Agronomic and Phytochimic Characteristics in Some Genotypes of Ocimum basilicum L.

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1 Agronomic and Phytochimic Characteristics in Some Genotypes of Ocimum basilicum L. M. Macchia 1, A. Pagano 1, L. Ceccarini 1, S. Benvenuti 1, P.L. Cioni 2, and G. Flamini 2 1 Dipartimento di Agronomia e Gestione dell Agroecosistema, Pisa University, Italy. 2 Dipartimento di Chimica Bioorganica e Biofarmacia, Pisa University, Italy. Keywords: cultivar, growth stage, germination, linalool, eugenol, methyl eugenol Abstract The recent news regarding the true or presumed toxicity of basil has raised a certain interest and necessity to better understand the agronomic characteristics and chemical composition of the numerous botanical accessions cultivated in our country. The aim of these trials was to investigate the morphological characteristics and the chemical composition of ten genotypes. The results showed many differences among genotypes: four phenotypes were identified on the basis of habit, life characteristics and plant height. The composition of the essential oils included different chemotypes, but all the cultivars were characterized by high content of linalool. Differences in chemical composition were also found during the different growth stages and they could suggest different harvest times according to the use. INTRODUCTION The genus Ocimum includes over 60 herbaceous and bush species, which differ in shape, growth habit, color and essential oil composition (Bown, 1999); in addition, the morphological differences among the species have been accentuated by centuries of cultivation in numerous diverse areas of the world. At present, the species Ocimum basilicum is renowned for its gastronomic uses, in particular for preparation of pesto, a sauce originally created in Liguria but now famous throughout the world (Minuto, 2000.) Numerous different chemotypes of basil (Ocimum basilicum L), which vary in essential oil composition depending on the geographic area of provenance, have been identified and classified (Vernin, 1984; Marotti et al., 1996). The essential oil is used mainly by the food and cosmetic industries, but it is also endowed with antimicrobial and antifungal activity (Prasad et al., 1985). However, reports concerning the toxicity of basil due to the presence of compounds derived from the phenylpropane biosynthetic pathway have appeared (Miele et al., 2001). The purpose of this research was thus to assess this possibility, and also to improve agronomic and chemical knowledge on the numerous botanical accessions grown in Italy. MATERIALS AND METHODS Plant Material Seeds of 10 cultivars of Ocymum basilicum, obtained from S.A.I.S. (Società Agricola Italiana Sementi), were sown in May in 30 cm diameter pots with capacity of over 5 litres and filled with same substrate (Humin Substat N3). Plants were grown outdoors under full sunlight and they were watered daily. The harvest of whole plants was performed at the vegetative stage, at the beginning and at the end of the flowering stage. The morphological characteristics were observed for each basil cultivar. A representative sample obtained for each harvest was used for essential oil extraction and analysis. Essential Oils Analyses The essential oils were obtained by hydrodistillation for two hours in a Clevengerlike apparatus of the dried crushed material (100 g). GC/EIMS analyses were performed with a Varian CP-3800 gas-chromatograph equipped with a DB-5 capillary column (30 m x 0.25 mm; coating thickness 0.25 mm) and a Varian Saturn 2000 ion trap mass detector. Proc. I st IC on Labiatae Eds.: B. Ruffoni et al. Acta Hort. 723, ISHS

2 Analytical conditions: injector and transfer line temperatures at 220 and 240 C respectively; oven temperature was programmed from 60 C to 240 C at 3 C/min; carrier gas helium at 1 ml/min; split ratio 1:30. Identification of the constituents was based on comparison of the retention times with those of authentic samples, comparing their Linear Retention Indices relative to the series of n-hydrocarbons, and by computer matching against commercial (NIST 98 and ADAMS 95) and home-made library mass spectra built up from pure substances and components of known essential oils and MS literature data (Adams, 1995). Moreover, the molecular weights of all the identified substances were confirmed by GC/CIMS, using MeOH as CI ionizing gas. The experimental data were statistically evaluated using analysis of variance (ANOVA). Significantly different means were separated at 0.05 and 0.01 probability level by the Least Significant Difference (LSD) test (Gomez and Gomez, 1984) RESULTS AND DISCUSSION Morphological Characteristics On the basis of the main morphological characteristics recorded during the trial period, the cultivars under examination were identified and grouped as follows: Genovese (cultivars 1, 5, 10): characterized by upright growth habit, roughly cm tall, branching, with "spoon-like oval shape leaves, fairly crinkly, intensely green, with clearly visible veins. Ornamental (cultivars 2, 4): characterized by the presence of anthocyanin pigmentation both on the leaves and stem; the leaves were oval elongate, with accentuated veins; colour varies from intense green (cv. 2) to ruby red (cv. 4). Height at flowering time was about 25 cm. Flowers were pink, in contrast to all other cultivars, which had white flowers. Small leaf (cultivars 3, 6, 9): small plants with a more compact growth habit, variable height ranging from 20 to 30 cm, with small lanceolate, brilliant green leaves. In particular, in cv. 9, the leaves had an intense lemon-like aroma. Large leaf (cultivars 7, 8): the plants had large oval leaves, with a crinkly lamina that felt thicker to the touch. Chemical Characterization Essential oil yield showed significant differences in relation both to the cultivar and the plant phenologic stage at harvest (Fig. 1). The cultivars which, on average, were richest in essential oil were 1, 7, 9 and 10, while those with significantly lower percentages were 2, 3, 4 and 8. In general, the phenologic stage giving the highest yield was the post-flowering stage, but some showed identical yields, regardless of the different sampling stages. The different chemotypes highlighted by the investigation on the ten basil cultivars revealed a considerable variability in chemical composition of this species (Table 1). Structures deriving from the mevalonic acid pathway through the isopentenyl pyrophosphate as well as compounds deriving from the phenyl-propane pathway were found. The terpenes included hydrocarbon monoterpenes, oxygenated compounds (mainly oxides, alcohols and aldehydes) and also sequiterpenes, The latter were mainly represented by hydrocarbons and oxygenated compounds). Among the phenyl-propane derivatives, eugenol and methyl-eugenol were observed, sometimes in notable percentages. Taking into consideration the compounds of greatest interest both from the quantitative and qualitative point of view, the cultivars examined can thus also be classified in terms of their chemical composition. For cv. 3, in all stages sampled, and for cv. 8, but only during the growth stage, no compound was recorded in a sufficient amount to characterize a precise chemotype. 144

3 Linalool Examination of the percentages of linalool in the various cultivars and in the three developmental stages of O. basilicum plants showed that linalool was the main compound present in at least two out of the three sampled phenological stages, with the exception of cultivar 9, in which linalool was absent (Fig. 2 a). In cultivars 2, 3, 7 and 8 the percentage of linalool increased progressively from the vegetative growth stage to the flowering stage, and then to the post-flowering one, rising from 14.2% in cv. 3 to an elevated value of 78.7% in cv. 7. In 1, 4 and 10, the percent increase in linalool ceased during the flowering stage, decreasing thereafter during post-flowering. The only exception to these trends was observed in cv. 6 Fine Verde, where the greatest linalool concentration was recorded during the vegetative growth stage, thereafter decreasing progressively during the flowering and post-flowering stages. Eugenol During the vegetative growth stage the cultivars in which the highest values of eugenol were recorded proved to be cv. 10, which showed 18.5%; cv. 3 with 13.2%; cv. 7 with 6.2%; and cv. 5 with 4.9% (Fig. 2 b). During the flowering stage the greatest concentrations were found in cv. 5 with 35.2%, cv. 6 with 29.6%, cv. 10 with 29.0%, cv. 7 with 24.3% and cv. 2 with 10.3%. During post-flowering, the highest eugenol content was found in cv. 10 with 9.0%, and cv. 1 with 4.2%. Taking into account the variations in eugenol content during the phenologic cycle, it can be noted that with the exception of cv. 1, the phenologic stage that consistently showed the greatest accumulation of this compound was the flowering stage, while concentrations generally declined during postflowering to values not exceeding 1%. Methyl Eugenol During the growth stage, the cultivar with the highest percentage of methyl eugenol was cv. 2 (7.9%), followed by cv. 8 (6.2%), while this compound was absent in 6, 7, 9 and 4 (Fig. 2 c). During the flowering stage, cv. 2 again had the greatest quantity of methyl eugenol (6.7%), while a marked decrease was observed in the other cultivars. During the post-flowering stage, only cultivars 2, 8 and 10 presented methyl eugenol percentages below 1%. These findings demonstrate that, as for the previously examined compounds, the amount of methyl eugenol also varies in relation to the developmental stage of the plant material used for the extraction of the essential oil, with quantities decreasing to virtually zero in the transition from the growth stage to the after flowering stage. CONCLUSIONS Our study highlighted marked differences both in morphological characteristics and chemical composition of the various botanical accessions under study. From the economic importance point of view, the two Genovese accessions were clearly those of greatest interest: a careful description however, may allow previously ignored varieties to be included in the national varietal lists and/or in regional repertories of varieties. Such lists were drawn up initially only for investigative purposes, but they could be officialized in the near future and could be made usable for commercial purposes with the introduction of specific regulations. A region such as Liguria, which is particularly interested in basil growing and processing, could utilize the data furnished in the present report both for inclusion of varieties in the above-mentioned varietal lists and also to promote better utilization of the product, with particular attention to the chemical characteristics displayed by these accessions in their different phenological stages. In this perspective, it is important to note that in addition to the morphological differences characterizing the various accessions tested, the chemical analyses performed in our study revealed that the chemical composition likewise differed according to the phenologic stage. This suggests that 145

4 different times and manner of harvesting could be adopted depending on the type of utilization and characterization desired. According to another paper (Marotti et al.,1996), it is interesting to note that in some cases the cultivars with similar morphological characteristics, such as has plants of the 2 Ornamental and 4 Large leaf groups, can be ascribed to the same chemotype. Conversely, in the case of 1 Genovese and 3 Small leaf groups, the chemical features were very different, but the morphological characteristics were quite similar. The problem concerning the presence of methyl eugenol in processed basil products is no major drawback for two reasons. Firstly, on the basis of the detected amounts of methyl eugenol, the quantities that should be ingested to reach hazardous levels are so high that even continuous and intense use of the various pesto sauces would not in any way represent a health threat. Secondly, we can confirm that by varying the harvesting time a virtually methyl eugenol-free product can be obtained, although the visual characteristics of the product could involve a darker and less brilliant color green. Literatured Cited Adams, R.P Identification of essential oil components by Gas Chromatography / Mass Spectroscopy. Allured Publ. Corp., CarolStream, Illinois. Bown, D La biblioteca della natura. Piante aromatiche. Dorling Kindersley & R.C.S. Libri S.P.A., Milano Gomez, K.A.and Gomez, A.A Statistical procedures for agricultural research. John Wiley & Sons Ed, New York, 680 pp. I.S.T.A International rules of seed testing. Seed Science and Technology Marotti, M., Piccaglia, R. and Giovanelli, E., Differences in essential oil composition of basil (Ocimum basilicum L.) italian cultivars related to morphological characteristics. J. Agric. Food Chem. 44: Miele, M., Pondero, R., Ciarallo, G. and Mazzei, M Methyl eugenol in Ocimum basilicum L. cultivar Genovese gigante. J. Agric. Food Chem. 49: Minuto, G Il basilico oggi. Quaderni di Agricoltura: il basilico (Ediz. Microart s- Recco). Prasad, G., Kumar, A., Singh, A.K., Bhattacharya, A.K., Singh, K. and Sharma, V.D Antimicrobial activity of essential oils of some Ocimum species and clove oil. Fitoterapia, 57 (6): Vernin, G. and Metzger, J Analysis of Basil oil by GC-MS data bank. Perfum. Flavor.,*:

5 Tables Table 1. Chemical charaterization of Basil cultivars. Cultivars Phenologic Stage a Chemotype 1: Gecom 1-3 linalool, tau-cadinol 2: Dark Opal 1-2 linalool, eugenol,methyl eugenol linalool 3: Greco a Palla : Red Rubin 1-3 linalool, trans α-bergamotene 5: Genovese Classico 1-3 linalool, trans-α-bergamotene, tau-cadinol eugenol, linalool 6: Fine Verde 1- linalool, trans-α-bergamotene linalool, eugenol tau-cadinol, linalool 7: Toscano a Foglie di lattuga 8: Extra Nano a foglia grande : Limoncino 1-3 neral, geranial 10: Genovese 1-2 a 1=vegetative stage; 2=flowering stage; 3=post-flowering stage. linalool, trans-α-bergamotene, tau-cadinol linalool, eugenol, trans α-bergamotene linalool, trans-α-bergamotene - linalool, 1,8-cineole, trans-α-bergamotene linalool, tau-cadinol c tau-cadinol, linalool, trans-α-bergamotene, cis-γ-cadinene Figures 0.8 MDS(P<0.01)= (%) 0 Fig. 1. Influence of the cultivar and the plant phenologic stage on basil essential oil yield. 147

6 a Linalool (%) MDS (P<0.01)=0.44 Fig. 2a. Effect of cultivar and phenologic stage of basil on linalool content. b Eugenol (%) MDS (P<0.01)=0.49 Fig. 2b. Effect of cultivar and phenologic stage of basil on eugenol content. 148

7 c Methyleugenol (%) MDS (P<0.01)=0.79 Fig. 2c. Effect of cultivar and phenologic stage of basil on methyl eugenol content. 149