1 In ternationa l Scholars Journa ls African Journal of Food Science Research ISSN 2375-0723 Vol. 9 (6), pp. 001-007, June, 2021. Available online at www.internationalscholarsjournals.org © International Scholars Journals Author(s) retain the copyright of this article. Full Length Research Paper Seasonal variation of fixed and volatile oil percentage of four Eucalyptus spp. related to lamina anatomy Kh. S. Emara1 and A. Emad Shalaby2* 1 Department of Agricultural Botany, Faculty of Agriculture, Cairo University, Giza, Egypt, 12613. 2 Department of Agricultural Biochemistry, Faculty of Agriculture, Cairo University, Giza, Egypt, 12613. Accepted 21 January, 2021 This experiment was conducted during the four seasons: Spring, summer, autumn and winter of two successive annual cycles; 2008/2009 and 2009/2010 (starting from May 2008). Four Eucalyptus species were under investigation; Eucalyptus camaldulensis Dehnh., Eucalyptus cinerea F. Muell. ex Bentham, Eucalyptus citriodora Hook. and Eucalyptus globulus Labill. Seasonal variations in the amount of fixed and volatile oils in Eucalyptus spp. matured leaves were investigated. It was determined that the amount of total lipids and essential oils significantly varied by the seasons (P < 0.01). The amount of total lipids in Eucalyptus spp. reached its peak mostly in spring. But the amounts of essential oils in different species were determined to be higher in summer, autumn and spring seasons, than in winter. Furthermore, the amount of total lipids and essential oils was higher in E. camaldulensis and E. cinerea than in other species. The anatomical investigation in the four studied Eucalyptus species, in relation to lipids percentage indicated that, the best lipids percentage amounts in this study were exhibited in E. cinerea and E. camaldulensis, for spring and winter; and were in agreement with these species highest lamina thickness. In general, fluctuation in lipids percentage is more correlated to the internal structure of lamina (duct average diameter, ducts total numbers, and open ducts numbers) in the same season; whereas, among seasons, it is thought that metabolism contributed more greatly. Cuticle thickness is true correspondence to seasonal environmental fluctuation, since it increases in all species, by shifting up from spring to summer then decrease to winter. Essential oils secretion which coincided with lipids percentage may be due to environmental stress influence over metabolism rather than structural adaptation. Key words: Eucalyptus, seasons, fixed and volatile oils, lamina, anatomy, glands. INTRODUCTION Eucalypti species are mostly evergreen trees of immense size; around 800 species belong to the Magnoliopsidous family, Myrtaceae and native mainly to Australia and some neighboring islands (Bailey, 1958; Cronquist, 1981; Heywood, 1993; Brooker, 2000). They are grown for their ornamental values, as windbreaks, for timber and fuel, and for oil, distilled from leaves, which is secondary compounds with pleasant aroma used as fragrance components in soap, detergents *Corresponding author. E-mail: dremad2009@yahoo.com. Tel: +202-0101203313. Fax: +202-37742600. and toiletries, and also have pharmacological properties (Santos et al., 2008). Eucalyptus oil is being extracted in many countries like China, India, South Africa, Portugal, Brazil and Tasmania on commercial scale. In 1992, the world Eucalyptus oil production was estimated to be 4000 tons of which 60 to 70% was consumed in medicinal market (Bhatti et al., 2007). Eucalyptus leaves oil amounting to less than 20% is enough in the commercial point of view, and only 10% of these accounts for the entire world production of essential oil (Peter, 2000). Essential oils are complex mixtures which comprised primarily of the lower classes of terpenes (Julia, 1992; Langenheim, 1994). The secondary compounds are probably co-evolution result from interaction of plants 2 Table 1. The effect of different seasons on the total lipids content (%) of the four studied Eucalyptus spp. during the two successive annual cycles, 2008/2009 to 2009/2010. Season Species E. camaldulensis E. cinerea E. citriodora E. globulus Spring 18.0 b 18.75 a 16.0 a 13.0 a Summer 13.2 c 13.5 d 11.0 c 11.75 c Autumn 13.0 c 13.75 c 10.75 d 11.25 d Winter 19.0 a 15.75 b 14.75 b 12.75 b LSD at 0.01 0.431 0.183 0.236 0.174 Values with different superscript letters within the same column are significantly different (P < 0.01). versus pathogens, herbivores, pollinators (Croteau et al., 2000). Although monoterpenes predominate in most essential oils, many also contain sesquiterpenes. Many sesquiterpenes play defensive roles in plant-insect and plant-fungal interactions (Samuelsson, 1999). Oil concentration is limited by gland capacity and density, which in turn is linked to leaf area reduction and increment in its thickness; thicker leaves were more likely to be smaller in area than thinner leaves, suggesting the expansion of leaves in one plane to a degree mutually exclusive of expansion in the other. Aside glands, probable sink of hydrocarbon monoterpenes in the leaves may be the leaf cuticle through which significant portion of monoterpenes is volatilized (King et al., 2006). Thicker epidermis and presence of two or more layers of palisade cells in leaves are the xeromorphic features adapting to water limited environments like drought (Iftikhar et al., 2009). Mediterranean climate is characterized by the incidence of two stress periods; summer drought and winter cold (Nahal, 1981). Seasonal variations in the composition and concentration of sequestered oils have been observed (Silvestre et al., 1997). There are several reports that the concentration of terpenoids in aromatic plants varies during the day and seasons (Hendriks et al., 1997). The aim of this study is to investigate the effect of seasonal fluctuations over the year on oil yield, fixed and volatile, of perennial evergreen tree (Eucalyptus), related to varying anatomical features in order to suggest best species (among studied ones) and season for harvesting oil from leaves. MATERIALS AND METHODS This experiment was conducted during four seasons; spring, summer, autumn and winter of two successive annual cycles; 2008/2009 and 2009/2010, at the laboratories of Agricultural Botany and Agricultural Biochemistry Departments, Faculty of Agriculture, Cairo University. Collection of samples The used plant materials were four Eucalyptus species (Quattrocchi, 2000); red gum (Eucalyptus camaldulensis Dehnh.), Argyle apple ( Eucalyptus cinerea F. Muell. ex Bentham), Lemon- scented gum (Eucalyptus citriodora Hook.) and blue gum (Eucalyptus globulus Labill.). The leaf materials were gotten from the gardens of the Faculty of Agriculture, Cairo University. Fully expanded mature leaves were cut at the mid-date of each season (starting from May 2008) and gathered early in the morning. The leaves were pre-cooled (4°C) and wrapped in Kraft paper in bunches, and then transported under dry conditions to the laboratories within two hours. Seasonal effects on essential oils and lipids content (percentage) of different Eucalyptus species were investigated, and then correlated with lamina anatomical correspondence to affected by climatic fluctuation over the seasons. Extraction and determination of total lipids Lipids were extracted by a modified method described by Xu et al. (1998). The cells (ca. 5 g) were extracted twice with a mixture of distilled water (H2O), chloroform and methanol (8:10:20, v/v/v) and sonicated for 10 min using a microtop of Microson Ultrasonic cell disrupter. Then, the sonicated cells were filtered through GF/C Whatman glass microfiber (47 mm). Chloroform (10 ml) and distilled water (10 ml) were added sequentially to the filtrate and sonicated again for 10 min. The resultant solution was filtered under vacuum through Whatman glass filter microfiber. The filtrate was washed by 30 ml of 5% NaCl solution, and then, the chloroform layer was separated and dried over anhydrous sodium sulfate. The solvent was removed through evaporation at 40°C under reduced pressure. Then, the total lipids were weighed and stored at -20°C until analysis. Essential oil extraction Plant samples (100 g) were hydro-distilled in Clevenger-type apparatus (Council of Europe, 1997). The essential oil samples were stored in the dark at 4°C. The amount of oil (Table 1) obtained from plant material was calculated as: Oil (% v/w) = Observed volume of oil (ml)/Weight of sample (g) × 100 Anatomical studies Leaves were randomly chosen as specimens for anatomical study over the four successive seasons of the year. The micro technique procedures given by Nassar and El-Sahhar (1998) were applied. Specimens were killed and fixed for at least 48 h in formalin aceto alcohol (FAA) solution. Then, materials were washed in 50% ethyl 3 Table 2. The effect of different seasons on essential oil content (%) of the four studied Eucalyptus spp. during the two successive annual cycles, 2008/2009 to 2009/2010. Season Species E. camaldulensis E. cinerea E. citriodora E. globulus Spring 1.78c 1.35c 0.60c 0.10c Summer 2.5a 1.95a 1.8a 0.66a Autumn 1.92b 1.50b 1.05b 0.23b Winter 0.98d 1.0d 0.25d 0.05d LSD at 0.01 0.033 0.086 0.033 0.028 Values with different superscript letters within the same column are significantly different (P < 0.01). alcohol and dehydrated in normal butyl alcohol series before being embedded in paraffin wax (mp 52 to 54°С). Transverse sections, which were cut on a rotary microtome to a thickness of 20 , were stained with safranin/light green before mounting in Canada balsam. Slides were examined; measurements of different tissues were recorded using light microscope with micrometer eye piece and micrometer stage, and then photomicrographed using Microscope Olympus AX70 made in Japan. The following anatomical characters were measured: Cuticle thickness (µ), lamina thickness (µ), opens ducts number (in 40 x field), ducts total number [in 10x (eye lens) Х 4x (object lens) field] and duct diameter (µ). Statistical analysis Data were subjected to an analysis of variance. Combined analysis was used for the two annual seasonal cycles. Means were compared by calculating the least significant difference (LSD) values at alpha 1% according to Snedecor and Cochran (1982). RESULTS AND DISCUSSION Chemical studies The amount of total lipids obtained from various Eucalyptus spp. during different seasons was investigated, and it was observed that values were between 10.75 to 19.0%. It scored the minimum level in autumn season and increased significantly, till it reached the maximum at spring season in all studied species, except for E. camaldulensis which had maximum value in winter (Table 1). Species E. camaldulensis and E. cinerea reflect best lipids amount (19 and 18.75%, respectively). The variations of total lipid contents were affected by the species, age, water temperature, nutritional condition and seasonal variation, as confirmed by Folch et al. (1957), Gill and Weatherley (1984), Lathi (1987) and Christiansen et al. (1989). Recent studies have shown that lipids and fatty acids composition was influenced by seasonal variations (Yılmaz et al., 1995). The same trend shows that, the variations of the levels of lipids are the results of irregular seasonal variations and water temperature was determined by Dutta et al. (1985), Agren et al. (1987) and Yılmaz et al. (1995). From Table 2, it can be seen that the content of essential oils in different seasons varied from 0.05% during winter to 2.5% during summer season. Best essential oils percentages were estimated in E. camaldulensis and E. cinerea (2.5 and 1.95% respectively) in the summer season. Data from Table 2 indicated that, the level of these components reached its maximum in all studied plant species during the summer season and had the lowest value during winter, and this may be due to the physical and chemical stress on plant especially during summer drought. This stress led to plant secretion to different defense components called secondary metabolites, as protecting agents, especially terpenoid compounds (essential oils). These results are in agreement with the results obtained by Samuelsson (1999). As to the significance estimates, more consistent trend in percentages variation of Eucalyptus spp. during different seasons are present for essential oils than for lipids, and this further confirm the formers are due mainly to the effect of environmental condition on plant synthesis. Wildy et al. (2000) investigated four promising Eucalyptus species reported 0.01 to 13.0% for oil production, from Western Australia at six locations. Also, Zafar et al. (2003) reported similar results; that is, 0.58 to 1.47%, regarding oil potential for the different Eucalyptus species. These variations might be attributed to different agro-climatic regions and soil composition in the districts of Punjab. Anatomical studies Aiming to explain the seasonal differences affecting lipids content (%) of the four studied species, microscopical counts and measurements of certain characters were recorded to investigate the internal differences in lamina of these species. The most important criteria in this study were to compare fluctuations in glands (number and size) over seasons, in random specimen’s descent by half meter from the top, in addition to measure the cuticle and lamina thickness as correspondence to heat/cold stress. Microphotographs of transverse sections illustrating four studied Eucalyptus spp. (Plate 1) showed that 4 Plate 1. Transverse sections in leaves of the four studied Eucalyptus spp. (X = 9). mesophyll of adult vertically positioned lamina is often isobilateral, with cells containing tannin abundant in all unlignified tissues and bicollateral vascular bundles. Epidermis coated by wax lamina characterized by the presence of secretory cavities, which are found below the epidermis on either sides of the lamina. Cavities are schizolysigenous in origin; lined with distinct epithelium when young which soon is obliterated by compression. Cavities are responsible for secreting oily substances. Data presented in Table 3 and Plate 1 for the leaf specimens subjected to histological investigation cleared that as typical correspondence to seasonal environmental fluctuation, cuticle thickness increase in all species, by shifting from spring to summer, then decrease to winter, 5 Table 3. Different tissue counts and measurements (µ) in lamina of the four studied Eucalyptus spp. during the two successive annual seasonal cycles, 2008/2009 to 2009/2010 (average of 5 readings). Season Measurement (µ) Species E. camaldulensis E. cinerea E. citriodora E. globulus Average cuticle thickness 18.33 10.83 9.17 9.17 Average lamina thickness 466.67 323.33 423.33 213.33 Spring Open ducts number 7 6 1 2 Ducts total number 9 6 3 3 Duct average diameter 135 152.5 125 85 Average cuticle thickness 25 13.33 15 10.83 Average lamina thickness 456.67 290 400 290 Summer Open ducts number 5 3 4 3 Ducts total number 7 5 4 3 Duct average diameter 157.5 172.5 115 150 Average cuticle thickness 16.67 9.17 15 10 Average lamina thickness 430 290 400 333.33 Autumn Open ducts number 5 5 4 3 Ducts total number 6 6 4 3 Duct average diameter 147.5 172.5 105 117.5 Average cuticle thickness 13.33 8.33 15 6.67 Average lamina thickness 463.33 350 486.67 290 Winter Open ducts number 3 3 3 4 Ducts total number 5 5 5 5 Duct average diameter 187.5 140 125 110 except for E. citriodora which also increase in summer (from 9.17 µ) but score the same thickness throughout the rest two subsequent seasons (15 µ). Lamina thickness varied over the seasons (by shifting, in samples, from spring to winter) for various studied species by what could be named 3 types; decreasing- sustaining-increasing type as in E. cinerea and E. citriodora; increasing-increasing-decreasing type as in E. globulus and finally, decreasing-decreasing-increasing type as in E. camaldulensis. In harmony with estimated lipid amount percentage, E. cinerea and E. camaldulensis exhibited their highest lamina thickness in spring and winter. In a trial to investigate the association between lipids percentage and the most direct contributed anatomical features to it, average diameter and number of glands were studied. In the same season, referring to Table 3, Plate 1 and Figure 1, it could be realized that, average diameter, total number of ducts, and also, open ducts number have positive relation with lipids percentage, for all species understudied. Over seasons, for each individual species, it could be determined that, open ducts number (also total number) showed limited range. Relation over seasons could not be distinguished as clear as in the same season, due to inconsistent expression of these features. It could be interpreted that, physiological balance between anabolism and catabolism activities, due to seasonal growth rate, has the principal role in determining the amount of stored lipids (within cavities) over seasons. Also, it could be noticed that, mostly, duct average diameter increases by removing from spring to summer samples, then decreased over autumn to winter. As coincide to this relation, E. citriodora decrease in summer and increase in winter, whereas, duct average diameter of E. camaldulensis increase in both summer and winter samples. The anatomical investigation in the four studied Eucalyptus species, in relation to lipid percentage indicated that, fluctuation in lipids percentage is more correlated to internal structure of lamina (ducts average diameter, ducts total numbers and open ducts numbers) in same season, whereas between seasons it was thought that, metabolism contributed more greatly. Best lipid percentage amounts in E. cinerea and E. camaldulensis in spring and winter, and were in agreement with these species highest lamina thickness. Cuticle thickness is true correspondence to seasonal environmental fluctuation, since it increases in all species, by shifting from spring to summer, and then decrease to winter. 6 Figure 1. Magnified portion of transverse sections in lamina of the most higher studied Eucalyptus spp. in lipids amount for optimal season (X = 28). As regards essential oils, it was clear that they coincides lipids percentage since their highest amount was estimated in summer, and then autumn, whereas lowest amounts were in winter, and spring, for all studied species. This may confirm that, essential oils secretion is due to environmental stress influence over the metabolism rather than structural adaptation. The previously mentioned structure of Eucalyptus lamina is in agreement with those given by Metcalfe and Chalk (1950) as well as by Bailey (1958), King et al. (2006), Santos et al. (2008) and Iftikhar et al. (2009). Conclusion These studies confirmed that, Eucalyptus spp. Is a potential source of oil (volatile and fixed) around the year, with regard to significant fluctuation in oils yield due to subsequent seasonal climatic conditions. Putting economical factor into consideration, E. camaldulensis and E. cinerea showed best amount of total lipids and essential oils than other studied species. For purposes mostly industrial, yielding these two species in spring and winter was best (biggest lipid percentages (%)), whereas, for other usages, mostly medical, extracting rich essential oil yield is more preferable in summer and autumn. The anatomical investigation indicated that, fluctuation in lipids percentage was more correlated to internal structure of lamina (ducts average diameter, ducts total numbers, and open ducts numbers) in same season, whereas between seasons it was thought that, metabolism contributed more greatly. Cuticle thickness is a true correspondence to seasonal environmental stress; same applies to essential oils secretion, which increased from spring to summer, and then decrease in winter. REFERENCES Agren J, Mute P, Hannınen O, Harranen J, Pentila I (1987). Seasonal variations of lipid fatty acids of Boreal freshwater fish species. Comp. Biochem. Physiol., 88B: 905-909. Bailey LH (1958). The Standard Cyclopedia of Horticulture. The Macmillan Company, New York, USA, p. 3640. Bhatti HN, Iqbal Z, Chatha SA, Bukhari IH (2007). Variations in Oil Potential and Chemical Composition of Eucalyptus crebra Among Different Districts of Punjab–Pakistan. Int. J. Agric. Biol., 9(1): 136- 138. Brooker MIH (2000). A new classification of the genus Eucalyptus L’Hér. (Myrtaceae). Aust. Syst. Bot., 13: 79-148. Christiansen JS, Ringo E, Farkas T (1989). Effect of sustained exercise on growth and body composition of first feeding fry of Arctic charr, Salvelinus alpines (L.). Aqua., 79: 329-335. Council of Europe (1997). Council of Europe, European Pharmacopoeia 3rd ed, Council of Europe, Strasbourg, France, p. 253. Cronquist A (1981). An Integrated System of Classification of Flowering Plants. Columbia Univ. Press, N.Y., U.S.A., p. 1282. Croteau R, Kutchan TM, Lewis NG (2000). Natural products (Secondary metabolites). American Society of Plant Physiologists, Rockville, Maryland, p. 25. Dutta H, Das AB, Farkas T (1985). Role of environmental temperature in seasonal changes of fatty acid composition of hepatic lipid in an airbreathing Indian teleost, Channa punctatus (Bloch). Comp. Biochem. Physiol., 81(B): 341-347. Folch J, Lees M, Sladane-Stanley GHA (1957). Simple method for the isolation and purification of total lipids from animal tissues. J. Biol. 7 Chem., 226: 497-509. Gill HS, Weatherley AH (1984). Protein, lipid and caloric content of bluntnose minnow, pimephales notatus, rafinosque, during growth at different temperatures. J. Fish Biol., 25: 491-500. Hendriks I, Anderson WY, Engle G, Bos R, Woerdenbag (1997). J. Planta Med., 63: 356-362. Heywood V (1993). Flowering Plants of the World. BT Batsford Ltd. London, UK, p. 336. Iftikhar A, Abbas SQ, Hameed M, Naz N, Zafar S, Kanwal S (2009). Leaf anatomical adaptations in some exotic species of Eucalyptus l'hér (Myrtaceae). Pak. J. Bot., 41(6): 2717-2727. Julia L (1992). The Encyclopaedia of Essential Oils.1 st ed. Element Books Limited, Longmead Shaftebury, Dorset, p. 178. King DJ, Gleadow RM, Woodrow IE (2006). Regulation of oil accumulation in single glands of Eucalyptus polybractea. New Phytol., 172: 440-451. Langenheim JH (1994). Higher plant terpenoids: a phytocentric overview of their ecological roles. J. Chem. Ecol., 20: 1223-1280. Lathi E (1987). Total Lipid and Cholesterol of Liver and Muscle in Some Fish Species, Especially Vendace (Coregonus albula L.) in Finland. Arch. Hydrobiol., 110: 133-142. Metcalfe CR, Chalk L (1950). Anatomy of the Dicotyledons; Leaves, Stem and Wood in Relation to Taxonomy with Notes on Economic Uses. The Clarendon Press, Oxford, UK, 1: 790. Nahal I (1981). The Mediterranean climate from a biological viewpoint. Elsevier, Amsterdam, pp. 63-86. Nassar MA, El-Sahhar KF (1998). Plant Microtechnique. Academic Bookshop, Egypt, p. 224 (In Arabic). Peter SA (2000). The Eucalyptus oil industry. Felton Grimmwade Bickford Pvt. Ltd. 61 – 69. Clarinda Road Oakleigh South Vic. 3169 Australia. Health, 29: 368-371 Quattrocchi U (2000). CRC World Dictionary of Plant Names. CRC Press, Washington D.C., USA, p. 2298. Samuelsson G (1999). Drugs of Natural Origin, A Textbook of Pharmacognosy, Swedish Pharmaceutical Press, p. 256. Santos LD, Thadeo M, Iarema L, Meira RM, Ferreira FA (2008). Foliar anatomy and histochemistry in seven species of Eucalyptus. R. Árvore, Viçosa-MG, 32(4): 769-779. Silvestre AJD, Cavaleiro JAS, Delmond B, Filliatre C, Bourgeois G (1997). Analysis of the variation of the essential oil composition of Eucalyptus globulus Labill from Portugal using multivariate statistical analysis. Ind. Crops Prod., 6: 27-33. Snedecor GW, Cochran WG (1982). Statistical Methods. The Iowa State Univ. Press., Ames., Iowa, USA, p. 507. Wildy D, T. John SP, Bartle JR (2000). Variation in composition and yield of leaf oil from alley framed oil mallees (Eucalyptus spp.) at a range of contrasting sites in Western Australia. For. Ecol. Manage., 134: 205-217 Xu X, Beardall J, Hallam DN (1998). Modification of fatty acid composition in halophilic antractic microalgae. Phytochem., 49: 1249- 1252. Yilmaz Ö, Konar V, Celik S (1995). Elazig Hazar Lake Capoeta some tissues of female and male members who umbla'nℵn capoeta total lipid and fatty acid compositions. Biochem. J., 20: 31-42. Zafar I, Hussain I, Hussain A, Ashraf MY (2003). Genetic variability to essential oil contents and composition in five species of Eucalyptus (NIAB), Faisalabad, Pakistan. Pakistan J. Bot., 35: 843-852.