Brassinosteroids and a pregnane glucoside from Daucus carota

Brassinosteroids and a Pregnane Glucoside fromDaucus carota

Jurgen Schmidt*, Andrea Porzel and Gunter AdamInstitute of Plant Biochemistry, Weinberg 3, D-06120 Halle/S., Germany

The brassinosteroids brassinolide, castasterone and 24-epi-castasterone could be isolated and identified fromseeds of Daucus carota. Furthermore, a new pregnanolone glucoside was identified as b-D-glucopyranosyl-(1→2)-b-D-glucopyranosyl-3b-hydroxy-5a-pregnane-20-one (sophorosylpregnanolone) by nuclear magneticresonance spectroscopy, liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry.© 1998 John Wiley & Sons, Ltd.

Phytochem. Anal. 9, 14–20 (1998)

Keywords: Daucus carota ssp. sativus L.; Apiaceae; brassinosteroids; b-D-glucopyranosyl-(1→2)-b-D-glucopyranosyl-3b-hydroxy-5a-pregnane-20-one; gas chromatography – mass spectrometry; liquid chromatography – electrospray mass spectrometry; nuclearmagnetic resonance spectroscopy.

INTRODUCTION

Brassinosteroids represent a new class of phytohormoneswith structural similarities to animal steroidal hormones(Cutler et al., 1991). They have been detected in manyhigher plants and therefore appear to be of ubiquitousoccurrence (Marquardt and Adam, 1991; Sakurai andFujioka, 1993; Adam et al., 1996). A wide spectrum ofphysiological responses are elicited by exogenous applica-tion of low doses of these compounds to intact plants or toexplants, although stimulation of cell growth is the mostprominent effect (Mandava, 1988). The high biologicalactivity of brassinosteroids suggests an important role in theregulation of physiological processes in plants. Recentmolecular genetic studies on dwarfed and de-etiolatedArabidopsis thaliana mutants provided strong evidence foran essential role of brassinosteroids in plant development(Clouse et al., 1996; Kauschmann et al., 1966; Li et al.,1996; Szekeres et al., 1996).

Continuing our investigations on the occurrence of suchcompounds in European cultivated plants, we have investi-gated seeds of Daucus carota ssp. sativus L. (Apiaceae).The brassinosteroids castasterone, 24-epi-castasterone andbrassinolide could be isolated and identified. Furthermore, anew naturally occurring pregnane-type bis-b-D-glucosidewas detected in trace amounts. The structure elucidation ofthe pregnanolone glucoside and the brassinosteroids isdescribed.

EXPERIMENTAL

Plant material. The seeds of Daucus carota ssp. sativus L. wereobtained from Saatgut GmbH, Quedlinburg, Germany.

w Correspondence to J. Schmidt (E-mail-c/o [email protected]).

Contract grant sponsor: Deutsche Forschungsgemeinschaft.

Table 1. 13C NMR chemical shifts of isolated sophorosyl-pregnanolone (4a), authentic sophorosylpregnanolone(4b) and 3b-hydroxy-5a-pregnan-20-one (5)a

Chemical shift, d ppm

Carbon 4a 4b 5

1 38.2 38.2 38.2

2 n.d.b 30.3 32.1

3 n.d. 80.2 71.8

4 n.d. 35.4 39.8

5 46.0 46.0 46.2

6 n.d. 29.8 29.9

7 n.d. 33.2 33.2

8 n.d. 36.8 36.9

9 55.9 55.6 55.7

10 36.7 36.8 36.6

11 n.d. 22.3 22.4

12 40.1 40.1 40.1

13 45.3 45.3 45.3

14 57.9 57.9 57.9

15 n.d. 25.3 25.4

16 n.d. 23.7 23.7

17 64.7 64.8 64.8

18 13.7 13.7 13.7

19 12.6 12.6 12.6

20 212.3 212.3 212.0

21 31.5 31.5 31.6

1' 101.3 101.3

2' 82.9 82.9

3' 77.8 77.8

4' n.d. 71.5

5' n.d. 77.9

6' 62.7 62.7

1" 105.1 105.1

2" 75.8 76.0

3" n.d. 77.6

4" n.d. 71.4

5" n.d. 78.1

6" 62.7 62.7a Spectra measured of compounds dissolved in CD3OD.b n.d.=not detected.

CCC 0958–0344/98/010014–07 $17.50 Received 17 December 1996© 1998 John Wiley & Sons, Ltd. Revised 3 March 1997

Accepted 24 March 1997

PHYTOCHEMICAL ANALYSIS, VOL. 9, 14–20 (1998)

Bioassay. The rice lamina inclinaton test was carried out using thecultivar Koshihikari as described by Arima et al., 1984.

Extraction, isolation and purification. The powdered seeds of D.carota (950 g) were extracted three times with methanol. Thecombined methanol extracts were evaporated to dryness in vacuo.The residue was partitioned three times between water andchloroform. The residue, after evaporation of the chloroform phase(41.15 g) was partitioned between n-hexane (500 mL) and 80%methanol (500 mL). The n-hexane phase was partitioned a secondtime with 80% methanol, and the combined 80% methanolfractions were concentrated (11.55 g).

The residue resulting from the 80% methanol fraction waschromatographed on a silica gel column (58 g). Elution wascarried out with chloroform (500 mL), chloroform:methanol (4 :1,v/v, 500 mL) and methanol (500 mL). The eluate with 20%methanol (5.05 g) was subjected to a second silica gel column(25.2 g). Elution was carried out stepwise with ten fractions(100 mL each) of methanol in chloroform (0, 2, 3, 4, 5, 7, 10, 15,30 and 50% methanol v/v). The fractions eluted with 4 and 15%methanol were biologically active. Fraction F I (4% methanol,235 mg) was evaporated and further purified by LH-20 Sephadexchromatography (bed volume 500 mL) with methanol :chloroform(4:1, v/v) as eluent. The eluates were collected in 10 mL fractions.Biological activity was found in fractions 24 to 34 (elutionvolume/total column volume 0.48–0.68), these fractions werecombined and evaporated. The residue (69 mg) was dissolved inchloroform:methanol (4 :1) and further purified by diethylamino-propyl (DEA) ion exchange chromatography (1.5 g; AnalytichemBondesil, Varian, Harbor City, CA, USA).

The residue (37 mg) was subjected to high pressure liquidchromatography (HPLC) using a Eurospher 80-C18 column(25038 mm i.d.; Knauer GmbH, Berlin, Germany) eluted at aflow-rate of 2 mL/min with acetonitrile :water (9 :11) for 40 min,followed by a linear gradient to acetonitrile :water (4 :1) over5 min, and held at this concentration of acetonitrile for a further25 min. Seventy fractions (2 mL each) were collected: those

fractions with activity (corresponding to a retention time (Rt) of27–28 min) were pooled, concentrated and examined by gaschromatography-mass spectrometry (GC-MS). Castasterone (1)and 24-epi-castasterone (2) were identified. The residue of thefraction eluted with 15% methanol (F II, 116 mg) was chargedonto a silica gel column (1.16 g). Elution was performed stepwise

Figure 1. Positive ion electrospray-mass spectrum of compound 4 obtained by liquid chromatography-massspectrometry.

STEROIDS IN DAUCUS CAROTA 15

© 1998 John Wiley & Sons, Ltd. Phytochem. Anal. VOL. 9, 14–20 (1998)

with six fractions (5 mL each) of methanol in chloroform (10,12.5, 15, 17.5, 20 and 50% methanol v/v). The fractions elutedfrom 12.5 to 17.5% methanol displayed biological activity andwere combined. The residue (90 mg) was dissolved in 2 mL andfurther purified by DEA ion exchange chromatography (2 g,Analytichem Bondesil).

The residue (59 mg) was chromatographed with a RP-18cartridge (1 g, Bond Elut LRC, Varian, Harbor City, CA, USA) inthree fractions by acetonitrile :water (7 :3; 9 :1; 100:0). Fractions1 and 2 which had biological activity were combined and theresidue (45 mg) further purified by preparative HPLC (see above).The fractions with activity (F IIa: Rt =9–12 min, 10 mg and F IIb:

Rt 15–22 min, 6 mg) were repeatedly purified by HPLC. FractionF IIb was charged onto a HPLC under the same conditions asdescribed above. The fraction with activity (Rt =16 min) wasconcentrated and examined by GC-MS. Brassinolide (3) could beidentified. Fraction F IIa was further separated via HPLC usingacetonitrile :water (1 :3) as solvent system. The fractions withRt =21–22 min (F IIa1, ca. 100 mg) displayed bioactivity and wereinvestigated by liquid chromatography-mass spectrometry (LC-MS) and proton nuclear magnetic resonance spectroscopy (1HNMR). A portion of fraction F IIa1 (ca. 50 mg) was acetylatedwith acetic anhydride in pyridine for 12 h at room temperature andanalysed by 1H and 1H, 1H COSY NMR.

Figure 2. Collision-induced dissociation-mass spectrum, obtained by liquid chromatography-mass spectrometry, of the[M-H]2 ion of m/z 641 of (a) the isolated compound 4, and (b) authentic compound 4.

J. SCHMIDT ET AL.16

Phytochem. Anal. VOL. 9, 14–20 (1998) © 1998 John Wiley & Sons, Ltd.

Enzymatic cleavage. To a portion of fraction F IIa1 (ca. 25 mg),1 mL aqueous buffer solution (0.1 M citric acid, 0.5 M Na2HPO4;pH 4.0) and b-D-glucosidase were added. After stirring the mixturefor 24 h at 37°C, the aglycone was extracted with n-hexane. The n-hexane layer was dried with Na2SO4, filtered, dried in vacuo andinvestigated by GC–MS after trimethylsilylation.

Preparation of a-acetobromosophorose. Sophorose (90 mg) wasadded to a solution of 2 mL acetic anhydride and 12 mL perchloricacid under constant stirring at 30°C as described previously(Houben/Weyl, 1960). After addition of 80 mg red phosphorus, themixture was cooled below 20°C and 0.3 mL bromine were addeddropwise. After addition of 0.2 mL water, the yellow-orangereaction mixture was kept at room temperature for 2 h and thentwice partitioned between 5 mL chloroform and 10 mL ice water.The combined chloroform layer was partitioned with 3 mL of asaturated NaHCO3 solution and dried under stirring with a mixtureof CaCl3, CaCO3, NaHCO3 and charcoal. After 30 min the mixturewas filtered and dried in vacuo.

Synthesis of sophorosyl-3b-hydroxy-5a-pregnane-20-one. 3b-Hydroxy-5a-pregnan-20-one (64 mg) in dichlorethane was reactedwith 140 mg a-acetobromosophorose in the presence of129 mg Ag2CO3: Celite for 10 min (Hartenstein and Satzinger,

1974). After cooling, filtration and washing with methanol andethyl acetate, the reaction products were dried in vacuo. The crudeproduct (167 mg) was chromatographed on Sephadex LH-20(200 ml) using methanol as eluent collecting 8 mL fractions.Fractions 9 and 10 (68 mg) were combined and deacetylated with70 mg K2CO3 in 5 ml methanol containing ca. 100 mL of water for1 h. After drying in vacuo the reaction product was partitionedbetween water and chloroform. The extracted product (31 mg) waschromatographed on a column of silica gel (3 g; 0.063–0.2 mm;Merck, Darmstadt, Germany) using a chloroform: methanolgradient system (0, 5, 10, 12, 14, 16, 18, 20, 25, 30, 50 and 100%methanol v/v) with 20 ml used for each step. From the fractionseluted with 12 to 14% methanol in chloroform, 8 mg sophorosyl-3b-hydroxy-5a-pregnane-20-one (4) could be isolated. Compound4: melting point 273–275°C; positive ion electrospray (ES)-MSm/z (relative intensity) 665 ([M+Na]+ , 51), 301 (67), 283 (100),257 (11), 189 (30), 135 (12); 1H NMR (see Table 1).

Spectroscopy. The 1H NMR-spectra were measured with a VarianUnity 500 spectrometer at 499.85 MHz using a Nalorac 3 mmmicrosample inverse detection probe. Deutero-chloroform wasused as solvent with tetramethyl silane (TMS) as internal standard.The positive and negative ion ES-MS were obtained using aFinnigan TSQ 7000 instrument (electrospray voltage — 4.5 kV;

Table 2. 1H NMR data of isolated sophorosylpregnanolone (4a) andauthentic sophorosylpregnanolone (4b)a

Chemical shift, d ppm (coupling constants, Hz)

Carbon 4a 4b

1 1.74/1.00 1.74/1.00

2 1.90/1.52 1.91/1.50

3 3.721 dddd (11.4/11.4/4.8/4.8) 3.721 dddd (11.4/11.4/4.8/4.8)

4 1.71/1.33 1.71/1.32

5 n.d.b 1.13

6 n.d. 1.31/1.31

7 n.d. 1.70/0.95

8 n.d. 1.40

9 0.727 ddd (12.4/10.8/4.1) 0.727 ddd (12.2/10.7/4.2)

10 — —

11 n.d. 1.62/1.35

12 2.11/1.64 2.01/1.44

13 — —

14 n.d. 1.19

15 n.d. 1.66/1.20

16 2.11/1.64 2.12/1.64

17 2.623 dd (9.2/9.2) 2.620 dd (9.1/9.1)

18 0.598 s 0.601 s

19 0.844 s 0.845 s

20 — —

21 2.104 s 2.104 s

1' 4.522 d (7.8) 4.524 d (7.8)

2' 3.381 dd (9.1/7.8) 3.391 dd (9.2/7.8)

3' 3.531 dd (9.1/8.6) 3.535 dd (9.2/8.7)

4' 3.28 3.305 dd (9.5/8.7)

5' 3.26 3.26

6'A 3.847 dd (11.9/2.2) 3.848 dd (11.9/2.2)

6'B 3.642 dd (11.9/5.6) 3.647 dd (11.9/5.5)

1" 4.566 d (7.8) 4.568 d (7.8)

2" 3.234 dd (9.0/7.8) 3.239 dd (9.0/7.8)

3" 3.36 3.371 dd (9.0/9.0)

4" n.d. 3.321 dd (9.4/9.0)

5" 3.27 3.27

6"A 3.834 dd (11.9/2.4) 3.837 dd (11.8/2.4)

6"B 3.691 dd (11.9/5.2) 3.695 dd (11.8/5.1)a Spectra measured of compounds dissolved in CD3 OD.b n.d.=not detected.



capillary temperature — 250°C; sheath gas — nitrogen) combinedwith a Consta-Metric (Thermo Separation Products, RivieraBeach, FL, USA) 4100 HPLC instrument fitted with aLiChrospher (Merck, Darmstadt, Germany) 100 RP18 column(10032 mm i.d.; 5 mm) elutedwith acetonitrile :water containing0.2% acetic acid (2 :3) at a flow-rate of 0.2 ml/min. Rt ofcompound 4 was 3.45 min. The collision-induced dissociation(CID) mass spectra of the [M-H]2 ion of 4 were measured underthe following conditions: collision energy — 25 eV; collision gas— argon; collision pressure — 231023 Torr.

Gas chromatography-mass spectrometry. The GC-electronimpact (EI)-MS measurements were carried out with a FisonsInstruments (Manchester, UK) MD-800 under the followingconditions: EI voltage — 70 eV; source temperature — 200°C;column — J&W DB-5MS, 15 m30.32 mm i.d., 0.25 mm filmthickness; injection temperature 260°C; column temperatureprogramme for the bismethylboronates of brassinosteroids (Rt of5a-cholestane=5.35 min) — 170°C for 1 min, then raised to290°C at a rate of 30°C/min and held on this temperature for20 min; column temperature programme for the pregnanolonetrimethylsilylether (Rt of 5a-cholestane=17.60 min) — 60°C for1 min, then raised to 110°C at a rate of 25°C/min and then with arate of 10°/min to 270°C and held at this temperature for 10 min;interface temperature — 300°C; carrier gas — helium; flow-rate— 1 mL/min; injection splitless. The relative retention times (RRt)were calculated with respect to 5a-cholestane.

Derivatization. The methylboronation of the brassinosteroids wascarried out with pyridine containing methaneboronic acid at 70°Cfor 30 min (Takatsuto et al., 1982). The trimethylsilylation of 3b-hydroxy-5a-pregnane-20-one was carried out withN,O-bis-tri-methylsilylacetamide.

GC-MS data of brassinosteroids. Castasterone bismethylbor-onate (1). RRt =1.81; EI-MS m/z (relative intensity) 512 (M+ , 14),441 (4), 399 (7), 358 (9), 329 (8), 287 (25), 155 (100), 85 (86).24-Epi-castasterone bismethylboronate (2). RRt =1.86; EI-MS m/z(relative intensity) 512 (M+ , 18), 441 (4), 399 (7), 358 (12),329 (10), 287 (21), 155 (100), 85 (88). Brassinolide bismethylbor-onate (3). RRt =2.04; EI-MS m/z (relative intensity) 528 (M+ , 3),457 (5), 415 (4), 374 (24), 344 (31), 332 (21), 177 (78), 155 (100),85 (97).

RESULTS AND DISCUSSION

Repeated silica gel column chromatography of the concen-trated 80% methanol extract of Daucus carota using severalmethanol :chloroform gradient systems led to two fractionseluted with 4% (fraction F I) and 15% (fraction F II)methanol which showed bioactivity in the rice lamina-inclination test (Wada et al., 1981). Fraction F I was furtherpurified via LH-20-Sephadex, DEA chromatography andpreparative HPLC. From the bioactive fractions resultingfrom the reversed-phase HPLC, castasterone (1) and 24-epi-castasterone (2) could be identified as theirbismethylboronates by GC-MS in comparison with authen-tic samples. A co-occurrence of these two 24-epimericbrassinosteroids was also detected in Ornithopus sativus,Beta vulgaris and Rheum rhabarbarum (Schmidt et al.,1993, 1994, 1995).

Fraction F II was further purified by silica gel chromato-graphy with a modified chloroform: methanol gradient,DEA ion exchange chromatography and repeated reversed-phase HPLC with several gradient systems(acetonitrile :water). Besides brassinolide (3), which wasalso identified as its bismethylboronate by GC-MS, abioactive fraction with a shorter HPLC retention time thanbrassinolide was found. This fraction was further purifiedby HPLC guided by the rice lamina-inclination bioassay. Anew pregnane-type glucoside 4 was isolated in traceamounts (ca. 100 mg) from this fraction. Compound 4displayed in the positive ion ES-MS, besides a [M+Na]+ -ion at m/z 665, significant ions at m/z301([aglycone+H2H2O]+ ), 283 (base peak), 257, 201,189, 161 and 135 (Fig. 1). The molecular weight of 642 isalso indicated by an [M-H]2 -ion at m/z 641 in the negativeion ES-MS obtained by LC-MS. The CID-MS of m/z 641showed significant ions at m/z 479 ([M-H-162]2 ), indicat-ing the loss of a hexose moiety, as well as m/z 221, 161([hexose-H-H2O]2 ) and 101 (Fig. 2). Key ions at m/z 221,161, 113 and 101 were also observed in the negative ion ES-MS of un-derivatized glucopyranosyl disaccharides such asthe (1→2)-b-D-glucose disaccharide sophorose (Mulroneyet al., 1995). Therefore, the positive and negative ion ES-MS can give complementary information concerning thestructure of the glucoside 4.

The 1H NMR-spectrum of 4 shows two singlets belong-ing to steroidal angular methyl groups (d 0.84 and 0.60), amethyl singlet at d 2.10 and two anomeric proton doublets.Because of its 1H, 1H COSY cross-peaks with four signals inthe high-field region, the multiplet at d 3.51 is assigned to asteroidal methine proton. Multiplet pattern (dddd) andvicinal coupling constants (11.4/11.4/4.8/4.8 Hz) indicatethat this signal belongs to H-3a of the aglycone. Because ofthe limited amount of 4 (ca. 100 mg) no 13C NMR spectrumcould be recorded. However, from correlation peaks of one-bond (HMQC) and long-range (HMBC) 1H, 13C twodimensional correlation experiments, a number of 13C (aswell as 1H) chemical shifts could be obtained (Tables 1 and2). The 13C NMR data (Table 1) of the aglycone correspondto those of 3b-hydroxy-5a-pregnan-20-one (5). Unfortu-nately, because of severe signal overlapping in the glycoside1H region, the very strong solvent signal, and the poorsignal-to-noise ratio of the HMQC spectrum, 1H and 13CNMR data of the glycoside moieties could not be deter-mined completely. However, the measured 13C chemicalshift of d 82.9 for C-2 of one of the sugar moieties suggestsa 1→2 intersugar linkage (Besso et al., 1982) and thus the

Table 3. Key ions in the mass spectra of derivatized compound5 obtained by GC-MS

relative abundance (%)a

3b-hydroxy-5a-pregnane-20-one-TMSi-etherb

Ion (m/z) authentic sample after enzymatic cleavage

390 (M+) 17 19

375 ([M-Me]+ 100 100

372 ([M-H2O]+) 8 7

333 ([M-Me-CH2CO]+) 12 12

300([M-TMSiOH]+) 31 33

285 ([M-Me-TMSiOH]+) 17 17

261 11 12

257 12 13

243 13 16

215 32 34

159 18 43

155 24 46

142 21 33a normalized to the most abundant peak above m/z 100.b relative retention time (RRt) related to 5a-cholestane was 0.97.

J. SCHMIDT ET AL.18


signal at d 82.9 is assigned to C-2'. The assignments of theother sugar 1H signals (Table 2) are made based on the 1H,1H COSY correlations.

The assumption of a 1→2 intersugar linkage wasconfirmed by an 1H, 1H COSY spectrum of the peracety-lated derivative of 4. One of the anomeric proton signals (d4.77, H-1") shows a cross-peak to a signal at d 4.94 (H-2"),whereas the other anomeric proton (d 4.55, H-1') correlateswith a signal at d 3.65 (H-2'). The high shielding of the latterproton indicates that this position is not acetylated andtherefore has to be the branching point of the sugar chain.The observation of seven acetyl singlets additional to thesinglet of Me-21 in the region d 2.11–1.98 indicates the twosugars to be hexoses.

Enzymatic cleavage of the glucoside 4 with b-D-glucosidase led to 3b-hydroxy-5a-pregnane-20-one (5)which was identified by GC-MS in comparison with anauthentic sample (Table 3). Therefore, on the basis of thesedata we propose for the new glucoside the structure of b-D-glucopyranosyl - (1→2) - b - D - glucopyranosyl -3b - hydroxy - 5a - pregnane - 20 - one (sophorosyl -pregnanolone; 4). The structure of the native pregnaneglucoside 4 was confirmed also by comparison (LC-MS,LC-MS–MS, 1H NMR) with authentic 4 (Fig. 2, Tables 1and 2) synthesized by the Konigs–Knorr method froma-acetobromosophorose and 3b-hydroxy-5a-preg-nane-20-one.

Pregnane-type glucosides, assumed to be biogenetic

precursors of cardenolides, are described as naturallyoccurring constituents especially in members of the Ascle-piadaceae and Apocynaceae (Hegnauer, 1964, 1989;Deepak et al., 1989). On the other hand, the metabolictransformation of brassinosteroids to pregnane-20-one typecompounds in cell suspension cultures of Ornithopussativus has been demonstrated recently (Kolbe et al.,1996).

While sophorosylpregnenolone has been detected inNerium odorum (Yamauchi et al., 1972), the correspondingpregnanolone derivative 4 is a new naturally occurringcompound. 3b-Hydroxy-5a-pregnane-20-one (5) wasfirstly identified as the aglycone of glucosides fromXysmalobium undulatum (Asclepiadaceae) as a constituentof an higher plant (Tschesche and Snatzke, 1960). To thebest of our knowledge, the finding of sophorosylpregnano-lone (4) in Daucus carota represents the first detection of apregnane derivative in a plant species of the Apiaceaefamily. Both compound 5 and its sophorosyl derivative 4were shown to be inactive in the rice lamina-inclinationbioassay. Therefore, the bioactive principle of the corre-sponding HPLC fraction needs further investigation.

Acknowledgements

The authors are grateful to Mrs. Helga Menzky for skilful technicalassistance, Mrs. Christine Kuhnt for the GC-MS measurements and theDeutsche Forschungsgemeinschaft (DFG) for financial support.

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Brassinosteroids and a pregnane glucoside from Daucus carota

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