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  Lignoids in Seedlings of   Virola sebifera Ana Paula Danelutte, Alberto Jose´ Cavalheiro 1 and Massuo Jorge Kato* 1 1 Instituto de Quı´mica, Universidade de Sa˜o Paulo, 05599-970 Sa˜o Paulo, SP, Brazil 2 Instituto de Quı´mica, Universidade Estadual Paulista, 14800-900, Araraquara, SP, Brazil Quantitative analysis carried out by high performance liquid chromatography indicated the accumulationofa major secondarycompoundinseedlingsof   Virola sebifera  whichwasisolatedandidentifiedasthelignanhydroxy-otobain. This lignan occurs only in trace amounts in the seeds, where cyclolignans (aryltetralones)are by far the major components. In addition to hydroxy-otobain, only hydroxy-aryltetralones weredetected in the seedlings, indicating a selective process in the translocation of secondary compounds.Copyright  2000 John Wiley & Sons, Ltd. Keywords:  HPLC; cyclolignans; seedlings;  Virola sebifera ; Myristicaceae. INTRODUCTION Within the family Myristicaceae,  Virola sebifera  isone of the most widely spread species in South America. Thetrees bear fruits at a younger stage and produce a largernumber of seeds than most of the Myristicaceae species(Howe and Smallwood, 1982). Additionally, its seeds arecapable of resisting dehydration to some extent whilstretaining a high percentage of germination (Kato, 1995).These factors possibly represent clues as to its widedistribution in South America forests and also in thecerrados (Rodrigues, 1980). V. sebifera  is chemically one of the most investigatedspecies of the family. Analyses carried out on differentparts of the adults plants have revealed a wide variety of secondary compounds. The bark of   V. sebifera  has beenfound to contain hallucinogenic alkaloids (McKeena  et al. , 1984; Kawanishi  et al. , 1985) as well as   -sitosterol(Corothie and Nakano, 1969; Kawanishi and Hashimoto,1987) and diepoxylignan (von Rotz  et al. , 1987). Di-benzylbutyrolactone and dibenzylbutane lignans as wellas diepoxylignan were identified as the major compoundsin the leaves (Lopes  et al. , 1983; Martinez  et al. , 1999).Representatives of the former two types of lignans havealso been observed in the pericarps (Lopes  et al. , 1983,1984a) which also accumulated polyketides (Kato  et al. ,1985) and cyclolignans (Lopes  et al. , 1982, 1984b). Previous investigations concerning secondary metabo-lism in seedlings of species of the Myristicaceae haveshown that in  V. venosa  the major lignans, cubebin anddihydrokusunokinin, which accumulated in the seeds werenot present in the seedlings, which accumulated instead apolyketide (Kato  et al. , 1992). The major compoundidentified in the roots of the seedling was shown to be thelignan sesamin, a minor compound in the seeds. In the caseof   V. surinamensis , the major compounds identified in theseedlings were butenolides, but these were not detected inthe seeds (Lopes  et al. , 1994). The chemistry of seedlings is a particularly importantaspect associated with the reproduction of tropicalspecies, and it has been assumed that secondarymetabolites are translocated from the seeds to the tissuesof the seedlings as part of the defence strategy againstherbivores (Janzen, 1978). Since species of the Myristi-caceae are representative of the tropical rain forest andtheir seeds are a rich source of lignans and neolignans, wehave addressed in the present study the development of an HPLC method to evaluate the composition of secondary metabolites in seedlings of   V. sebifera . EXPERIMENTALPlant material.  Mature seeds of   Virola sebifera  wereobtained from trees growing at Estac¸a˜o Experimental deAraraquara (Instituto Florestal, Araraquara, Brazil): avoucher specimen has been deposited in the herbarium of the Instituto de Biocieˆncias (Universidade de Sa˜o Paulo,Sa˜o Paulo, Brazil). Seeds were germinated on moistenedsand and the seedlings were kept under greenhouseconditions and analysed at an age of 2 months. Spectral determinations.  1 H and  13 C NMR spectra wereobtained using a Bruker (Rheinstetten, Germany) modelAC-200 spectrophotometer at 200 and 50 MHz, respec-tively: tetramethylsilane (TMS) was used as a internalreference and deuterochloroform as a solvent. Massspectra were measured ona Finnigan (Bremen, Germany)INCOS 50B (direct injection; 70 eV) instrument, coupledwith a Varian (Palo Alto, CA, USA) model 3400 gaschromatograph. IR spectra were recorded from potassiumbromide discs on a Nicolet (Madison, WI, USA) FT-IR510 spectrophotometer. Sample preparation.  The various parts of the seedlings(seeds, leaves, hypocotyls, epicotyls and roots) werefreeze dried for 24 h and milled, weighed and extractedthree times with dichloromethane; the combined extracts PHYTOCHEMICAL ANALYSIS Phytochem. Anal.  11 , 383–386 (2000)Copyright  2000 John Wiley & Sons, Ltd. * Correspondence to: Dr. M. J. Kato, Instituto de Quı´mica, Universidade deSa˜o Paulo, 05599-970 Sa˜o Paulo, SP, Brazil. E-mail: majokato@iq.usp.brContract/grant sponsor: PADCT.Contract/grant sponsor: CAPES.Contract/grant sponsor: FAPESP.Contract/grant sponsor: CNPq.  Received 2 August 1999 Revised 15 January 2000 Accepted 28 January  were evaporated to dryness under vacuum. Part of eachextract was suspended in methanol:water (95:5), filteredin a Sep Pak C 18  cartridge (Waters, Milford, MA, USA)and concentrated under vacuum to dryness. The resultingfractions were suspended in methanol:tetrahydrofuran(9:1) at a concentration of 1 mg/mL, and an aliquot(10   L) was analysed by HPLC. Each sample preparationwas repeated three times and the average values for eachdetermination are presented. HPLC analysis.  The analyses were performed using aHewlett-Packard (HP; Waldbronn, Germany) series 1050liquid chromatograph coupled with a UV detector, HP3395 integrator and a Shimadzu (Tokyo, Japan) modelSIL-9A automatic injector. A Taxsil  (MetachemTechnologies, Torrance, CA, USA) column (250  4.6 mm i.d.; 5   ) with a pre-column was employed. Themobile phase consisted of a gradient mixture of HPLC-grade methanol, acetonitrile and water (details shown inTable 1) at a flow rate of 0.6 mL/min. The UV absorbancewas monitored at 280 nm. Cyclolignans  1, 2, 5  and  6 ,hydroxy-otobain ( 3 ) and otobain ( 4 ) (see Fig. 1) wereisolated from seed and seedling leaf extracts. Solutionscontaining 0.001, 0.05, 0.25, 0.5 and 1.0 mg/mL of eachof the compounds  1–6  were prepared in methanol:aceto-nitrile:water (42:18:10) containing 100   g/mL of methylferulate as internal standard, and an aliquot (10   L) fromeach was injected onto the HPLC. Isolation of the lignans 1–6.  The dichloromethaneextracts of seedling leaves (2.5 g) were suspended inmethanol:water (4:1) and filtered over a layer of Celite.The filtrate was partitioned with dichloromethane, driedwith anhydrous sodium sulphate, filtered and thenconcentrated undervacuum(yield720 mg).Thedichloro-methane extract was submitted to column chromatogra-phy over silica gel 60 H (10–40   ; Merck, Darmstadt,Germany catalogue no. 7736) eluted with a gradient of hexane:ethyl acetate to yield fractions  1–44 . Fractions  1– 6   were bulked and purified by preparative TLC[hexane:dichloromethane:acetone (20:78:6:0.4)] fol-lowed by HPLC using a C 18  column eluted withmethanol:water (91:9) to yield otobain ( 4 ; 1.0 mg); thebulked fractions  10–13  were purified by preparative TLC[(hexane:dichloromethane:acetone (20:78.6:0.4)] to yieldhydroxy-otobain ( 3 ; 14.0 mg); bulked fractions  21–22 and  23–26   were separately purified by preparative TLC[(dichloromethane:acetone (95:5)] to yield cyclolignans 1  (14.0 mg) and  2  (2.1 mg), respectively.The dichloromethane extract of seeds of   V. sebifera (80.0 mg), prepared as described above, was submitted topreparative TLC [hexane:ethyl acetate:isopropanol(70:30:1)], furnishing fractions at  R f   values 0.35 and0.20. These fractions were further purified by preparativeTLC [fraction  R f   0.35 with hexane:ethyl acetate:isopro-panol (70:30:1); fraction  R f   0.20 with hexane:dichloro-methane:acetone (20:78.6:0.4)] to yield, respectively,  6 (25.7 mg) and  5  (12.7 mg). (  )-Hydroxy-otobain (3).  (8  R , 7   R , 8  S  )-7  -Hydroxy-3,4:3  ,4  -bis(methylenedioxy)-2,7  -cyclolignan,     21D  –15.2  (CHCl 3 ; c 1.59).  1 H-NMR (CDCl 3 ), 200 MHz,   :0.83 ( d  ,  J   = 6.7 Hz, H-9  ), 0.97 ( d  ,  J   = 6.5 Hz, H-9), 1.5( dq ,  J   = 4.5; 13.4 Hz, H-8  ), 1.78–1.84 ( m , H-8), 2.2 ( sl ,OH-7  ), 2.47 ( dd  ,  J   = 11.8; 17.0 Hz, 1H-7), 2.74 ( dd  ,  J   = 4.0; 17.0 Hz, 1H-7), 5.51, 5.68 (2 d  ,  J   = 1.4 Hz,CH 2 O 2 ), 5.88 ( s , CH 2 O 2 ), 6.54 ( d  ,  J   = 7.9 Hz, H-5),6.64 ( d  ,  J   = 8.0 Hz, H-6), 6.64 ( d  ,  J   = 7.9 Hz, H-5  ), 6.71( d  ,  J   = 1.2 Hz, H-2  ), 6.74 ( dd  ,  J   = 1.9; 8.0 Hz). MS  m/z (relative intensity) 324 ([M]  , 100), 268 (17), 238 (79),209 (38), 202 (38), 187 (50), 135 (93). RESULTS AND DISCUSSION A comparison of the chemical composition of thedifferent parts of the seedlings (seeds, leaves, epicotyls,hypocotyls and roots) and in the seeds of   V. sebifera  waseffected by HPLC analysis. Preliminary evaluationscarried out using a reversed-phase column (Hypersil-ODS) and a normal phase column (data not shown) didnot allow a complete resolution between the cyclolignans 1  and  2 , and  5  and  6 . A total resolution was eventuallyobtained using a Taxsil column which has beenpreviously used to analyse taxanes in  Taxus  species andpodophyllotoxin lignans in extracts from  Podophyllum species (Bastos  et al. , 1995). The complete resolutionwas achieved using a ternary gradient system of methanol:acetonitrile:water, as shown in Table 1.Among the tissues analysed, the germinated seedspresented the greatest diversity of cyclolignans, andunder these conditions compounds  1–6  could beidentified in the chromatogram (Fig. 2). This profilewas very similar to that observed in mature, but non-germinated, seeds and in seeds that had been detachedafter establishment of the seedlings (data not shown).Figure 3 shows the chromatogram of the extract obtained Table 1. The gradient conditions used to analyse extracts of  Virola sebifera  by HPLC (for further chromato-graphic information see the Experimental section)                                         384 A. P. DANELUTTE  ET AL. Copyright  2000 John Wiley & Sons, Ltd.  Phytochem. Anal.  11 : 383–386 (2000)  from leaves of 2-month-old seedlings, indicating thepresence of a major compound 3 (9.76% of dry weight inthe leaves). This compound was isolated by chroma-tography and characterised by interpretation of itsspectrometric data as the lignan hydroxy-otobain ( 3 ).Compound  3  was also detected in hypocotyls (1.25%),epicotyls (0.14%) and roots (0.13%). This is the firstreport of hydroxy-otobain in  V. sebifera , but  3  haspreviously been isolated from seeds of   Myristica otoba  asan antioxidant compound (Wallace  et al. , 1963) and alsofrom seeds of   Diallyanthera otoba  (Kohen  et al. , 1966)and  Osteophloeum platyspermum  (Braz Filho  et al. ,1984). In the case of   V. sebifera , hydroxy-otobain occursonly in trace amounts in the seeds.Among the compounds occurring in the seeds, only thehydroxylated cyclolignans  1  and  2 , could be detected inthe seedlings (Fig. 4), while compounds  4–6  were notdetected at all in the hypocotyls, epicotyls, leaves androots. This profile did not change in seedlings whoseseeds had been detached after emergence of the firstleaves and which were subsequently allowed to groweither under natural light conditions or in the dark.The data obtained in this work clearly reveal thepreferential accumulation of hydroxylated cyclolignans,especially hydroxy-otobain ( 3 ), in the seedlings, but sofar there is no further evidence for specific translocationof compounds  1, 2  and  3  from seeds to the seedlings or of  de novo  biosynthesis of cyclolignans in the seedlingtissues. Acknowledgements This work wassupported byfinancial aid provided by PADCT,CAPESand FAPESP, and by fellowships from CNPq. REFERENCES                                                                                                                                                                                                                            LIGNOIDS IN  VIROLA SEBIFERA  385Copyright  2000 John Wiley & Sons, Ltd.  Phytochem. Anal.  11 : 383–386 (2000)                                                                                                                                                                                                                                                                                                                                                                                                386 A. P. DANELUTTE  ET AL. Copyright  2000 John Wiley & Sons, Ltd.  Phytochem. Anal.  11 : 383–386 (2000)
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