Comparison of methyl eugenol levels and eugenol O-methyltransferase gene structure in different Ocimum plant species

Ocimum tenuiflorum L. (Holy basil or Tulsi), belonging to Lamiaceae family, is an important medicinal plant, with particular religious significance to the Hindu community throughout the world. Tulsi plants are characterised by high levels of essential oils, containing phenylpropanoids, such as eugenol, methyl eugenol (ME), chavicol and estragole (methyl chavicol). Two chemotypes of Tulsi have been distinguished, based on high or low methyl eugenol:eugenol ratios. As methyl eugenol and methyl chavicol are classed as genotoxic carcinogens, it is important to ensure that the levels of these compounds in herbal products fall below the regulatory thresholds.

The levels of methyl eugenol in O. tenuiflorum are generally higher than in other Ocimum species. The conversion of eugenol to methyl eugenol is catalysed by eugenol O-methyltransferase (EOMT) enzyme. EOMT gene sequences have been isolated from a range of Ocimum species and from different chemotypes of O. tenuiflorum. Analyses of EOMT genomic and cDNA sequences revealed a 843 bp open reading frame and the presence of a 90-104 bp intron. Alignment of the protein sequences from several Ocimum species indicates a number of amino acid substitutions that may be correlated with methyl eugenol content and reflect differences in enzyme activity and substrate specificity. Later, the levels of ME along with other three secondary metabolites - eugenol, chavicol and estragole have been determined by HPLC.

Full length EOMT cDNA were prepared from O. tenuiflorum, O. gratissimum and O. basilicum plants and are being used to study the properties of the plant EOMT enzyme after expression in bacteria. Along with protein study, the Gateway cloning system was successfully used to prepare four binary vectors [O. basilicum (EOMT), O. basilicum (CVOMT), O. tenuiflorum and O. gratissimum] for future plant transformation experiments.