Essential Oil Composition from Twelve Varieties of Basil ( Ocimum spp ) Grown in Colombia

Foi analizada a composicao do oleo esencial das partes aereas de doze variedades de alfavaca (Ocimum spp). A analise foi feita por cromatografia gasosa-espectrometria de massas (CG-EM) e encontrou-se que dez das amostras foram caracterizadas pela presenca de uma alta porcentagem de cinamato de metila (35-80%), o qual define o quimiotipo destas variedades. Somente uma das variedades apresentou o quimiotipo cariofileno e mais uma o quimiotipo linalol. As variedades "crespa morada", "virgen pequena", e "virgen morada" apresentam o subtipo cinamato > linalol. A "blanca compacta" e a "crespa blanca" tem o subtipo cinamato de metila > linalol > 1,8-cineol. A "castilla morada" tem o subtipo cinamato de metila > metil eugenol. O subtipo linalol > cinamato de metila foi apresentado pela "verde de folhas grandes". Cariofileno > metil eugenol foi o subtipo da alfavaca "clavo". Cinamato de metila (constituindo mais do 62% do total do oleo esencial) foi o quimiotipo para as outras variedades: "canela", "querendona morada", "dulce de castilla" e "zancona morada".


Introduction
Ocimum spp (basil) is an annual, aromatic, branched herb, 30 to 150 cm high.The plant has been used for medicinal purposes as a digestive stimulant and for treatment of insomnia and constipation.The oils of basil have been used principally in the food and cosmetic industries. 1 They have wide applications as spices in sauces, confectionery products and sausages.Some of their volatile compounds have been suggested as allelopathic agents. 2 The genus Ocimum undergoes abundant cross-pollination resulting in large numbers of subspecies and varieties, which differ in essential oil composition and morphological characteristics.Therefore, the taxonomy of the group is difficult.][5][6][7][8][9][10][11][12][13] According to the chemical composition and geographical origin, Lawrence in 1980 and Boniface in 1987 classified them into three large groups: European type, Exotic or Reunion type and African type. 14On the basis of more than 200 analyses of oils extracted from O. basilicum, Lawrence 8 established four essential oil chemotypes (methyl chavicol, linalool, methyl eugenol and methyl cinnamate) and also numerous subtypes.According to the biosynthetic origins of major compounds, he classified them as chemotypes with single or dual biosynthetic pathways.Other chemotypes such as eugenol-rich and thymol-rich in O. gratissimum, 10,11,15 sesquiterpenes-rich in O. canum and O. sanctum, 15 and terpinen-4-ol-rich, 10 have been reported.However, there is little data available about the chemical characterization of the essential oils of the Ocimum species growing in Colombia, except those reported by Murillo and Viña. 16The purpose of the present work was to determine the essential oil chemotype of twelve basil varieties cultivated in Ibagué, Tolima-Colombia, in order to provide data, either for practical use or for basic research needs.This study will contribute to the knowledge of a local product that could improve the use of Colombian basil.The system of chemotype classification used was the one proposed by Grayer 17 which is based on the combination of major chemical components rather than the sole dominant compound, defining a major component as one with content close to 20%.
There are 26 major components of essential oils (1% or more of the total in at least one variety).
The chemical composition profile of the varieties of this group indicates a prevalence of the cinnamic acid biogenetic pathway, although the biogenetic pathway from mevalonic acid makes a high contribution in the profiles of two varieties (n= 6, 7).

Varieties rich in monoterpene
The varieties that make up this group were identified as "profile 2".Comparison of the composition profiles of the varieties inside this group (Figure 2), shows a decreasing monoterpene content (50-44%) and a small  decrease in the sesquiterpene content (18-13.4%)as well as an ever-increasing phenylpropanoid content (30-41.4%),except n=9 which shows the lowest content of sesquiterpenes (10%) and a high content of phenylpropanes (41%).It could be said that the mevalonate pathway is the predominant biosynthetic route in this group.The essential oils belonging to this composition profile are oxygenated terpene-rich oils (n=8, n= 9 are the richest varieties, 48 and 48.7% respectively).From the contribution of the terpenoid content as a whole (both monoterpenes and sesquiterpenes) and the high content of oxygenated terpenes (43.6%), "little virgin" (n=7), a phenylpropanoidrich variety (profile 1), also resembles the varieties named "profile 2".

Chemotypes
According to Grayer´s chemotype classification system, the chemotypes for the 12 varieties utilized in this investigation were established (Table 2).Ten of them exhibited the "methyl cinnamate" chemotype with methyl cinnamate as the most abundant Methyl cinnamate, methyl eugenol and methyl chavicol, which are compounds belonging to the shikimate pathway, prevailed in n=1,2,4 and 5 and it could be said that only one biosynthetic pathway is operating for these oils.
"Purple castle" (n=3), presented two major components: methyl cinnamate (52.2%) and methyl eugenol (24%); therefore, the "methyl cinnamate > methyl eugenol" subtype was defined for this sample.Moreover, all the oils of this group (except oil from n=4) were characterized by relatively high contents of methyl chavicol (6-12%).On the other hand, n=4 seemed somehow differentiated, having a considerable amount of 1,8-cineol (11%) and linalool (13%), belonging to the mevalonate pathway.The five remaining varieties can be ascribed to those plants in which essential oil constituents are produced by two different biosynthetic pathways: three varieties (n=6,7,11) presented two major components, methyl cinnamate and linalool, and the "methyl cinnamate> linalool" subtype was defined for them; two varieties (n=9,10) presented three major components which defined the "methyl cinnamate > linalool > 1,8-cineol" subtype.Actually, methyl cinnamate has a biosynthetic origin from cinnamic acid whereas linalool and 1,8-cineol follow another biogenetic pathway from mevalonic acid.About 80% of the total of the studied varieties had a high content of methyl cinnamate (35-80%).Hence, extraction of essential oil from them could be important for the chemical industry.Distinct types of basil on the international market include European and Egyptian oils (two main types) and a Bulgarian or Cinnamon basil oil, rich in methyl cinnamate (as a source of methyl cinnamate).In 1987, Simon 2 initiated a study to develop a methyl cinnamate rich basil, as this type was of the interest to the perfume industry.These authors increased the methyl cinnamate content (by 15%); but, even so, they were lower than those reported for methyl cinnamate chemotypes in the present study.
rich in methyl cinnamate (74%) which could be classified as "profile 1", according to this study.However, this oil presented qualitative and quantitative differences with respect to the components found for this profile.Lachowiczs 4 identified both methyl cinnamate and linalool as the main components (ratio 1:1), together with small amounts of both methyl chavicol and 1,8-cineol, in hydrodistilled oil from a cinnamon basil.That hydrodistilled oil is similar to those called "profile 2" in our study, but these latter ones have methyl cinnamate to linalool in a 2:1proportion.
The "large green leaves" variety (n=8) is the variety richest in monoterpenes (50%) and presented a high content of linalool (33%) which defined its chemotype as "linalool".The presence of methyl cinnamate as another main component (25.2%) suggests a dual biosynthetic origin and defined the "linalool > methyl cinnamate" subtype for this variety.The essential oil composition of this variety of O. basilicum resembles the composition of two varieties reported by Lawrence 18 for the same chemotype and genus.Pushpangadan and Bradu 19 described the same chemotype, linalool > methyl cinnamate, for O. basilicum var.Purpurascens.The "clove" variety (n=12), of dual biosynthetic origin rich in sesquiterpenes, contains caryophyllene (43%, mevalonate pathway) and methyl eugenol (26%, shikimate pathway).Therefore, this variety is characterized as "caryophyllene" chemotype and "caryophyllene > methyl eugenol" subtype.This "sesquiterpene" oil notably differs from those previously described by Sanda 10 for O. canum and O. sanctum, which showed the same chemotype.On the other hand, it is important to mention that Lawrence 15 described a variety of O. sanctum which he defined as "methyl eugenol" chemotype that presents caryophyllene as the second major component.

Plant material
The 12 basil varieties used in this study are listed in Table 2, as well as the common and scientific names of each one.The aerial parts of the different basils utilized in this investigation were manually harvested, under optimal phytosanitary conditions, from plants growing on small farms located in urban and suburban areas of Ibagué, Tolima-Colombia.The samples were dried for a week (30-40°C).Identification of the species was done by the Toli Herbarium of Tolima University, using the taxonomy keys determined by Moreno. 20Voucher specimens (006981-00692) have been deposited in the Toli Herbarium.

Essential oil extraction
Leaves, flowers and stems of plant material (50 g) were subjected to distillation in water (1:20), in a modified Clevenger apparatus 21 for 2h.The essential oils were dried over anhydrous Na 2 SO 4 , stored in a dark glass bottle and kept at 4°C until analysis.Tetradecane was used as internal standard.

Conclusions
Concerning the chemical composition of Ocimum varieties growing in Ibagué, Tolima-Colombia it could be stated that the "methyl cinnamate" chemotype was found for 80% of the varieties.The essential oils of Ocimum americanum, with the exception of "clove" basil, were rich in phenylpropanoids and a single biosynthetic origin is indicated for them as all of them showed a high methyl cinnamate content."Clove", a variety of O. americanum, was the only basil of this region that was rich in sesquiterpenes; moreover, its chemotype indicates a dual biosynthetic origin.The essential oils of O. minimum had a dual biosynthetic origin which showed methyl cinnamate and linalool in a 2:1 proportion.Methyl cinnamate, linalool, 1,8-cineol, methyl chavicol, eugenol, methyl eugenol, caryophyllene, caryophyllene oxide and tcadinol were common compounds in all the oils of Ocimum varieties grown in Ibagué.

Figure 3 .
Figure 3.Chemical composition of Colombian clove basil (12), based on the compound group classification.