Production of podophyllotoxin from roots and plantlets of Hyptis suaveolens cultivated in vitro

BACKGROUND: Hyptis suaveolens was an important source of food and medicines in pre-hispanic Mιxico and is actually used popularly to treat respiratory and skin diseases, fever, pain, and cramps, between other ailments. In 2008 the presence of podophyllotoxin (PTOX) was reported in this plant. OBJECTIVE: To establish in vitro cultures of H. suaveolens able to produce PTOX. MATERIALS AND METHODS: Explants of H. suaveolens were cultivated in Murashige and Skoog (MS) medium supplemented with different concentrations of the phytohormones 6-benzylaminopurine (6-BAP), 2,4-dichlorophenoxyacetic acid (2,4-D), 1-naphthaleneacetic acid (NAA) and kinetin (Kin), in order to induce the production of podophyllotoxin. Root cultures without hormones were also established and the quantification of PTOX was performed by HPLC analysis. RESULTS: The presence of growth regulators during in vitro cultivation of H. suaveolens, provoked morphological variations in explants, and induced the accumulation of different levels of PTOX. Roots grown without phytohormones accumulated PTOX at 0.013% dry weight (DW), while in three of the callus cultures cell lines growing together with roots, PTOX accumulated at concentrations of 0.003, 0.005 and 0.006% DW when NAA was combined with either Kin or BAP. In wild plant material PTOX was present in trace amounts in the aerial parts, while in the roots it was found at 0.005% DW. CONCLUSION: This study demonstrated that although it is possible to obtain PTOX in a variety of in vitro cultures of H. suaveolens, in vitro roots grown without the addition of growth regulators were better producers of PTOX.