Analysis of hairy root culture of Rauvolfia serpentina ... - CIMAP Staff

Analysis of hairy root culture of Rauvolfia serpentina ORIGINAL RESEARCH 599Figure 2. DART mass spectrum of the hairy roots of Raulvolfia serpentina.Table 1. Elemental compositon of vomilenine (1) and reserpine (2) in the DART mass spectrum ofRauvolfia serpentinaMolecular formulaCompound (as M + H) Calculated Measured Error (mmu)1 C 21 H 23 N 2 O 3 351.17086 351.17192 +1.062 C 33 H 41 N 2 O 9 609.28121 609.28368 +2.47a mass difference of 0.002 between the measured andthe theoretical m/z value of their molecular weights. Theelemental composition for the peaks at m/z 351 and609 were determined using the Mass Center Software(Table 1). The molecular formula corresponding tothe molecular weight calculated for the two alkaloidsvomilenine and reserpine confirmed their identity. Thedetection and identification of reserpine in the hairyroots of R. serpentina is quite interesting as this is thefirst report of expression of this important moleculein the hairy root culture of this plant. Thus, itappears that DART would be a very useful techniquefor detection and characterization of reserpine in thehairy root cultures of R. serpentina without the use ofany solvent or sample preparation.CONCLUSIONThe DART mass spectrometric technique has beenapplied for the first time for the profiling of alkaloidsthat were expressed in the hairy roots of Rauvolfiaserpentina. It produced an instantaneous response whenthe intact roots were placed directly between the DARTsource and the mass spectrometer. Two alkaloids weredetected and they were characterized as vomilenineand reserpine. The results were confirmed by comparingtheir measured molecular formula with the calculatedones. The expression of reserpine is quite interesting asit is the first report of its occurrence in the hairy rootscultures of Rauvolfia serpentina.AcknowledgmentsGrateful acknowledgement is made to SophisticatedAnalytical Instrument Facility, Central Drug ResearchInstitute, Lucknow, where the mass spectral studieswere carried out. The CIMAP communication numberof this manuscript is 2007-63J.REFERENCESBanerjee S, Madhusudanan KP, Khanuja, SPS and Chattopadhyay, SK.Analysis of cell cultures of Taxus wallichaiana using direct analysisin real time (DART) mass spectrometric technique. BiomedicalChromatography 2007 (DOI: 10.1002/bmc.919).Benjamin BD, Roja G and Heble MR. Agrobacterium rhizogens mediatedtransformation of Rauvolfia serpentina: Regeneration and alkaloidsynthesis. Plant Cell, Tissue and Organ Culture 1993; 35: 253–257.Benjamin BD, Roja G and Heble MR. Alkaloid synthesis by rootcultures of Rauvolfia serpentina transformed by Agrobacteriumrhizogenes. Phytochemistry 1994; 35: 381–383.Christey MC and Braun RH. Production of hairy root cultures andtransgenic plants by Agrobacterium rhizogenes-mediated transformation.Methods in Molecular Biology 2005; 286: 47–60.Cody RB, Laramee JA and Durst HD. Versatile new ion sourcefor the analysis of material in open air under ambient condition.Analytical Chemistry 2005; 77: 2297–2302.Guillon S, Tremouillaux-Guiller J, Pati PK, Rideau M and Gantet P.Harnessing the potential of hairy roots: dawn of a new era. Trendsin Biotechnology 2006; 24: 403–409.Hooykass PJJ, Klapwijk PM, Nuti PM, Shilperoot RA and Rorsch A.Transfer of the Agrobacterium tumefaciens Ti-plasmid to a virulentAgrobacteria and Rhizobium explanta. Journal of General Microbiology1977; 98: 477–484.Klyushnichenko VE, Yakimov SA, Tuzova TP, Syagailo YV,Kuzovkina IN, Wulfson AN and Miroshnikov AI. Determinationof indole alkaloids from R. serpentina and R. vomitoria by highperformanceliquid chromatography and high-performance thinlayerchromatography. Journal of Chromatography A 1995; 704:357–362.Copyright © 2008 John Wiley & Sons, Ltd. Biomed. Chromatogr. 22: 596–600 (2008)DOI: 10.1002/bmc