, fenchol. On the contrary, sabinene often showed the highest concentration in the oldest leaves, which can be explained very well by a higher production in the early season. 1140 Natural Product Communications Vol. 3 (7) 2008 Grassi et al. Also the concentrations of sesquiterpenes, having lower vapor pressures than monoterpenes, were reduced in the older tissues. Thus, the data do not support the loss of fenchol simply by evaporation from disrupted essential oil glands. Hydrodistillation and solvent extraction: Dried leaves and inflorescences (20 – 30 g) were hydrodistilled in a modified Clevenger apparatus for 2 h. The solvent extractions were performed with CH2Cl2 for 15 min in an ultrasonic bath. Other possible reasons for the great semi-quantitative changes in the large leaves could be either metabolism to a glycoside, similar to that of menthone in the case of Mentha [8,9] or another conversion or degradation. In fact, yellowish leaves were characterized, apart from color, by a sticky surface. Finally, it cannot be excluded that different oil gland types are involved in this compositional variation of the older leaf material. Gas chromatography and identification: The essential oil compounds were analyzed using a HP 6890 gas chromatograph coupled with a HP 5972 MSD (Hewlett-Packard, Palo Alto, CA, USA) equipped with a DB-5MS capillary column (30 m x 0.25 mm i.d.; 0.25 µm film thickness). The injector was set at 250°C with a split ratio of 100:1. Helium was used as carrier gas (average velocity 42 cm/s). A gradient temperature program to 280°C was established and the obtained retention times and mass spectra were compared with published data [10] and with the WILEY library [11]. Compound concentrations were calculated from the GC peak areas of the total ion current (TIC) plot. It is concluded that the chemical intra-individual variation of the fenchone-fenchol chemotype of H. suaveolens is mainly based on the abrupt loss of fenchol during the last leaf developmental stages. Experimental Plant material: The plants were taken from research cultivation blocks in El Pacùn, district San Vicente and at the Finca Lutesia, Los Planes Renderos, district in San Salvador. The samples were collected at the full flowering stage and dried before further processing. Acknowledgments – Special thanks to Fidel and Saùl and their families in El Salvador for their great support during the cultivation. The work was financially supported by the Austrian Academy of Science and performed in cooperation with the Salvadorian counterpart APROCSAL (Asociación de promotores comunales salvadoreños. References [1] Grassi P. (2003) Botanical and chemical investigations in Hyptis spp. (Lamiaceae) in El Salvador. Thesis, University of Vienna [2] Pico B, Nuez F. (2000) Minor crops of Mesoamerica in early sources (II). Herbs used as condiments. Genetic Resources and Crop Evolution, 47, 541-552. Guzman DJ. (1975) Especies Utiles de la Flora Salvadoreña. Tomo 1. 3ª ed., Ministerio de educación, Dirección de publicaciónes, San Salvador, El Salvador, 85-86. Heinrich M. (1992) In: Advances in Labiate Science, Harley RM, Reynolds T. (Eds), Royal Botanical Gardens, Kew, 475-488. Grassi P, Nuñez MJ, Varmuza K, Franz C. (2005) Chemical polymorphism of essential oils of Hyptis suaveolens from El Salvador. Flavour and Fragrance Journal, 20, 131-135. Murray MJ. 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