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

BIOMEDICAL CHROMATOGRAPHYBiomed. Chromatogr. 22: 596–600 (2008)596 Published online ORIGINAL 18 January RESEARCH 2008 in Wiley InterScienceORIGINAL K. P. Madhusudanan RESEARCH et al.(www.interscience.wiley.com) DOI: 10.1002/bmc.974Analysis of hairy root culture of Rauvolfia serpentinausing direct analysis in real time mass spectrometrictechniqueK. P. Madhusudanan, 2 Suchitra Banerjee, 1 Suman P. S. Khanuja 1 and Sunil K. Chattopadhyay 1 *1Central Institute of Medicinal and Aromatic Plants, PO CIMAP, Lucknow-226015, India2 Sophisticated Analytical Instrument Facility, Central Drug Research Institute, Lucknow-226015, IndiaReceived 28 September 2007; accepted 2 November 2007ABSTRACT: The applicability of a new mass spectrometric technique, DART (direct analysis in real time) has been studiedin the analysis of the hairy root culture of Rauvolfia serpentina. The intact hairy roots were analyzed by holding them in the gapbetween the DART source and the mass spectrometer for measurements. Two nitrogen-containing compounds, vomilenine andreserpine, were characterized from the analysis of the hairy roots almost instantaneously. The confirmation of the structures of theidentified compounds was made through their accurate molecular formula determinations. This is the first report of the applicationof DART technique for the characterization of compounds that are expressed in the hairy root cultures of Rauvolfiaserpentina. Moreover, this also constitutes the first report of expression of reserpine in the hairy root culture of Rauvolfiaserpentina. Copyright © 2008 John Wiley & Sons, Ltd.KEYWORDS: DART MS; analysis; hairy root; Rauvolfia serpentinaINTRODUCTION*Correspondence to: S. K. Chattopadhyay, Central Institute ofMedicinal and Aromatic Plants, P.O. CIMAP, Lucknow-226015, India.E-mail: chattsk@yahoo.comAbbreviations used: DART, direct analysis in real time.Plant-based molecules have become the drugs of choiceinternationally in recent years due to their effectivetherapeutic values. Reserpine is one such importantmolecule, considered to be a potent antihypertensiveagent that acts by depleting stores of noradrenalinein sympathetic neurons. In India, Rauvolfia has beenemployed for centuries for the relief of various centralnervous system disorders, both psychic and motor,including anxiety states, excitement, maniacal behaviorassociated with psychosis, schizophrenia, insanity,insomnia and epilepsy. Extracts of the roots are valuedfor the treatment of intestinal disorders, particularlydiarrhea and dysentery and also as an antihelmentic.Mixed with other plant extracts, they have been usedin the treatment of cholera, colic and fever. It alsopossesses depressant action on central nervous systemand produces sedation.Reserpine is normally obtained from the roots of3–4-year-old Rauvolfia serpentina plants (Ruyter et al.,1991). Such a long gestation period between plantingand harvesting along with uncertainty in the supply ofraw materials due to environmental and/or pathologicalconstraints and sample-to-sample variation in the contentof the active principle have led to the applicationof different biotechnological tools in order to find aneffective alternative source of this important drug molecule.In addition to this, since the root is the source ofthe desired phytomolecule, indiscriminate uprootingand killing of the whole plant is undeniably the causeof the present endangered status of this importantmedicinal plant.Amongst the different techniques adopted so far toconserve this endangered medicinal plant, Agrobacteriumrhizogenes-mediated ‘hairy root’ cultures of differentRauvolfia species have attracted the major attention.These genetically transformed ‘hairy root’ cultures havealready provided much evidence of their suitability asan alternative production source, and offer the advantagesof genetic stability and consistency in the productyield over a longer period of time, faster growth rate,autonomy for auxin biosynthesis and minimal nutritionalrequirements, leading to cost reduction and thepotential to synthesize secondary metabolites similar tothose of the parent plants (Guillon et al., 2006).Separation of the active components from naturalproducts has always been associated with problems oftedious and complex solvent extraction processes forisolation, separation and identification of the targetedmolecule due to the enormous chemical complexity ofthe extracts. Moreover, the clonal nature of individualhairy root lines due to the disparity in copy numbersand insertion sites of Ri T-DNA has made it obligatoryCopyright © 2008 John Wiley & Sons, Ltd.Biomed. Chromatogr. 22: 596–600 (2008)DOI: 10.1002/bmc

Analysis of hairy root culture of Rauvolfia serpentina ORIGINAL RESEARCH 597to screen and select the best performer amongst a wider,independently generated, heterogeneous background,as reported in earlier analogous studies (Christey andBraun, 2005). Chemical profiling of a large number ofindependently generated hairy root clones through theapplication of conventional complex solvent extractionprocess is undeniably a herculean task.In order to profile the hairy root clones of Rauvolfiaserpentina quickly and with great accuracy for the characterizationof the indole alkaloids expressed in them,we have utilized a new mass spectrometric ionizationtechnique, DART (direct analysis in real time). DARTis coupled to a time-of-flight mass analyzer that providesselectivity and accurate elemental composition assignmentthrough exact mass measurement. The DARTtechnique has been found to be extremely useful fordirect detection of chemicals on surfaces withoutrequiring sample preparation such as solvent extraction.In this communication, we report the identificationof two alkaloids, vomilenine and reserpine, that wereexpressed in the hairy root cultures of R. serpentina bydirectly analyzing the hairy roots using the DARTspectrometric method. The structures of the two alkoidswere confirmed as vomilenine (1) and reserpine (2) byexact measurement of their molecular formulae. Tothe best of our knowledge, this is the first report ofchemical profiling of the hairy roots of plant tissueculture origin using DART technique. Moreover, thisis also the first report of expression of reserpine in thehairy root culture of R. serpentina.EXPERIMENTALInduction and establishment of hairy roots. Rauvolfiaserpentina plants (8–10 weeks), maintained under in-vitroconditions on semisolid MS (Murashige and Skoog, 1962)medium supplemented with 2.0 mg/L 6-benzylaminopurine(BAP) and 0.1 mg/L α-naphthalene acetic acid (NAA), wereused as the explant source. The young leaves were inoculatedthrough pricking with a 48-h-old suspension cultureof A. rhizogenes strain A 4 (a kind gift from Professor D.Tepfer, INRA, Versailles Cedex, France), grown in liquidYMB (Hooykass et al., 1977) medium (O.D. 600 = 0.9–1.0) andco-cultured on hormone-free, semi-solid MS medium. After48 h of co-cultivation with the bacterial strain, the explantswere transferred on to the same medium containing 1.0 g/L ofCephalaxin (Ranbaxy, India) under dark conditions. Similartypes of explants, pricked with a sterile needle devoid of thebacterial suspension, were cultured under uniform conditionsas controls. The emerging hairy roots were subsequentlytransferred to the half and full strengths of the MS mediumcontaining 3% (w/v) sucrose for their further proliferation.Once established, the individual hairy root clones were transferredto liquid MS medium with the same concentrationof antibiotic and incubated on a rotary shaker in the dark at25 ± 1°C under constant agitation (80 rpm). The antibioticconcentration was progressively lowered and finally completelyomitted after 4 months. Roots excised from in-vitrogrown complete plantlets were cultured under identical conditionsin liquid MS medium supplemented with 1.0 mg/Lindole-3-butyric acid (IBA) to serve as control roots.Characterization of hairy roots. Polymerase chain reactionanalysis was carried out to prove the transformed nature ofthe independently generated hairy root clones following apublished procedure (Rahman et al., 2004).DART mass spectrometryThe mass spectrometer used was a JMS-100 TLC (AccuTof)atmospheric pressure ionization time-of-flight mass spectrometer(Jeol, Tokyo, Japan) fitted with a DART ion source.The mass spectrometer was operated in positive-ion modewith a resolving power of 6000 (full-width at half-maximum).The orifice 1 potential was set to 28 V, resulting in minimalfragmentation. The ring lens and orifice 2 potentials were setto 13 and 5 V, respectively. Orifice 1 was set to a temperatureof 100°C. The RF ion guide potential was 300 V. Data acquisitionwas from m/z 10 to 1050. The DART ion source wasoperated with helium gas flowing at approximately 4 L/min.The gas heater was set to 300°C. The potential on the dischargeneedle electrode of the DART source was set to 3000 V, electrode1 to 100 V and the grid to 250 V. The sample (hairyroot) was positioned in the gap between the DART sourceand mass spectrometer for measurements. Exact mass calibrationwas accomplished by including a mass spectrum of neatpolyethylene glycol (1:1 mixture PEG 200 and PEG 600)in the data file. The mass calibration was accurate to within± 0.003 u. Using the Mass Center software, the elementalcomposition could be determined on selected peaks.RESULTS AND DISCUSSIONEmergence of hairy roots could be seen after four weeksof bacterial inoculation of the leaf explants with the A.rhizogenes strain A 4 at 81.25% relative transformationfrequency. In accordance with an earlier report (Benjaminet al., 1993), most of the emerging roots of the presentstudy tend to form calli in the half-strength, hormonefree,liquid basal MS medium during further sub-culturing.However, on the basis of potential to grow without callusingalong with maximum elongation and branchingbehavior, one particular hairy root clone could be selectedfor further studies out of 30 independent hairyroot lines. The selected hairy root clone exhibited anRol-A-positive result, which indicated its transformednature. The biosynthetic potential of this particularhairy root clone has been evaluated through applicationof the newly developed DART technique.Mass spectrometry is a very important tool for thedetermination of molecular weight and structure determinationof organic molecules. It has also been utilizedin the field of biotechnology as one of the core methodsfor characterization of different compounds. TheCopyright © 2008 John Wiley & Sons, Ltd. Biomed. Chromatogr. 22: 596–600 (2008)DOI: 10.1002/bmc

598 ORIGINAL RESEARCHK. P. Madhusudanan et al.different ion sources that have been used in massspectrometry include electron ionization, chemical ionization,fast atom bombardment, field desorption/fieldionization, electrospray ionization and matrix-assistedlaser desorption ionization.DART, a new ionization source, has recently beendeveloped by Cody and Laramee (Cody et al., 2005). InDART, the sample is not directly exposed to highvoltages, laser beams, radiation or plasma. DART iscoupled with a time-of-flight mass spectrometer. DARTis based on the atmospheric pressure interaction oflong-lived electronic excited state atoms or vibronicexcited state molecules with atmospheric gases and thesample. Helium or nitrogen is used for producingthe ionizing plasma. The gas flows through a chamberwhen an electric discharge (corona discharge) producesions, electrons and excited state metastable atoms andmolecules. These metastable species, in turn, generateprotonated gaseous water clusters in ambient air. Thewater clusters on proton exchange with the sample giverise to [M + H] + ions of the sample. Thus, proton transferis the dominant mechanism in positive ionization. Innegative ionization mode, oxygen water clusters arereported to be responsible for ionizing the sample. TheDART gas flow is towards the mass spectrometer inletorifice across a gap of 5–25 mm. This gap is opened tothe laboratory room and the material to be analyzed isheld in this gap in the stream of warm gas. The DARTion source has been used to analyze an extremely widerange of analytes including drugs (tablets, formulationsetc.), metabolites in body fluid, explosives, forensic substances,synthetic-organic and organo-metallic compounds,food, spices, beverages, fatty acids in bacteria andalso materials on surfaces such as paper or currency.Recently, we have utilized the DART technique forthe detection and identification of taxoid molecules thatwere expressed in the cell culture of Taxus wallichiana(Banerjee et al., 2007).The hairy root cultures of different Rauvolfia specieshave been explored in the past in the search for analternative source of reserpine. However, in the firstreport, 40-day-old hairy root cultures of R. serpentinaproduced only trace amounts of serpentina, while allthe other targeted indole alkaloids, i.e. reserpine, ajmalineand raucaffricine, remained absent (Benjamin et al.,1993). The same research group subsequently revealedthat pure hairy root cultures without the problemof callusing could never be obtained in R. serpentinaand the mixed hairy root + callus cultures produced onlyajmaline (0.045% dry weight [DW]) and serpentine(0.007% DW; Benjamin et al., 1994). Klyushnichenkoet al. (1995) later reported yohimbine as the majoralkaloid on a backdrop of small unidentified impuritiesin the hairy root cultures of R. serpentina and R.vomitoria. Sheludko et al. (2002a,b), on the other hand,have reported the synthesis indole alkaloid of raumaclinegroup and a new alkaloid of the sarpagine group in thehairy root cultures of R. serpentina. Recently Sudhaet al. (2003) have also reported the production ofajmalicine and ajmaline in the hairy root cultures ofR. micrantha.As DART is a new ionization technique and itssensitivity is quite high, we have decided to analyze thehairy roots of R. serpentina by this technique as it hasnot yet been applied for this purpose. The intact hairyroots of R. serpentina were held in front of the DARTsource. A mass spectrum was instantly obtained (Fig. 2).The peaks at m/z 351 and 609 were assigned to the [M+ H] + of two nitrogen-containing compounds. The twonitrogen containing compounds (alkaloids) were characterizedas vomilenine (1) and reserpine (2) (Fig. 1) onthe basis of their exact mass measurement which gaveFigure 1. Chemical structures of vomilenine (1) and reserpine (2).Copyright © 2008 John Wiley & Sons, Ltd. Biomed. Chromatogr. 22: 596–600 (2008)DOI: 10.1002/bmc

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

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