4-Demethyl simmondsin from
Simmondsia chinensis
M. Van Boven
a,*, R. Busson
b, M. Cokelaere
c, G. Flo
c, E. Decuypere
daLaboratory of Toxicology and Food Chemistry,Katholieke Uni6ersiteit Leu6en,Van E6enstraat4,B-3000Leu6en,Belgium bLaboratory of Medicinal Chemistry,Katholieke Uni6ersiteit Leu6en,Minderbroedersstraat,B-3000Leu6en,Belgium
cInterdisciplinary Research Center,Katholieke Uni6ersiteit Leu6en Campus Kortrijk,Uni6ersitaire Campus, B-8500Kortrijk,Belgium
dLaboratory of Physiology and Immunology of Domestic Animals,Katholieke Uni6ersiteit Leu6en,B-3001He6erlee,Belgium
Accepted 12 May 2000
Abstract
Jojoba seed meal (Simmondsia chinensis) contains approximately 15% glucosides identified as simmondsin, 5-demethyl simmondsin, didemethyl simmondsin, along with simmondsin 2%-ferulate, 4-demethyl simmondsin 2% -feru-late, and 5-demethyl simmondsin ferulate. The new simmondsin derivative was isolated from jojoba meal by a combination of column chromatography and preparative HPLC and identified by 2D-NMR and L-SIMS as 2-(cyanomethylene)-3,4-dihydroxy-5-methoxycyclohexylb-D-glucoside or 4-demethyl simmmondsin. © 2000 Elsevier Science B.V. All rights reserved.
Keywords:2D-NMR; 4-Demethyl simmondsin; HPLC; Jojoba
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1. Introduction
Jojoba [Simmondsia chinensis(Link) Schneider], from the family of Buxaceae or Simmondsiaceae) grew originally in the Sonora desert and is now being cultivated in Arizona, California, Mexico and other semi-arid countries. After removing the oil from the seeds, a protein rich meal remains. This meal has been described as toxic to rodents and chickens (Elliger et al., 1973, 1974a). The
symptoms described as toxic are due to the food intake inhibition caused by jojoba constituents. Simmondsin has been identified as the most im-portant inhibitor (Elliger et al., 1973, 1974a; Booth et al., 1974; Cokelaere et al., 1992a,b; Erhan et al., 1997). Elliger et al. (1974b) have also isolated and characterised simmondsin-2%-ferulate as well as 5-demethyl simmondsin and 4,5-didemethyl simmondsin in the meal. This study deals with the isolation and structure determina-tion of 4-demethyl simmondsin in jojoba meal. Although 4- and 5-demethyl simmondsin 2% -feru-lates already were described (Van Boven et al., 1995) 4-demethyl simmondsin was not previously isolated as a pure compound.
* Corresponding author. Tel.: +32-16-323411; fax: + 32-16-323505.
E-mail address:[email protected] (M. Van Boven).
2. Materials and methods
2.1. Plant material
Jojoba meal was obtained from EMEC Agro Industries (Antwerp, Belgium).
2.2. Silylation
Derivatives were prepared by adding 1 mg of 4-and 5-demethyl simmondsin or mixtures of both to 1.0 ml of Tri-SIL. After 60 min reaction at room temperature the reaction mixture was examined by GC and GC-MS.
2.3. Gas chromatography
A Chrompack 9000 gas chromatograph equipped with a flame ionisation detector was used for the analysis. Separations were made by a 40 m×0.25 mm i.d. glass capillary column with a chemically bonded phase (Chrompack, The Netherlands) of a phenyl (50%) dimethylpolysiloxane (50%) gum or CP-Sil 24 (0.25 mm film). Samples of 2 ml were injected by means of a split/splitless injector. To protect the column a special insert glass liner (Chrompack Cat. No. 729814) was used. Injector and detector temperatures were set at 295°C. He-lium was used as carrier gas at 40 cm/s (set at 60°C).
2.4. Thin layer chromatography(TLC)
TLC was performed on silica gel plates (Poly-gram Sil G/UV254-Machery-Nagel, Germany) us-ing a mixture of methanol and chloroform (20/80, v/v) as the solvent. The spots were visualised by spraying the plates with 1-naphthol reagent. The naphthol reagent was prepared by adding 10.5 ml of a 15% ethanolic solution of 1-naphthol to a mixture of 40.5 ml ethanol, 4 ml water and 6.5 ml sulfuric acid. After spraying, the plates were heated at 100°C in an oven for 5 min.
2.5. High performance liquid chromatography
(HPLC)
HPLC was performed with a Merck Hitachi-6200 apparatus. Samples were injected into a
Rheodyne injector (Model 7125, Berkeley, CA) supplied with a 200 ml injector loop. A stainless steel Si 60 (5 mm particle size) column was used, 25 cm×1.0 cm i.d. (E. Merck, Darmstadt, Germany) for analytical purposes and another Si 60 column (10 m particle size, 25×1.0 cm) was used as a preparative column.. The flow rate of the solvent, a mixture of acetonitrile and water 90/10 (v/v), was 1.0 ml/min for the analyti-cal system and 3.0 ml/min for the preparative separation. The column eluate was monitored at 217 nm with a Hitachi Model L-3000 photo diode array detector. All solvents used were analytical grade.
2.6. Spectroscopy
2.6.1. Nuclear magnetic resonance spectroscopy
1H and13C NMR spectra were performed on a
Varian UNITY-500 NMR spectrometer operating at 125.591 MHz for 13C and at 499.40 MHz for 1H NMR using the standard pulse sequences and
standard software (vnmr× 5.3a). Spectra were recorded at 27°C in 5 mm tubes in CD3OD solution and chemical shifts are reported in ppm relative to tetramethylsilane (TMS) as internal reference, or in the absence of TMS to the CH3OH resonance set at 3.30 ppm versus TMS for proton or set at 49.0 ppm for carbon spectra. The coupling patterns of the 1H NMR spectra were elucidated by two dimensional (2D) corre-lated spectroscopy (COSY) or by homonuclear spin decoupling techniques. The 13C assigments were made by using inverse 2D-heterocorrelation experiments such as GHSQC (gradient heteronu-clear single quantum correlation spectroscopy) and GHMBC (gradient heteronuclear multiple quantum correlation spectroscopy).
2.6.2. Mass spectrometry.
Liquid surface-assisted secondary ionisation mass-spectrometry (L-SIMS) was performed with a Kratos Concept 1H instrument using a 7-keV Cs beam. The products were dissolved in glycerine.
2.7. Isolation of4- and 5-demethyl simmondsin
from jojoba meal
Part of the isolation is already described by Van Boven et al. (1994). Jojoba meal was ex-tracted twice with hexane to eliminate any re-maining residues of oil. The deoiled meal (1 kg) was extracted with acetone for 12 h by means of a Soxhlet apparatus. After evaporation of the solvent, a brown residue (40 g) was obtained. The residue was taken up in methanol and added to 100 g of silica gel (0.2 – 0.5 mm). The solvent was removed under vacuum and the silica gel was loaded into a silica gel column (length 30 cm, 6 cm i.d.), containing a suspension of silica gel (0.040 – 0.063 mm) in chloroform. The column
was first eluted with 1 l of chloroform, which was discarded. The column was further eluted with acetone. Fractions (100 ml) were collected and analysed by TLC until simmondsin and analogous compounds were eluted completely. The fraction eluting after simmondsin (16 g of a mixture of demethyl simmondsins together with some simmondsin and some didemethyl sim-mondsin) was collected separately and filtered over activated carbon resulting in a yellow solu-tion. After evaporation of the acetone, the crude residue was further purified on another silica gel column (60 cm×6 cm i.d.), containing a suspen-sion of 0.5 kg of silica gel (0.040 – 0.063 mm) in chloroform. The crude residue was first dissolved in methanol and absorbed into silica gel (0.2 – 0.5 mm, 100 g) which was loaded into the silica gel column. Elution was performed with of a mixture of methanol and chloroform (20/80, v/v). The column output was passed through a UV instru-ment for direct monitoring eluting compounds at adequate wavelengths, chosen to keep
ab-Table 1
1H chemical shifts (ppm) for simmondsin ferulates in CD 3ODa
H4 2.97 dd(3,9) 3.44 dd(3,9) 2.96 dd(3,9) 3.33 dd(3,9)
3.70 q(3,3.5,4) 4.18 q(3,3.5,4) 3.60 q(3,3.5,4) H5 4.28 q(3,3.5,4)
H6A 1.74 ddd(3.5,4,15) 1.69 ddd(3.5,4,15) 1.76 ddd(3.5,4,15) 1.53 ddd(3.5,4,15) H6B 2.42 ddd(4,4,15) 2.50 ddd(4,4,15) 2.22 ddd(4,4,15) 2.42 ddd(4,4,15)
s
3.20 dd(7.8,9) 3.20 dd(7.8,9)
H2% 4.83 dd(7.8,9) 4.85 dd(7.8,9)
3.35 dd(9,9)
3.39
H3% dd(9,9) 3.62 dd(9,9) 3.59 dd(9,9)
3.36 dd(5.2,9.9) 3.30 dd(5.2,9.9)
H4% 3.44 dd(5.2,9.9) 3.43 dd(5.2,9.9)
m(9,5.2,2.2)
3.69 dd(5.2,12) 3.68 dd(5.2,12)
H6%A 3.71 dd(5.2,12) 3.70 dd(5.2,12)
3.86 dd(2.2,12) 3.85 dd(2.2,12) H6%B 3.82 dd(2.2,12) 3.84 dd(2.2,12)
Table 2
13C chemical shifts (ppm) in CD3OD
3
Carbon no.a 2 7 8
76.9
1 77.8 78.4 78.1
2 166.5 166.8 166.0 166.9
72.1 70.2
CN 117.6 117.4 117.1
58.3
aCarbon numbers correspond to the numbers of Fig. 1.
sorbances in scale. All peaks were collected sepa-rately, examined for purity by HPLC and TLC, and concentrated. The peak eluting just after sim-mondsin delivered 5-demethyl simsim-mondsin after crystallisation as already described (Van Boven et al., 1994). When the mother liquor was submitted to the described analytical HPLC method a peak consisting of two incompletely resolved com-pounds was observed. This HPLC method was adapted, as described in the experimental section, to separate both products in sufficient quantities to allow NMR and mass spectrometry.
3. Results and discussion
3.1. Isolation procedure
The above described HPLC method led to the isolation of two distinct demethyl simmondsins with retention times of 111 and 137 min, respec-tively. The compound with Rt=137 min was identified as the previously described 5-demethyl simmondsin. The compound with Rt=111 min was further examined by NMR and mass spec-troscopy. The method allows one to isolate 5 mg of the new compound in a single injection. The
described TLC procedure, however, did not allow the separation of these compounds. Both isomers appeared as violet spots after spraying the plates with the 1-naphthol reagent. However, the iso-mers could be separated by the described gas chromatographic procedure. The isolated 4-demethyl simmondsin appeared as one single peak with Rt=137 min and Rt=111 min for 5-demethyl simmondsin.
3.2. Structure elucidation by NMR spectroscopy
The 1
H-NMR chemical shifts of 5-demethyl simmondsin and the new isolated compound are represented in Table 1 along with the spectral data from 4-demethyl simmondsin 2%-ferulate and 5-demethyl simmondsin 2%-ferulate.
The 1H and 13C NMR spectral data of sim-mondsin (1), 5-demethyl simmondsin (2), 4,5 didemethyl simmondsin (4), simmondsin 2% -feru-late (5), 4-demethyl simmondsin 2%-ferulate (8), 5-demethyl simmondsin 2%-ferulate (7) have been discussed in detail by Van Boven et al. (1993, 1994, 1995). The 1H and 13C spectra of the iso-lated new product correspond perfectly with the already described spectra of 4-demethyl sim-mondsin 2%-ferulate (Tables 1 and 2) like the 1
H and13C spectra of 5-demethyl simmondsin corre-spond with the spectra from 5-demethyl sim-mondsin 2%-ferulate. 1H spectra for the 5- and 4-demethyl simmondsin isomers are identical ex-cept for the H4and H5values. The presence of the
b-glucose moiety was shown by the presence of the signals betweend3.30 and 3.85 and the signal at 4.38 (1H, d, J=7.8Hz), proving the equatorial position of the anomeric proton. The coupling constant of 9 Hz between H3 an H4 confirms a diaxial relationship. H5 shows an equatorial cou-pling constant of 3 Hz with H4.
The 13
C shifts represented in Table 2 prove in an exact way the presence and place of the methoxy substituent in the new isolated product. Replacement of a methoxy substitution by a hy-droxyl substitution implicates for the aliphatic carbon atom an upfield shift of about 10 ppm as described by Wehrli and Wirthlin (1978). This is demonstrated in the mentioned compound when the spectrum is compared with the spectrum of
simmondsin (Van Boven et al., 1994) with ab-sorbances at 86.4 for C4 and at 76.5 for C5.
Structures of the different simmondsins and simmondsin ferulates are represented in Fig. 1.
3.3. Mass spectrometry
Due to intense decomposition, electron impact ionisation mass spectrometry of compounds (2) and (3) did not result in distinct mass spectra. On the other hand, the L-SIMS technique, which bombards with Cs ions, provided distinct spectra. As described by Rinehart (1982) and Barber et al. (1982), the use of glycerol as a solvent in the L-SIMS technique can give rise to both (M+H)+ and (M+Na)+ ions, as well as fragment ions. In the present case both 5-demethyl simmondsin and the isolated isomer 4-demethyl simmondsin show a nearly identical mass spectrum (Van Boven et al., 1994) with (M+H)+ at m/z362.
Acknowledgements
We thank G. Jansen (Pharmaceutical Chem-istry, KULeuven) for recording the mass spectra. This work was supported by the Onderzoeksfonds KULeuven (OT/35/99).
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