Essential Oil in the Taxonomy of Ocimum selloi Benth #

A composição química do óleo essencial das folhas e flores de dois acessos de Ocimum selloi Benth., cultivados na Universidade Fed eral de Viçosa foi analisada. Para o acesso A o componente prin ci pal foi identificado como estragol, o qual representa 94,95% e 92,54% do óleo das folhas e das flores, respectivamente. O óleo obtido das folhas e flores do acesso B é constituído de 65,49% e 66,18% de metil eugenol, respectivamente. Para ambos os acessos, diversos constituintes químicos, presentes em quantidades menores, foram identificados. As diferenças fenotípicas e químicas observadas en tre os dois acessos estudados indicam a existência de duas variedades quimicamente distintas de Ocimum selloi Benth.

The ge nus Ocimum (Labiatae) com prises 160 spe cies and is found through out the trop i cal and sub-tropical re gions of the world 1 .The larger ge netic di ver sity of this ge nus is found in Brazil 1 .Sev eral spe cies of this ge nus has com mer cial util ity as a source of es sen tial oil for the phar ma ceu tic, food, fla vour and perfumary in dus tries 2 .
The spe cie Ocimum selloi Benth known as "alfavaquinha", "anis" and "elixir-paregorico", is orig i nated from South Amer ica, and is used in the tra di tional med i cine as antiinflammatory, an alge sic and antiespasmodic 3 .
In the germplasm col lec tion kept at the Fed eral Uni ver sity of Viçosa, ac ces sions of this spe cie were found to have dif fer ent mor pho log i cal char ac ter is tics.The ac ces sion hav ing more vig orous plants, with pink co rol las and leaves with an anis-like odour was called "A".The ac ces sion formed by smaller plants, with darker co rol las, red dish ca lyx and leaves with out anis-like odour was called "B".Thus a chem i cal anal y sis of the es sen tial oil of these two ac ces sions was car ried out in or der to see if the phenotypic vari abil ity ob served was also pres ent at mo lec u lar level.In for ma tion about the chem i cal com po si tion of the oil could then be use ful in the clas si fi ca tion of these plants.

Plant ma te rial
Seeds of Ocimum selloi , accessions A and B, from the germplasm collection of the "Grupo Entre-Folhas-Viçosa (MG)", were cultivated at the Federal University of Viçosa, beginning November 1994 and harvested while in full bloom in April 1995.The aerial parts were collected and the leaves and flow ers were sep a rated.

Es sen tial oil ex trac tion
Steam distillation was carried out by passing steam in a 2 L round-bottoned flask containing the fresh plant material (100 g of leaves or 80 g of flowers).A total of 850 mL of distillate was collected, and the oil was extracted with diethyl ether (3 x 80 mL).The combined organic extract was dried over MgSO 4 and concentrated under reduced pressure in a rotary evaporator at 30 °C, and the essential oil obtained was weighted.The extractions were carried out in triplicates, and for each extraction the material (leaves and flowers) were obtained from one sin gle spec i men.

Es sen tial oil anal y sis
The major constituent of the essential oil from each plant group was purified by column chromatography on silica gel, using hexane or hexane:diethyl ether (8:1) as a solvent for groups A and B respectively.The compounds isolated were identified by means of their MS, IR and 1 H-NMR data.
IR spectra were recorded with a Perkin Elmer 599B double beam grating spectrophotometer; 1 H-NMR spectra were recorded with a Bruker WH300 (300 MHz) in strument, using tetramethylsilane as internal standard.Minor constituents were identified by combined GC-MS analysis.These were performed by a model 5890 A GC system with mass-selective detector Model 5970 (Hewlett-Packard).GC conditions were: direct injection of 1.0 µL sample diluted 10:1 with CHCl 3; fused sil ica col umn HP-1(30 m x 0.25 mm x 0.33 µm); he lium as a car rier gas (1.0 mL/min); oven pro gram 80 °C (5 °C/min, 1 min hold) to 250 °C, in jec tor and de tec tor tem pera ture of 150 °C and 225 °C re spec tively.All spec tra were recorded in the elec tron im pact ion iza tion mode at 70 ev.The com par i sons of the ac quired spec tra were made with those found in a Wiley li brary by an in te grated pro gram.
A quan ti ta tive anal y sis of the ma jor chem i cal con stit u ents of the es sen tial oil was car ried out us ing a Shimadzu GC-14A in strument, equipped with a FID de tec tor, a Carbowax 20M cap il lary col umn of 30 m, i.d.0.25 mm and the fol low ing tem per a ture program: 60 °C (1 min) ris ing to 160 °C at the rate of 5 °C; in jec tor and de tec tor tem per a tures of 150 °C and 220 °C re spec tively.Carrier gas used was hy dro gen at 1.0 mL/min.Estragole and methyleugenol, pu ri fied pre vi ously, were used as stan dards, and the anal y sis were car ried out in trip li cates.

Re sults and Dis cus sion
The es sen tial oil con tent in the fresh leaves and flow ers of ac ces sion A were 0.311 ± 0.043% (av er age ± stan dard devi a tion) and 0.474 ± 0.036%, re spec tively, and for ac cession B 0.210 ± 0.022% and 0.398 ± 0.015%.
Chromatograms of the es sen tial oils from the leaves and flow ers of both ac ces sions of Ocimum selloi are shown in Figs. 1 and 2. The com pounds iden ti fied are listed in Table 1, and the rel a tive area (%) of each com pound is also pre sented.
The ma jor com pound found in the oil from the leaves and flow ers of ac ces sion A was iden ti fied by GC/MS as estragole (Fig. 3) This oil was sub mit ted to fractioning in a col umn chro ma tog ra phy on sil ica gel and the ma jor compound was ob tained in a pure form.The IR spec trum of this com pound was iden ti cal to the cor re spond ing data for estragole re ported in the lit er a ture 4 .The 1 H-NMR spectrum con firmed the pro posed struc ture.
The struc ture for some of the mi nor con stit u ents were pro posed by com par i son of their mass spec trum with the mass spec trom e ter data bank (Ta ble 1).All these compounds have al ready been iden ti fied from sev eral other spe cies in this ge nus 5 .Al though the lack of pure ref er ences sam ples makes it dif fi cult to have com plete con fi dence in the pro posed iden ti fi ca tion, this is not a prob lem for the pres ent in ves ti ga tion, as for the differenciation be tween two chemotypes only the ma jor oil com po nents are con sidered 6 .
The es sen tial oil of ac ces sion B was also sub mit ted to a col umn chro ma tog ra phy pu ri fi ca tion and the ma jor constit u ent was iso lated.The struc ture for this com pound was ini tially pro posed from GC/MS data as methyleugenol (Fig. 3) and was fur ther con firmed by anal y sis of its IR and 1 H-NMR data.
The char ac ter iza tion of the mi nor con stit u ents was also car ried out by GC/MS as pre vi ously men tioned (Ta ble 1).
With pure samples of estragole and methyleugenol available to be used as standards, a quantitative analysis of these two components in the oil from the leaves and flowers was carried out by gas chromatography,  using the calibration curve method 7 .The percentage of estragole in the oil from the leaves and flowers of accession A was 94.95 ± 1.02% (av er age ± stan dard de vi a tion) and 92.54 ± 0.78% re spec tively.For ac ces sion B the percent age of methyleugenol in the oil from the leaves and flow ers was 65.49± 3.43% and 66.18 ± 1.97% re spec tively.
For each accession the chemical composition of the oil from the leaves and flowers were very similar.For accession B, the major difference observed was the presence of a reason able amount of compounds , 2 and 9 in the flow ers, while in the leaves these com pounds were pres ent in trace amounts.Also com pound 12 was not present in the leaves and was the sec ond ma jor con stit u ent in the oil from the flow ers.On the other hand com pound 11 was the sec ond ma jor con stit u ent in the leaves and was not present in the flow ers.
For ac ces sion A the chem i cal com po si tion of the oil from the leaves and flow ers were very sim i lar in qual i ta tive terms (Ta ble 1).
Be tween the two ac ces sions, the ma jor dif fer ence in the oil chem i cal com po si tion was the pres ence of estragole as the ma jor con stit u ent in the leaves and flow ers of ac ces sion A and its ab sence in ac ces sion B. On the other hand, the major com pound found in the oil from the leaves and flow ers of ac ces sion B was methyleugenol.This com pound was found in very small amount in the oil of plants from ac cession A.
The chem i cal com po si tion of the es sen tial oil for dif ferent Ocimum spe cies has been shown to vary greatly.For exam ple, cam phor amounts to 70% of the es sen tial oil of O. kilimadjaricum 8 and 65% in the case of O. canun 5 .The chem i cal com po si tion of vol a tile oil from O. basilicum culti vated in dif fer ent parts of the world has been shown to vary enor mously 5,9 .
Ribeiro et al. 10 has shown the ex is tence of two chemotypes of O. nudicaule Benth.The one clas si fied as the anisifolia va ri ety has estragole as the ma jor con stit u ent of its es sen tial oil, the other that has no estragole was clas sified as the nudicaule va ri ety.
Also the spe cie Ocimum basilicum known as Sweet basil or French ba sil has two populary rec og nized races.One is rich in eugenol and the other is rich in estragole 11 .Thus, the dif fer ence in chem i cal com po si tion found for the two ac ces sions stud ied is not unique.
Con sidering that the plants in ves ti gated in this work were cul ti vated un der the same con di tions, the mor pho logi cal and chem i cal dif fer ences ob served could not be due to eco log i cal fac tors, but should be a con se quence of genetical dif fer ence be tween the two groups of plants 10 .These re sults sug gest that the two ac ces sions stud ied should con sti tute two tax o nomic va ri et ies of Ocimum selloi.A ge netic in ves ti ga tion of these two ac ces sions is un der way at the mo ment in or der to cor rob o rate, or not, this hy poth e sis.
Fig ure 3. Ma jor con stit u ents from the es sen tial oil of Ocimum selloi Benth.
Ar ti cle # Taken from the MSc dissertation presented by E.R.M. at the Federal University of Viçosa on February 1996.

Fig ure 1 .
Fig ure 1. Chromatogram of the es sen tial oil from the leaves of Ocimum selloi Benth.: (a) ac ces sion A and (b) ac ces sion B.

Fig ure 2 .
Fig ure 2. Chromatogram of the es sen tial oil from the flow ers of Ocimum selloi Benth.: (a) ac ces sion A and (b) ac ces sion B. Ta ble 1. Com po si tion of the es sen tial oil from the leaves and flow ers of Ocimum selloi Benth.