fatty acid from Gevuina oil is the palmitoleic C16:1â11 isomer, present in high quantities. ... when cultivated in myristoleic acid (C14:1Î9)-supplemented media, ...
Monounsaturated Fatty Acid Isomers of Gevuina avellana Mol. Nut Oil and Its UV Radiation Filter Behaviour F. Medel1, G. Medel2, P. Jil1, H. Palma3 and R. Mansilla1 1 Facultad de Ciencias Agrarias 2 Facultad de Ciencias Forestales 3 Facultad de Ciencias Universidad Austral de Chile (UACh) Chile Keywords: Gevuina avellana, monounsaturated isomers, UV radiation filter Abstract The research programme for Gevuina avellana Mol. (Gevuin; Chilean hazelnut), has developed genotypes with interesting results for phytotherapy and cosmetic purposes. The physical and chemical characteristics of nut oil have potential uses as skin care, for restoring treatments (turgor, elasticity, wound healing) and as UV radiation filter. From 2003 to 2007, nut oil of selected clones was analyzed to determine the cis-monounsaturated fatty acids (cis-MUFAs) composition and UV filter quality of the nut oil. HPLC and GC-MSD methods were used to determine the fatty acid content profile. UV absorbance from different samples was analyzed with a Milton Roy Array 3000 spectrophotometer. 12 cis-MUFAs were detected, four of them being only traces. Gevuin nut oil is composed of a great amount of unusual cis-MUFAs having between 14 to 24 carbons, with four different groups (C16, C18, C20, and C22). The effect of the positional double bond within the acyl chain (Δ/ω), its molecular weight and carbon number together with the nature of its skin, could explain the UV filter behaviour in function of the different absorbance of the cis-MUFAs. Gevuina oil has an absorbance range starting at 350 nm up to a maximum at 250 nm, with a peak around 280 to 260 nm, with differential responses among clones. The unique chemical composition of cis-MUFAs differentiates Gevuin from other seeds oils in respect to its UV filter behaviour. INTRODUCTION The research program on Gevuina avellana Mol. (Gevuin, Chilean hazelnut) a native tree from Chile has developed high yielding and quality clones for edible nuts with important attributes in nutrition, with its oil having special interest for phytotherapy purposes. The physical and chemical characteristics of nut oil have several uses as skin care, for restoring treatments and as a UV radiation filter. There is strong empirical evidence concerning the extraordinary performance of Gevuin nut oil in giving protection from sun damaging radiation (Medel and Medel, 2000; Medel, 2001a,b). The evidence for variability among clones with respect to the amount and quality of fatty acid composition in nut oil (Medel and Carrillo, 2005) is highly interesting for genetic improvement, especially in relation to its UV filter properties. Chronic exposure to the sun causes damage such as skin rash and malignant melanoma, and the protective action from synthesis products and the active mechanisms which allow for the blocking of the solar UV radiation have been reviewed by June (2006). An adequate knowledge about human skin in relation to its composition and functions is necessary due to its complexity as a physical and biological barrier allowing for homeostasis (Nicolaides, 1974; Kitson and Thewalt, 2000; Curcuff et al., 2001). The skin has at least two barriers with protective functions: the stratum corneum physical barrier and a biochemical barrier in the epidermis and dermis that preserve the homeostasis (Hikima et al., 2005). The stratum corneum is a complex material characterized by very low permeability to water and other molecules (Kitson and Thewalt, 2000). Its permeability barrier is mediated by a mixture of ceramides, sterols and free fatty acids arranged as extra cellular lamellar by layers in the stratum corneum. The lamellar body secretor system plays a central role in mediating Proc. VIIth Intern. Congress on Hazelnut Eds.: L. Varvaro and S. Franco Acta Hort. 845, ISHS 2009
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changes in barrier function, and bulk fatty acids for barrier homeostasis are necessary (Mao-Qing et al., 2003). The objective of this study was to advance the general knowledge of the nature of Gevuin oil, with particular attention to its cis-monounsaturated fatty acids (cis-MUFAs) isomers composition. Also to gain a better understanding of the mechanisms that give organic and natural Gevuin oil the properties of a UV filter. In this work we present and discuss new experimental results obtained from 2003 to 2007. MATERIALS AND METHODS Several experiments were carried out at Valdivia (Chile) in the 2003 to 2007 growing seasons. During these five years, 90 samples of two kilos of nuts were harvested for a group of selected Gevuina avellana Mol. clones and then processed for chemical analysis (Medel and Carrillo, 2005). After oil Soxhlet extraction (INN-Chile, 1999) fatty acid methyl esters (FAME) were prepared by reacting the oil in n-hexane with a methanolic solution. Fatty acids (FAs) were determined in a Hewlett Packard 5890 (II) with a flame ionization detector. GC-MSD (GC/MS/FID Varian 3800/Saturn 2000) technique was applied to confirm results by NIST Mass Spectral Library of individual and certified fatty acid methyl esters. Cis-MUFAs, polyunsaturated (PUFAs), saturated fatty acids (SFAs) and unsaturated/saturated ratio were determined. UV absorbance from two genotypes was analyzed with a Milton Roy Array 3000 spectrophotometer, with the aim of obtaining more information that would provide a better understanding of the role of certain fatty acids in relation to their UV filter behaviour. We compared Gevuina oil with Olea europeae var. europeae (olive) and Helianthus annuus L. (sunflower) oils. RESULTS AND DISCUSSION Composition and Synthesis of Gevuin cis-MUFAs More accurate analytical processing for kernel oil from selected Gevuina avellana Mol. (Gevuin) clones, has confirmed average values (Table 1) from previous reports (Medel and Carrillo, 2005). The nuts have a 50% fat content, high in MUFAs and low for PUFAs and SFAs, resulting in a high unsaturated/saturated ratio. The 12 cis-MUFAs described show differences with other reports indicating only 3 to 8 isomers (Karmelic, 1982; Masson and Mella, 1985; Bertoli et al., 1998; Romero et al., 2004), which is explained in part by the determination of trace-compounds (≤0,1%) (Table 2). Thus, Gevuin nut oil has an elevated number of unusual cis-MUFAs composed of 14 to 24 carbons in the acyl chain, with four different groups (C16, C18, C20 and C22). Aitzetmuller (2004) also reported an elevated number of cis-MUFAs isomers (11, and two as traces), indicating that Gevuina nut oil is very different from other known edible oils, belonging to the same group as the seed oils from the genus Grevillea (Plattner and Kleiman, 1977) and other Proteaceae species (Vickeri, 1971). A very rare fatty acid from Gevuina oil is the palmitoleic C16:1∆11 isomer, present in high quantities. This isomer has been found in only a few plants (Aitzetmuller, 2004) but occurs in some fish species, some animals (Brockerhoff and Ackman, 1967), insects (Roelofs et al., 2002), in micro-algae and diatom species (Tonon et al., 2004). It was also found in myxobacteria Cytophaga hutchinsonii by Walker (1969), who named it as “cispalmitvaccenic”). The cis-11-octadecenoic acid (C18:1∆11, vaccenic acid), is found in a wide variety of species including animals and bacteria (Kuemmel and Chapman, 1968; De Mendoza et al., 1982), and in the seed oils of a few species from Asclepiaceae (Chisholm and Hopkins, 1960), Araceae, Umbelliferae, and in fruit pulps (Saglik et al., 2002). Gevuin C20:1 and C22:1 MUFAs isomers are founded in Cruciferae species (Fehling et al., 1990), in fish oils and in marine mammal predators (Brockerhoff and Ackman, 1967; Iverson et al., 2004). Formation of this and other unusual MUFAs is in part possible because soluble desaturases insert a double bond into the acyl-ACP chain that can vary from the standard ∆918:0-ACP in their substrate specificity, regiospecificity, or both (Voelker and Kinney, 620
2001). Tonon et al. (2004) identified a desaturase activity over palmitic acid to form ∆11 palmitoleic acid through the expression of Thalassiossira pseudonana (micro-algae) genes into Saccharomyces cereviseae. ∆11 desaturation is also a common feature in the synthesis of sex pheromones of insects (Roelofs et al., 2002). Accordingly, Aitzetmuller (2004) indicates that in Gevuina a highly active desaturase could transform saturated fatty acids into monounsaturated ones with a ∆11 double bond, regardless of the acyl chain length. However, Schneiter et al. (2000) reported that wild-type cells of S. cerevisiae, when cultivated in myristoleic acid (C14:1Δ9)-supplemented media, synthesized the C16:1∆11 fatty acid through a carbon elongation, while this kind of biosynthetic pathway is operative to form vaccenic acid from 16:1Δ9 (Kuemmel and Chapman, 1968; De Mendoza et al., 1982). Also, fatty acid elongase enzymes allow for the formation of monoenes beyond the usual C18 length, through C20:1 and C22:1 isomers (Salas et al., 2005), as Aitzetmuller (2004) suggests for Gevuina. These occur in Cruciferae (Fehling et al., 1990; Salas et al., 2005) and presumably during oil synthesis of fish species (Stoffel and Ahrens, 1960). Probably, the biosynthesis of C20:1∆9 and C22:1∆11 acids in Gevuin oil is achieved by a desaturation mechanism on acyl-chain substrates longer than C:18, or by carbon elongation (as C22:1∆11 could be formed from C20:1∆9 substrates). Cis-MUFAs and Its UV Radiation Filter Behaviour The particular compositions of cis-MUFAs from Gevuin oil and the nature of its skin serve as a basis to discuss its UV filter behaviour. Its action would be located in the epidermis and stratum cornea, which is composed of dead cells with tightly packed structures and keratinized proteins. The dynamics of some cis-MUFAs as enhancers for a propylene glycol penetration through the skin for topically applied drug treatments, have been studied (Taguchi et al., 1999). Taguchi et al. suggest that penetration increases as the position of the double bond is shifted towards the hydrophylic end (carboxyl head). Fatty acids with a lower ∆/ω rate can induce a structural disorder in the stratum corneum, which in turn allows for an elicited disordering of the epidermis, even when this structure becomes more hydrophilic in depth. Rowat et al. (2006), indicate that oleic acid extracts a fraction of the endogenous stratum corneum membrane components, promoting phase separation in the membrane system and creating more permeable oleic acid-rich domains, this being a plausible mechanism that explains how oleic acid enhances transdermal penetration. Table 3 presents the series of Gevuin oil MUFAs in relation to its greater or lesser penetration behaviour through the epidermis, based on the experiments of Taguchi et al. (1999), who concluded that the percutaneous penetration effect is a function of the ∆/ω relationship in terms of the double bond position, considering also a range for the molecular volume and carbon chain length. Gevuin oil has a number of the fatty acids cited by Taguchi et al. (1999) (oleic, gondoic, palmitoleic and erucic), but also other isomers. Single fatty acids of this broad blend of cis-MUFAs have different penetration ranges, from very high (C20:1∆9, C18:1∆9 and C22:1∆11) to very low (C16:1∆11), which allows for an explanation of its solar UV filter and skin regeneration behaviour. What makes this oil different to other oils from plant species is its abundance of palmitoleic ∆11, having an elevated ∆/ω rate and therefore penetrating just a little, remaining in the superficial layer of the stratum corneum or in the skin pores (Kitson and Thewalt, 2000). This feature does not occur with the nut oil of Macadamia, a relative to Gevuina, which has a minimum content of palmitoleic ∆11 but an elevated content for C16:1∆9 (Vickery, 1971). On the other hand, the penetration behaviour of Gevuina oil could allow it to remain in the strata cornea, a considerable part of the “antioxidant lipid complex”, like the lipid soluble pro-vitamin A (β-carotene), vitamin E (α-tocotrienol) and sterols, which could explain in part the UV filter performance of Gevuin oil. Comparative UV Absorbance Behaviour from Three Seed Oils All of the above leads to the suggestion of an absorbance potential for a wide range of UV radiation, which was tested through spectrophotometer analysis, contrasting 621
seed oils from Olea europeae var. europeae (olive) and Helianthus annus L. (sunflower) with Gevuin oil (Fig. 2). Gevuin oil has an absorbance range starting from 350 nm up to a maximum at 250 nm, with a peak around 280 to 260 nm, although there are differential responses among clones, as previously reported by Medel and Carrillo (2005). Gevuin oil exhibits a greater filter performance against UVB radiation and in part of the UVA and UVC radiation. Previous work reported a high content of α-tocotrienol in Gevuina oil and this may also provide photo-protection to the skin via absorption of hazardous UVB radiation due to its ability to absorb rays at maximum 292 nm (Krol et al., 2000). Few synthetic sunscreen products have this wide range of absorbance, including the Avobenzones (butyl methoxydibenzolmethane), which have photo-stability problems and there is a potential for degradation of other ingredients in commercial sunscreens. Gevuin nut shell (pericarp) extract can have a similar range of UV filter behaviour (Franco et al., 2001), but it yields a considerably lower amount than cotyledonar seed oil. The differential response of Gevuin oil when compared with the other two oils in relation to its solar radiation protection, measured as UVB absorbance, can be interpreted as a function of its chemical composition. Seed oil samples from olive and sunflower (without palmitoleic ∆11 content) had 55 and 15% of oleic acid, respectively. Sunflower oil contains a high content (52.8%) of the cis-polyunsaturated linoleic (C18:2∆9,12) fatty acid, which is much lower in the other two oils (8%, approximately). The last does not have UV filter power, while olive oil cannot remain in the very superficial layers of the skin because it penetrates deeply into the skin (Taguchi et al., 1999; Rowat et al., 2006). CONCLUSIONS The results of this study has provided basic data that enables us to characterize Gevuin oil as an extraordinary natural and organic UV radiation filter. However, its UV filter behaviour and other important properties, such as its healing power, are determined in qualitative and quantitative terms, by an adequate balance mainly among the components in the “antioxidant lipid complex” of the oil. Assessing the availability of a combination of molecules with biological activity for the protection and regeneration of skin was the objective of the research. It was found that the tested clonal selections varied in this characteristic. ACKNOWLEDGEMENTS For economic support and assistance to the 7th International Congress on Hazelnut (Viterbo, Italy) (ISHS), we thank the Genetic and Production Improvement Program of Gevuina, FONDECYT Project n°1060192, and also from the following bureau from UACh: Vicerrectoría Académica, Dirección de Estudios de Postgrado, Dirección de Investigación y Desarrollo, Escuela de Graduados de la Facultad de Ciencias Forestales and Facultad de Ciencias Agrarias. Literature Cited Aitzetmuller, K. 2004. Chilean hazelnut (Gevuina avellana) seed oil. JAOCS 81:721-723. Bertoli, C., Fay, L.B. and Lambelet, P. 1998. Characterization of chilean hazelnut (Gevuina avellana Mol.) seed oil. JAOCS 75(8):1037-1040. Brockerhoff, H. and Ackman, R.G. 1967. Positional distribution of isomers of monoenoic fatty acids in animal glycerolipids. J. Lipid Res. 8:661-666. Chisholm, M.J. and Hopkins, C.Y. 1960. 11-octadecenoic acid and other fatty acids of Asclepias syriaca seed oil. Can. J. Chem. 38(6):805. Curcuff, P., Fiat, F. and Minondo, A.M. 2001. Ultraestructure of the human stratum corneum. Skin Pharmacol. Appl. Physiol. 14(1):4-9. De Mendoza, D., Garwin, J.L. and Cronan Jr., J.E. 1982. Overproduction of cis-vaccenic acid and altered temperature control of fatty acid synthesis in a mutant of Escherichia coli. J. of Bacteriology 151(3):1608-1611. Fehling, E., Murphy, D.J. and Mukherjee, K.D. 1990. Biosynthesis of triacylglycerols containing very long chain monounsaturated acyl moieties in developing seeds. Plant 622
Physiol. 94:492-498. Franco, D., Moure, A., Sineiro, J., Domínguez, H. and Núñez, M.J. 2001. Extracto natural de cáscara de Gevuina avellana como antioxidante/filtro UV para uso alimentario y cosmético. Oficina Española de Patentes y Marcas. ES2157847A1. Hikima, T., Tojob, K. and Maibacha, H. 2005. Skin metabolism in transdermal systems. Skin Pharmacol. and Physiol. 18:153-159. Instituto Nacional de Normalización-Chile, INN. 1999. Norma chilena oficial. Granos o semillas de oleaginosas. Determinación del extracto al éter de petróleo denominado contenido graso. NCh 485 Of. 88. Santiago, Chile. 11p. Iverson, S.J., Field, C., Bowen, W.D. and Blanchard, W. 2004. Quantitative fatty acid signature analysis: a new method of estimating predator diets. Ecol. Mon. 74:211-235. Life Science. The Helios 10-01/02. Jun, L. 2006. Tocotrienols: vitamin E beyond tocopherols in skin protection. Davos Life Science. The Helios 10-01/02. Karmelic, J. 1982. Recolección e industrialización de avellana chilena. INTEC. 87p. Krol, E.S., Kramer-Strickland, K.A. and Liebler, D.C. 2000. Photoprotective actions of topically applied vitamin E. Drug Metab. Rev. 32:413-420. Kitson, N. and Thewalt, J.L. 2000. Hypothesis: the epidermal permeability barrier is a porous medium. Acta Dermato – Venereologica 80(208):12-15. Kuemmel, D.F. and Chapman, L.R. 1968. The 9-hexadecenoic and 11-octadecenoic acid content of natural fats and oils. Lipids 3(4):313.-316. Mao-Qiang, M., Elias, P.M. and Feingold, K. 2003. Fatty acids are required for epidermal permeability barrier function. J. Clin. Invest. 92:791-798. Mason, L. and Mella, M. 1985. Materias grasas de consumo habitual y potencial en Chile. Departamento de Química y Tecnología de Alimentos, Universidad de Chile. Medel, F. 2001a. Gevuina avellana: potential for commercial nut clones. Acta Hort. 556:521-528. Medel, F. 2001b. Genetic and production improvement of Gevuina avellana Mol. in Chile: selected clones for nut production. Nucis Newsletter 10:17-20. Medel, F. 2005. Clonal selection in Gevuina avellana for nutritional and phytotherapy purposes. Acta Hort. 686:625-630. Medel, F. and Carrillo, T. 2005. Variability of total fat and fatty acids in nut oil from Gevuina avellana clones. Acta Hort. 686:631-640. Medel, F. and Medel, R. 2000. Gevuina avellana Mol.: características y mejoramiento genético de un frutal de nuez nativo para el mercado internacional. Rev. Frutícola 21:37-47. Nicolaides, N. 1974. Skin lipids: Their biochemical uniqueness. Science 186:19-26. Plattner, R.D. and Kleiman, R. 1977. Grevillea robusta Seed Oil: A Source of Omega-5 Monoenes. Phytochemistry 16:255-256. Roelofs, W.L., Liu, W., Hao, G., Jiao, H., Rooney, A.P. and Linn Jr., C.E. 2002. Evolution of moth sex pheromones via ancestral genes. PNAS 99:13621-13626. Romero, N., Robert, P., Masson, L., Ortiz, J., Pavez, J., Garrido, C., Foster, M. and Dobarganes, C. 2004. Effect of α-tocopherol and α-tocotrienol on the perfomance of Chilean hazelnut oil (Gevuina avellana Mol.) at high temperature. J. Sci. Food. Agric. 84:943-948. Rowat, A.C., Kitson, N. and Thewalt J.L. 2006. Interactions of oleic acid and model stratum corneum membranes as seen by 2H NMR. Int. J. Pharm. 307(2):225-231. Saglik, S., Alpinar, K. and Imre, S. 2002. Fatty acid composition of Dracunculus vulgaris Schott (Araceae) seed oil from Turkey. J. Pharm. Pharmaceut. Sci. 5(3):231-239. Salas, J.J., Martínez-Force, E. and Garcés, R. 2005. Very long chain fatty acid synthesis in sunflower kernels. J. Agric. Food Chem. 53:2710-2716. Schneiter, R., Tatzer, V., Gogg, G., Leitner, E. and Kohlwein, S.P. 2000. Elo1pdependent carboxy-terminal elongation of C14:1Δ9 to C16:1Δ11 fatty acids in Saccharomyces cerevisiae. J. of Bacteriology 182(13):3655-3660. Stoffel, W. and Ahrens, E.H. 1960. The unsaturated fatty acids in menhaden body oil: the 623
C18, C20 and C22 series. J. Lipid Research 2(1):139-146. Taguchi, K., Fukushima, S., Yamaoka, V., Takeuchi, Y. and Suzuki, M. 1999. Enhancement of propylene glycol distribution in the skin by high purity cisunsaturated fatty acids with different alkyl chain lengths having different double bond position. Biol. Pharm. Bull. 22(4):407-471. Tonon, D.H., Qing, R., Li, Y., Larsen, T.R. and Graham, I.A. 2004. Identification of a fatty acid ∆11-desaturase from the microalga Thalassiossira pseudonana. FEBS Letters 263:23-24. Vickery, J.R. 1971. The fatty acid composition of the seed oils of Proteaceae. Phytochemistry 10:123-130. Voelker, T. and Kinney, A.J. 2001. Variations in the biosynthesis of seed-storage lipids. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52:335-361. Walker, R.W. 1969. Cis-11-hexadecenoic acid from Cytophaga hutchinsonii lipids. Lipids 4(1):15-18.
Tables
Table 1. Mean values of fatty acids and unsaturated/saturated ratio in Gevuin nut oil.
* Mean value from 90 samples (2003 to 2007).
Table 2. Mean values of cis-monounsaturated fatty acids in Gevuin nut oil.
Traces (
