- Email: [email protected]
Norlignans and homoisoflavanones from two South African Drimiopsis species (Hyacinthaceae: Hyacinthoideae)
Biochemical Systematics and Ecology 34 (2006) 588e592 www.elsevier.com/locate/biochemsyseco
Norlignans and homoisoflavanones from two South African Drimiopsis species (Hyacinthaceae: Hyacinthoideae) Chantal Koorbanally a, Dulcie A. Mulholland a,b,*, Neil R. Crouch a,c a
Natural Products Research Group, School of Chemistry, University of KwaZulu-Natal, Durban 4041, South Africa b School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK c Ethnobotany Unit, South African National Biodiversity Institute, PO Box 52099, Berea Road, 4007 Durban, South Africa Received 24 November 2004; accepted 6 December 2005
Keywords: Drimiopsis burkei; Drimiopsis maculata; Hyacinthaceae; Norlignan; Homoisoflavanone; ()-(Z)-1,3-bis(4-Hydroxyphenyl)-1,4-pentadiene (()-Nyasol); 5,7-Dimethoxy-3-(4-hydroxybenzyl)-4-chromanone; 7-O-Methyl-3,9-dihydropunctatin; 5,7-Dihydroxy-3-(4-hydroxybenzyl)4-chromanone; 5,6-Dihydroxy-7-methoxy-3-(4-hydroxybenzyl)-4-chromanone; ()-(E)-1,3-bis(4-Hydroxyphenyl)-1,4-pentadiene
1. Subject and source The genus Drimiopsis Lindl. (Hyacinthaceae: Hyacinthoideae) is endemic to sub-Saharan Africa where it is represented by approximately 20 species, at least five of which occur in southern Africa (Jessop, 1972). Drimiopsis maculata Lindl. is the only South African species from this genus reportedly employed in ethnomedicine, preparations of the bulb being used by both Zulu and Xhosa practitioners to treat paediatric gastro-intestinal complaints (Hulme, 1954; Hutchings, 1989; Tyiso and Bhat, 1998). This species is widely distributed in southern, central and east Africa (Stedje, 1994; Kativu, 2000). Sheep toxicity tests using fresh flowering and seeding plant materials of D. maculata have proven negative (Van der Walt, 1944). Drimiopsis burkei Baker is a polymorphic taxon, considered by some authors (Muller-Doblies and Muller-Doblies, 1997) to consist of at least two subspecies. However, as cited type materials remain inaccessible to the taxonomic community, the systematic arrangement of Jessop (1972) is followed. D. burkei is found in a wide diversity of habitats, distributed across Botswana and Zimbabwe (Kativu, 2000), southwards to the Eastern Cape Province of South Africa (Jessop, 1972). 2. Previous work No previous phytochemical work appears to have been conducted on D. burkei. We have reported previously on the isolation of six methylxanthones, drimiopsins AeF, from the methylene chloride extract of the bulbs of D. maculata (Mulholland et al., 2004), and the known scillascillins, 30 ,5-dihydroxy-40 ,7-dimethoxyspiro[2H-1-benzopyran-3(4H), * Corresponding author. School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK. Tel.: þ44 1483 686850; fax: þ44 1483 686851. E-mail address: [email protected] (D.A. Mulholland). 0305-1978/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2005.12.011
C. Koorbanally et al. / Biochemical Systematics and Ecology 34 (2006) 588e592
589
70 -bicyclo[4.2.0]octa[1,3,5]-trien]-4-one and 30 ,5,7-trihydroxy-40 -methoxyspiro[2H-1-benzopyran-3(4H), 70 -bicyclo[4.2.0]octa[1,3,5]-trien]-4-one (Koorbanally et al., 2001). In order to isolate larger amounts of the methylxanthones for screening purposes, a further collection of D. maculata was made which yielded further interesting compounds. 3. Present study Bulbs of flowering material of D. burkei Baker were collected from Paris Dam, near Vryheid, KwaZulu-Natal, South Africa in September 1999 and a voucher specimen retained (Crouch 827, NH). The bulbs (678 g) were dried, chopped and successively extracted with dichloromethane and methanol on a mechanical shaker for 48 h. The dichloromethane extract (1.57 g) was separated using column chromatography over silica gel (Merck 9385, 100% MeCl2) to yield two compounds: the norlignan ()-nyasol, ()-(Z)-1,3-bis(4-hydroxyphenyl)-1,4-pentadiene 1 (4.8 mg) (Marini-Bettolo et al., 1985 (optical rotation not given); Brown and Tsui, 1996, [(þ)-isomer], named as hinokiresinol by these authors) and the homoisoflavanone 5,7-dimethoxy-3-(4-hydroxybenzyl)-4-chromanone 2 (10.3 mg), the structures of which were determined using 2D NMR and MS techniques, and by comparison of NMR data against literature data as given above. Compound 2 has been reported previously only as the acetate derivative (Bangani et al., 1999), so NMR data is given in Table 1. The methanol extract contained only sugars, so it was not examined further. Bulbs of D. maculata Lindl. were collected during March 1999 from a cultivated source in Kloof, KwaZulu-Natal, South Africa and a voucher specimen retained (Crouch 790, NH). The bulbs (3871 g) were extracted as described above. The dichloromethane extract (4.28 g) was chromatographed over silica gel (Merck 9385) using a dichloromethane/ethyl acetate step gradient to yield four compounds: the known homoisoflavanones (100% dichloromethane) 7-O-methyl-3,9-dihydropunctatin, 3 (15.3 mg) (Adinolfi et al., 1986; Koorbanally et al., 2001), 5,7-dihydroxy-3(4-hydroxybenzyl)-4-chromanone, 4 (12.5 mg), (Adinolfi et al., 1986; Pohl, 1999), the novel homoisoflavanone, 5,6-dihydroxy-7-methoxy-3-(4-hydroxybenzyl)-4-chromanone, 5 (10.1 mg), the six methylxanthones described earlier (10e20% ethyl acetate in dichloromethane) (Mulholland et al., 2004) and the norlignan, ()-(E)-1,3-bis(4hydroxyphenyl)-1,4-pentadiene, 6 (4.7 mg) (25% ethyl acetate in dichloromethane), which has been synthesised previously (Ameer et al., 1988), but has not been reported as a natural product. Structures of compounds 3, 4 and 6 were determined using 2D NMR and MS techniques and confirmed by comparison against literature values as referenced above. The IR spectra were recorded with a Nicolet Impact 400 D spectrometer on sodium chloride plates and calibrated against an air background. The HRMS was obtained using a Kratos high resolution MS 9/50 spectrometer. 1H and 13C Table 1 NMR data for compound 5, 5a and 2 (400 MHz) (J in Hz) 1
1
1
4.32 dd (3.9, 11.3) 4.21 dd (6.3, 11.3) 2.80 m
4.31 dd (3.8, 11.5) 4.17 dd (6.8, 11.5) 2.77 m
4.06 dd (4.5, 11.3) 4.25 dd (3.8, 11.3) 2.65 m
H (5) (CDCl3)
2a 2b 3 4 4a 5 6 7 8 8a 9a 9b 10 20 60 30 50 40 40 -OCH3 7-OCH3 5-OH 5-OCH3
H (5a) (CDCl3)
6.08 s 6.12 s 2.72 dd (10.2, 13.9) 3.12 dd (4.2, 13.9) 7.09 d (8.5) 6.77 d (8.4)
3.90 s 11.70 s
H (2) (CD3OD)
6.1 d (2.1) 6.11 d (2.1)
2.68 dd (10.3) 3.13 dd (4.3, 13.7) 7.07 d (8.1) 6.76 d (8.5) 3.76 s 3.86 s 12.03 s
3.03 dd (4.2, 13.0) 2.62 dd (2.9, 13.1) 7.01 d (8.6) 6.71 d (8.6)
3.81 s 3.81 s
13
C (5) (CDCl3)
13
C (5a) (CDCl3)
13
C (2) (CD3OD)
69.3
69.1
68.8
47.2 198.1 102.1 155.1 125.8 157.3 92.5 147.9 32.0
46.8 198.3 102.2 160.2 92.9 161.3 129.3 153.6 31.9
48.7 192.7 104.8 162.6 92.5 166.7 93.3 165.2 32.2
129.8 130.3 115.5 154.3
129.3 130.2 115.5 154.7 61.3 56.1
129.1 129.8 115.1 155.8
56.3
55.1 55.0
C. Koorbanally et al. / Biochemical Systematics and Ecology 34 (2006) 588e592
590
NMR spectra were recorded on a Varian Unity Inova 400 MHz NMR spectrometer. The UV spectrum was recorded in methylene chloride on a Varian DMS 300 UVevisible spectrometer. Optical rotations were measured at room temperature in methanol using a Perkin Elmer Polarimeter e Model 341. The CI positive HRMS of compound 5 gave an [M þ H] molecular ion peak at m/z 317.1030766, corresponding to a molecular formula C17H16O6 for 5. The 1H NMR spectrum showed the presence of a 3-benzyl-4-chromanone-type homoisoflavanone with coupled resonances ascribed to 2H-2 (4.32 dd, 4.21 dd), H-3 (2.80 m) and 2H-9 (2.72 dd, 3.12 dd). The two proton doublets at dH 6.77 and dH 7.09 (J ¼ 8.42 Hz) were assigned as H-30 /50 and H-20 /60 , respectively, of ring B and these assignments were confirmed using the HMBC spectrum. The base peak at m/z 107 resulting from hydroxybenzyl fragmentation in the mass spectrum indicated a hydroxybenzyl/hydroxytropylium ion indicating the presence of a hydroxyl group at C-40 (Wall et al., 1989). The proton NMR spectrum showed a singlet resonance at dH 6.12 which was indicative of only one proton on the Aring, which would have to be placed at either C-6 or C-8 on biosynthetic grounds, and one three proton methoxy group proton resonance at dH 3.90. The presence of a hydroxy group at C-5 was indicated by the appearance of the C-4 carbonyl resonance at dC 198.1, the downfield shift being a consequence of the chelating effects between the hydrogen of the hydroxy group at C-5 and the oxygen of the carbonyl group (Adinolfi et al., 1986). This was confirmed by the bathochromic shift that was seen when the UV spectrum was rerun on addition of AlCl3 (þ25 nm). No shift occurred upon the addition of NaOAc, indicating no hydroxy group was present at C-7, hence the methoxy group was placed at this position (Adinolfi et al., 1984, 1985). The 1H NMR spectrum acquired in CDCl3 showed the 5-OH proton resonance at dH 11.70. The NOESY spectrum indicated a correlation between the single proton resonance dH 6.12 and the methoxy group proton resonance but not the 5-hydroxy group proton resonance. This suggested that the single proton should be placed at C-8, but HMBC supporting evidence was inconclusive. The 40 -methoxy compound (5a) has been isolated from the related Ledebouria revoluta Lf. Jessop (Moodley et al., in press) with the proton at the C-6 position, confirmed by a correlation in the NOESY spectrum between H-6 and both the 5-OH and C-7 methoxy group proton resonances as well as an HMBC correlation from C-5 to H-6. The H-6 proton resonance of 5a occurred at dH 6.08, whereas the A-ring proton of compound 5 occurred at dH 6.12. NMR data for 5 and 5a are given in Table 1 for comparison purposes. The 13 C NMR data are significantly different for ring-A of the two compounds. A literature search indicated that compound 5 was a novel homoisoflavonoid and was named 5,6-dihydroxy-7-methoxy-3-(4-hydroxybenzyl)-4-chromanone.
3.1. Compound 1 ()-nyasol, ()-(Z)-1,3-bis(4-hydroxyphenyl)-1,4-pentadiene
[a]25 D ¼ 135.7 (c ¼ 0.14 g/100 mL).
3.2. Compound 2 5,7-dimethoxy-3-(4-hydroxybenzyl)-4-chromanone (10.3 mg), yellow vitreous solid, UVevisible lmax nm (log 3) in MeOH: 282 (3.67). HRMS: [M þ H] 315.1236874 (C18H18O5 þ H requires 315.123249), GCeMS: [Mþ] 314, 207, 181, 152, 137, 107, 77. IR nmax (NaCl) cm1: 3348, 2913, 2848, 1607, 1455, 1215, 1155. [a]25 D ¼ 25.00 (c ¼ 0.03 g/100 mL).
3.3. Compound 5 5,6-dihydroxy-7-methoxy-3-(4-hydroxybenzyl)-4-chromanone (10.1 mg), bright yellow, fine, needle-like crystals, m.p. 174e176 C. UVevisible lmax nm (log 3) in MeOH: 290 (4.04). HRMS: [M þ H] 317.1030766 (C17H16O6 þ H requires 317.102513), GCeMS: [Mþ] 316, 209, 107, 77. IR nmax (NaCl) cm1: 3387, 1640, 1511, 1376, 1221, 1099. [a]25 D ¼ 40.91 (c ¼ 0.044 g/100 mL).
3.4. Compound 6 ()-(E)-1,3-bis(4-hydroxyphenyl)-1,4-pentadiene
[a]25 D ¼ 4.76 (c ¼ 0.042 g/100 mL).
C. Koorbanally et al. / Biochemical Systematics and Ecology 34 (2006) 588e592
HO
R1 R2
O
591
5" 4"
6"
H
OH 3" 2"
1"
2 1
3 1'
R3
4 6'
H R1
R2
2
H
OMe H
3
OMe OMe H
OH
4
H
OH
5
H
OMe OH
OH
5a
OH
OMe H
OMe
OH
R3
H
2'
5
O
R4
3'
5'
R4
4'
OMe
OH
1 HO
5'
5" 4"
H 3"
4'
6'
6" 2
1'
3'
1" 3
2"
1
OH
2'
4 5
6
4. Chemotaxonomic significance The isolation of homoisoflavanones (compounds 2e5) was anticipated as they are common constituents of the Hyacinthoideae, and generally useful as subfamily chemotaxonomic markers (Pohl et al., 2000). However, this is the first reported isolation of norlignans (compounds 1 and 6) from the Hyacinthaceae. We have also subsequently isolated a further norlignan from Ledebouria ovatifolia (Bak.) Jessop (Langlois, 2003). Significantly, the three norlignans isolated to date are from taxa in the tribe Massonieae (subfamily Hyacinthoideae), from representatives of a clade circumscribed generically by Manning et al. (2004) as Ledebouria Roth. Their decision was based on a phylogenetic analysis employing trnLeF and rbcL molecular data. Although now known from Ledebouria (Langlois, 2003) and Drimiopsis, norlignans have not yet been isolated from Resnova Van der Merwe (Koorbanally et al., 2006), a genus also synonymised under Ledebouria by Manning et al. (2004). Were this so, the close relationship between the three genera would be confirmed chemotaxonomically. However, on the basis of definitive morphological characters (Muller-Doblies and Muller-Doblies, 1997; Stedje, 1998), generic distinctions would still be justified. Norlignans currently provide the only intra-tribal chemotaxonomic markers in the Hyacinthoideae; notably, no such markers are known at the tribal level. Compound 1 is the enantiomer of hinokiresinol, which has previously been isolated from Asparagus cochinchinensis Merrill (Asparagaceae) (Brown and Tsui, 1996) and prepared by enzymic hydrolysis of the diglucoside nyasoside isolated from Hypoxis nyasica Baker (Hypoxidaceae) (Marini-Bettolo et al., 1985). Accordingly, all isolations of 1 have been from the Liliaceae sensu lato, of which the Hypoxidaceae, Asparagaceae and the Hyacinthaceae were all formerly a part. Notably, the Z- or cis-isomer, 1, was isolated from D. burkei, and the E- or trans-isomer, 6, from its sister taxon D. maculata. This is the first report of a naturally occurring E-norlignan, although this particular compound has earlier been synthesised (Ameer et al., 1988). The E-isomer was found to be less stable than the Z-isomer, and started decomposing during acquisition of NMR spectra. Compound 2 has been reported previously from Schizocarphus nervosus (Burch.) Van der Merwe (syn. Scilla nervosa (Burch.) Jessop) (Hyacinthoideae) but was isolated as the acetate derivative resulting from acetylation of a non-separable mixture (Bangani et al., 1999). Compounds 3 and 4 have been isolated previously from D. maculata, Eucomis montana Compton, Ledebouria cooperi (Hook.f.) Jessop, Muscari comosum Mill., and D. maculata, L. ovatifolia, Resnova humifusa (Bak.) U. & D.M.-D. and L. revoluta, respectively.
592
C. Koorbanally et al. / Biochemical Systematics and Ecology 34 (2006) 588e592
Acknowledgements We are grateful to Mr. D. Jagjivan for running NMR spectra and Mr. John Hill for mass spectrometric analysis. CK gratefully acknowledges the National Research Foundation (NRF) postgraduate scholarship. The staff of the Mary Gunn Library (SANBI) kindly facilitated access to literature. This research was funded by the NRF and the University of Natal Research Fund. References Adinolfi, M., Barone, G., Belardini, M., Lanzetta, R., Laonigro, G., Parrilli, M., 1984. Phytochemistry 23, 2091. Adinolfi, M., Barone, G., Belardini, M., Lanzetta, R., Laonigro, G., Parrilli, M., 1985. Phytochemistry 24, 2423. Adinolfi, M., Lanzetta, R., Laonigro, G., Parrilli, M., 1986. Magn. Reson. Chem. 24, 663. Ameer, F., Drewes, S.E., Drewes, M.W., Roos, G.H.P., Watson, M.C., 1988. J. Chem. Soc. Perkin Trans. 1, 1425. Bangani, V., Crouch, N.R., Mulholland, D.A., 1999. Phytochemistry 51, 947. Brown, G.D., Tsui, W., 1996. Phytochemistry 43, 1413. Hulme, M.M., 1954. Wild Flowers of Natal. Shuter and Shooter, Pietermaritzburg. t. 13. Hutchings, A., 1989. Bothalia 19, 225. Jessop, J.P., 1972. J. S. Afr. Bot. 38, 151. Kativu, S., 2000. Kirkia 17, 150. Koorbanally, C., Crouch, N.R., Mulholland, D.A., 2001. Biochem. Syst. Ecol. 29, 539. Koorbanally, N.A., Crouch, N.R., Harilal, A., Pillay, B., Mulholland, D.A., 2006. Biochem. Syst. Ecol. 34, 114. Langlois, A., 2003. Unpublished Ph.D. dissertation, University of Natal, Durban, South Africa. Manning, J.C., Goldblatt, P., Fay, M.F., 2004. Edinb. J. Bot. 60, 533. Marini-Bettolo, G.B., Nicoletti, M., Messana, I., 1985. Tetrahedron 41, 665. Moodley, N., Crouch, N.R., Mulholland, D.A., Slade, D., Ferreira, D. South African Journal of Botany, in press. Mulholland, D.A., Koorbanally, C., Crouch, N.R., Sandor, P., 2004. J. Nat. Prod. 67, 1726. Muller-Doblies, U., Muller-Doblies, D., 1997. Feddes Repert. 108, 1. Pohl, T., 1999. M.Sc. dissertation, University of Natal, Durban, South Africa. Pohl, T., Crouch, N.R., Mulholland, D.A., 2000. Curr. Org. Chem. 4, 1287. Stedje, B., 1994. Nord. J. Bot. 14, 45. Stedje, B., 1998. Plant Syst. Evol. 211, 1. Tyiso, S., Bhat, R.B., 1998. Ver. Angew. Bot. 72, 92. Van der Walt, S.J., 1944. Onderstepoort J. Vet. Sci. Anim. Ind. 20, 75. Wall, M.E., Wani, M.C., Manikumar, G., Taylor, H., McGivney, R., 1989. J. Nat. Prod. 52, 774.
Norlignans and homoisoflavanones from two South African Drimiopsis species (Hyacinthaceae: Hyacinthoideae) Chantal Koorbanally a, Dulcie A. Mulholland a,b,*, Neil R. Crouch a,c a
Natural Products Research Group, School of Chemistry, University of KwaZulu-Natal, Durban 4041, South Africa b School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK c Ethnobotany Unit, South African National Biodiversity Institute, PO Box 52099, Berea Road, 4007 Durban, South Africa Received 24 November 2004; accepted 6 December 2005
Keywords: Drimiopsis burkei; Drimiopsis maculata; Hyacinthaceae; Norlignan; Homoisoflavanone; ()-(Z)-1,3-bis(4-Hydroxyphenyl)-1,4-pentadiene (()-Nyasol); 5,7-Dimethoxy-3-(4-hydroxybenzyl)-4-chromanone; 7-O-Methyl-3,9-dihydropunctatin; 5,7-Dihydroxy-3-(4-hydroxybenzyl)4-chromanone; 5,6-Dihydroxy-7-methoxy-3-(4-hydroxybenzyl)-4-chromanone; ()-(E)-1,3-bis(4-Hydroxyphenyl)-1,4-pentadiene
1. Subject and source The genus Drimiopsis Lindl. (Hyacinthaceae: Hyacinthoideae) is endemic to sub-Saharan Africa where it is represented by approximately 20 species, at least five of which occur in southern Africa (Jessop, 1972). Drimiopsis maculata Lindl. is the only South African species from this genus reportedly employed in ethnomedicine, preparations of the bulb being used by both Zulu and Xhosa practitioners to treat paediatric gastro-intestinal complaints (Hulme, 1954; Hutchings, 1989; Tyiso and Bhat, 1998). This species is widely distributed in southern, central and east Africa (Stedje, 1994; Kativu, 2000). Sheep toxicity tests using fresh flowering and seeding plant materials of D. maculata have proven negative (Van der Walt, 1944). Drimiopsis burkei Baker is a polymorphic taxon, considered by some authors (Muller-Doblies and Muller-Doblies, 1997) to consist of at least two subspecies. However, as cited type materials remain inaccessible to the taxonomic community, the systematic arrangement of Jessop (1972) is followed. D. burkei is found in a wide diversity of habitats, distributed across Botswana and Zimbabwe (Kativu, 2000), southwards to the Eastern Cape Province of South Africa (Jessop, 1972). 2. Previous work No previous phytochemical work appears to have been conducted on D. burkei. We have reported previously on the isolation of six methylxanthones, drimiopsins AeF, from the methylene chloride extract of the bulbs of D. maculata (Mulholland et al., 2004), and the known scillascillins, 30 ,5-dihydroxy-40 ,7-dimethoxyspiro[2H-1-benzopyran-3(4H), * Corresponding author. School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK. Tel.: þ44 1483 686850; fax: þ44 1483 686851. E-mail address: [email protected] (D.A. Mulholland). 0305-1978/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2005.12.011
C. Koorbanally et al. / Biochemical Systematics and Ecology 34 (2006) 588e592
589
70 -bicyclo[4.2.0]octa[1,3,5]-trien]-4-one and 30 ,5,7-trihydroxy-40 -methoxyspiro[2H-1-benzopyran-3(4H), 70 -bicyclo[4.2.0]octa[1,3,5]-trien]-4-one (Koorbanally et al., 2001). In order to isolate larger amounts of the methylxanthones for screening purposes, a further collection of D. maculata was made which yielded further interesting compounds. 3. Present study Bulbs of flowering material of D. burkei Baker were collected from Paris Dam, near Vryheid, KwaZulu-Natal, South Africa in September 1999 and a voucher specimen retained (Crouch 827, NH). The bulbs (678 g) were dried, chopped and successively extracted with dichloromethane and methanol on a mechanical shaker for 48 h. The dichloromethane extract (1.57 g) was separated using column chromatography over silica gel (Merck 9385, 100% MeCl2) to yield two compounds: the norlignan ()-nyasol, ()-(Z)-1,3-bis(4-hydroxyphenyl)-1,4-pentadiene 1 (4.8 mg) (Marini-Bettolo et al., 1985 (optical rotation not given); Brown and Tsui, 1996, [(þ)-isomer], named as hinokiresinol by these authors) and the homoisoflavanone 5,7-dimethoxy-3-(4-hydroxybenzyl)-4-chromanone 2 (10.3 mg), the structures of which were determined using 2D NMR and MS techniques, and by comparison of NMR data against literature data as given above. Compound 2 has been reported previously only as the acetate derivative (Bangani et al., 1999), so NMR data is given in Table 1. The methanol extract contained only sugars, so it was not examined further. Bulbs of D. maculata Lindl. were collected during March 1999 from a cultivated source in Kloof, KwaZulu-Natal, South Africa and a voucher specimen retained (Crouch 790, NH). The bulbs (3871 g) were extracted as described above. The dichloromethane extract (4.28 g) was chromatographed over silica gel (Merck 9385) using a dichloromethane/ethyl acetate step gradient to yield four compounds: the known homoisoflavanones (100% dichloromethane) 7-O-methyl-3,9-dihydropunctatin, 3 (15.3 mg) (Adinolfi et al., 1986; Koorbanally et al., 2001), 5,7-dihydroxy-3(4-hydroxybenzyl)-4-chromanone, 4 (12.5 mg), (Adinolfi et al., 1986; Pohl, 1999), the novel homoisoflavanone, 5,6-dihydroxy-7-methoxy-3-(4-hydroxybenzyl)-4-chromanone, 5 (10.1 mg), the six methylxanthones described earlier (10e20% ethyl acetate in dichloromethane) (Mulholland et al., 2004) and the norlignan, ()-(E)-1,3-bis(4hydroxyphenyl)-1,4-pentadiene, 6 (4.7 mg) (25% ethyl acetate in dichloromethane), which has been synthesised previously (Ameer et al., 1988), but has not been reported as a natural product. Structures of compounds 3, 4 and 6 were determined using 2D NMR and MS techniques and confirmed by comparison against literature values as referenced above. The IR spectra were recorded with a Nicolet Impact 400 D spectrometer on sodium chloride plates and calibrated against an air background. The HRMS was obtained using a Kratos high resolution MS 9/50 spectrometer. 1H and 13C Table 1 NMR data for compound 5, 5a and 2 (400 MHz) (J in Hz) 1
1
1
4.32 dd (3.9, 11.3) 4.21 dd (6.3, 11.3) 2.80 m
4.31 dd (3.8, 11.5) 4.17 dd (6.8, 11.5) 2.77 m
4.06 dd (4.5, 11.3) 4.25 dd (3.8, 11.3) 2.65 m
H (5) (CDCl3)
2a 2b 3 4 4a 5 6 7 8 8a 9a 9b 10 20 60 30 50 40 40 -OCH3 7-OCH3 5-OH 5-OCH3
H (5a) (CDCl3)
6.08 s 6.12 s 2.72 dd (10.2, 13.9) 3.12 dd (4.2, 13.9) 7.09 d (8.5) 6.77 d (8.4)
3.90 s 11.70 s
H (2) (CD3OD)
6.1 d (2.1) 6.11 d (2.1)
2.68 dd (10.3) 3.13 dd (4.3, 13.7) 7.07 d (8.1) 6.76 d (8.5) 3.76 s 3.86 s 12.03 s
3.03 dd (4.2, 13.0) 2.62 dd (2.9, 13.1) 7.01 d (8.6) 6.71 d (8.6)
3.81 s 3.81 s
13
C (5) (CDCl3)
13
C (5a) (CDCl3)
13
C (2) (CD3OD)
69.3
69.1
68.8
47.2 198.1 102.1 155.1 125.8 157.3 92.5 147.9 32.0
46.8 198.3 102.2 160.2 92.9 161.3 129.3 153.6 31.9
48.7 192.7 104.8 162.6 92.5 166.7 93.3 165.2 32.2
129.8 130.3 115.5 154.3
129.3 130.2 115.5 154.7 61.3 56.1
129.1 129.8 115.1 155.8
56.3
55.1 55.0
C. Koorbanally et al. / Biochemical Systematics and Ecology 34 (2006) 588e592
590
NMR spectra were recorded on a Varian Unity Inova 400 MHz NMR spectrometer. The UV spectrum was recorded in methylene chloride on a Varian DMS 300 UVevisible spectrometer. Optical rotations were measured at room temperature in methanol using a Perkin Elmer Polarimeter e Model 341. The CI positive HRMS of compound 5 gave an [M þ H] molecular ion peak at m/z 317.1030766, corresponding to a molecular formula C17H16O6 for 5. The 1H NMR spectrum showed the presence of a 3-benzyl-4-chromanone-type homoisoflavanone with coupled resonances ascribed to 2H-2 (4.32 dd, 4.21 dd), H-3 (2.80 m) and 2H-9 (2.72 dd, 3.12 dd). The two proton doublets at dH 6.77 and dH 7.09 (J ¼ 8.42 Hz) were assigned as H-30 /50 and H-20 /60 , respectively, of ring B and these assignments were confirmed using the HMBC spectrum. The base peak at m/z 107 resulting from hydroxybenzyl fragmentation in the mass spectrum indicated a hydroxybenzyl/hydroxytropylium ion indicating the presence of a hydroxyl group at C-40 (Wall et al., 1989). The proton NMR spectrum showed a singlet resonance at dH 6.12 which was indicative of only one proton on the Aring, which would have to be placed at either C-6 or C-8 on biosynthetic grounds, and one three proton methoxy group proton resonance at dH 3.90. The presence of a hydroxy group at C-5 was indicated by the appearance of the C-4 carbonyl resonance at dC 198.1, the downfield shift being a consequence of the chelating effects between the hydrogen of the hydroxy group at C-5 and the oxygen of the carbonyl group (Adinolfi et al., 1986). This was confirmed by the bathochromic shift that was seen when the UV spectrum was rerun on addition of AlCl3 (þ25 nm). No shift occurred upon the addition of NaOAc, indicating no hydroxy group was present at C-7, hence the methoxy group was placed at this position (Adinolfi et al., 1984, 1985). The 1H NMR spectrum acquired in CDCl3 showed the 5-OH proton resonance at dH 11.70. The NOESY spectrum indicated a correlation between the single proton resonance dH 6.12 and the methoxy group proton resonance but not the 5-hydroxy group proton resonance. This suggested that the single proton should be placed at C-8, but HMBC supporting evidence was inconclusive. The 40 -methoxy compound (5a) has been isolated from the related Ledebouria revoluta Lf. Jessop (Moodley et al., in press) with the proton at the C-6 position, confirmed by a correlation in the NOESY spectrum between H-6 and both the 5-OH and C-7 methoxy group proton resonances as well as an HMBC correlation from C-5 to H-6. The H-6 proton resonance of 5a occurred at dH 6.08, whereas the A-ring proton of compound 5 occurred at dH 6.12. NMR data for 5 and 5a are given in Table 1 for comparison purposes. The 13 C NMR data are significantly different for ring-A of the two compounds. A literature search indicated that compound 5 was a novel homoisoflavonoid and was named 5,6-dihydroxy-7-methoxy-3-(4-hydroxybenzyl)-4-chromanone.
3.1. Compound 1 ()-nyasol, ()-(Z)-1,3-bis(4-hydroxyphenyl)-1,4-pentadiene
[a]25 D ¼ 135.7 (c ¼ 0.14 g/100 mL).
3.2. Compound 2 5,7-dimethoxy-3-(4-hydroxybenzyl)-4-chromanone (10.3 mg), yellow vitreous solid, UVevisible lmax nm (log 3) in MeOH: 282 (3.67). HRMS: [M þ H] 315.1236874 (C18H18O5 þ H requires 315.123249), GCeMS: [Mþ] 314, 207, 181, 152, 137, 107, 77. IR nmax (NaCl) cm1: 3348, 2913, 2848, 1607, 1455, 1215, 1155. [a]25 D ¼ 25.00 (c ¼ 0.03 g/100 mL).
3.3. Compound 5 5,6-dihydroxy-7-methoxy-3-(4-hydroxybenzyl)-4-chromanone (10.1 mg), bright yellow, fine, needle-like crystals, m.p. 174e176 C. UVevisible lmax nm (log 3) in MeOH: 290 (4.04). HRMS: [M þ H] 317.1030766 (C17H16O6 þ H requires 317.102513), GCeMS: [Mþ] 316, 209, 107, 77. IR nmax (NaCl) cm1: 3387, 1640, 1511, 1376, 1221, 1099. [a]25 D ¼ 40.91 (c ¼ 0.044 g/100 mL).
3.4. Compound 6 ()-(E)-1,3-bis(4-hydroxyphenyl)-1,4-pentadiene
[a]25 D ¼ 4.76 (c ¼ 0.042 g/100 mL).
C. Koorbanally et al. / Biochemical Systematics and Ecology 34 (2006) 588e592
HO
R1 R2
O
591
5" 4"
6"
H
OH 3" 2"
1"
2 1
3 1'
R3
4 6'
H R1
R2
2
H
OMe H
3
OMe OMe H
OH
4
H
OH
5
H
OMe OH
OH
5a
OH
OMe H
OMe
OH
R3
H
2'
5
O
R4
3'
5'
R4
4'
OMe
OH
1 HO
5'
5" 4"
H 3"
4'
6'
6" 2
1'
3'
1" 3
2"
1
OH
2'
4 5
6
4. Chemotaxonomic significance The isolation of homoisoflavanones (compounds 2e5) was anticipated as they are common constituents of the Hyacinthoideae, and generally useful as subfamily chemotaxonomic markers (Pohl et al., 2000). However, this is the first reported isolation of norlignans (compounds 1 and 6) from the Hyacinthaceae. We have also subsequently isolated a further norlignan from Ledebouria ovatifolia (Bak.) Jessop (Langlois, 2003). Significantly, the three norlignans isolated to date are from taxa in the tribe Massonieae (subfamily Hyacinthoideae), from representatives of a clade circumscribed generically by Manning et al. (2004) as Ledebouria Roth. Their decision was based on a phylogenetic analysis employing trnLeF and rbcL molecular data. Although now known from Ledebouria (Langlois, 2003) and Drimiopsis, norlignans have not yet been isolated from Resnova Van der Merwe (Koorbanally et al., 2006), a genus also synonymised under Ledebouria by Manning et al. (2004). Were this so, the close relationship between the three genera would be confirmed chemotaxonomically. However, on the basis of definitive morphological characters (Muller-Doblies and Muller-Doblies, 1997; Stedje, 1998), generic distinctions would still be justified. Norlignans currently provide the only intra-tribal chemotaxonomic markers in the Hyacinthoideae; notably, no such markers are known at the tribal level. Compound 1 is the enantiomer of hinokiresinol, which has previously been isolated from Asparagus cochinchinensis Merrill (Asparagaceae) (Brown and Tsui, 1996) and prepared by enzymic hydrolysis of the diglucoside nyasoside isolated from Hypoxis nyasica Baker (Hypoxidaceae) (Marini-Bettolo et al., 1985). Accordingly, all isolations of 1 have been from the Liliaceae sensu lato, of which the Hypoxidaceae, Asparagaceae and the Hyacinthaceae were all formerly a part. Notably, the Z- or cis-isomer, 1, was isolated from D. burkei, and the E- or trans-isomer, 6, from its sister taxon D. maculata. This is the first report of a naturally occurring E-norlignan, although this particular compound has earlier been synthesised (Ameer et al., 1988). The E-isomer was found to be less stable than the Z-isomer, and started decomposing during acquisition of NMR spectra. Compound 2 has been reported previously from Schizocarphus nervosus (Burch.) Van der Merwe (syn. Scilla nervosa (Burch.) Jessop) (Hyacinthoideae) but was isolated as the acetate derivative resulting from acetylation of a non-separable mixture (Bangani et al., 1999). Compounds 3 and 4 have been isolated previously from D. maculata, Eucomis montana Compton, Ledebouria cooperi (Hook.f.) Jessop, Muscari comosum Mill., and D. maculata, L. ovatifolia, Resnova humifusa (Bak.) U. & D.M.-D. and L. revoluta, respectively.
592
C. Koorbanally et al. / Biochemical Systematics and Ecology 34 (2006) 588e592
Acknowledgements We are grateful to Mr. D. Jagjivan for running NMR spectra and Mr. John Hill for mass spectrometric analysis. CK gratefully acknowledges the National Research Foundation (NRF) postgraduate scholarship. The staff of the Mary Gunn Library (SANBI) kindly facilitated access to literature. This research was funded by the NRF and the University of Natal Research Fund. References Adinolfi, M., Barone, G., Belardini, M., Lanzetta, R., Laonigro, G., Parrilli, M., 1984. Phytochemistry 23, 2091. Adinolfi, M., Barone, G., Belardini, M., Lanzetta, R., Laonigro, G., Parrilli, M., 1985. Phytochemistry 24, 2423. Adinolfi, M., Lanzetta, R., Laonigro, G., Parrilli, M., 1986. Magn. Reson. Chem. 24, 663. Ameer, F., Drewes, S.E., Drewes, M.W., Roos, G.H.P., Watson, M.C., 1988. J. Chem. Soc. Perkin Trans. 1, 1425. Bangani, V., Crouch, N.R., Mulholland, D.A., 1999. Phytochemistry 51, 947. Brown, G.D., Tsui, W., 1996. Phytochemistry 43, 1413. Hulme, M.M., 1954. Wild Flowers of Natal. Shuter and Shooter, Pietermaritzburg. t. 13. Hutchings, A., 1989. Bothalia 19, 225. Jessop, J.P., 1972. J. S. Afr. Bot. 38, 151. Kativu, S., 2000. Kirkia 17, 150. Koorbanally, C., Crouch, N.R., Mulholland, D.A., 2001. Biochem. Syst. Ecol. 29, 539. Koorbanally, N.A., Crouch, N.R., Harilal, A., Pillay, B., Mulholland, D.A., 2006. Biochem. Syst. Ecol. 34, 114. Langlois, A., 2003. Unpublished Ph.D. dissertation, University of Natal, Durban, South Africa. Manning, J.C., Goldblatt, P., Fay, M.F., 2004. Edinb. J. Bot. 60, 533. Marini-Bettolo, G.B., Nicoletti, M., Messana, I., 1985. Tetrahedron 41, 665. Moodley, N., Crouch, N.R., Mulholland, D.A., Slade, D., Ferreira, D. South African Journal of Botany, in press. Mulholland, D.A., Koorbanally, C., Crouch, N.R., Sandor, P., 2004. J. Nat. Prod. 67, 1726. Muller-Doblies, U., Muller-Doblies, D., 1997. Feddes Repert. 108, 1. Pohl, T., 1999. M.Sc. dissertation, University of Natal, Durban, South Africa. Pohl, T., Crouch, N.R., Mulholland, D.A., 2000. Curr. Org. Chem. 4, 1287. Stedje, B., 1994. Nord. J. Bot. 14, 45. Stedje, B., 1998. Plant Syst. Evol. 211, 1. Tyiso, S., Bhat, R.B., 1998. Ver. Angew. Bot. 72, 92. Van der Walt, S.J., 1944. Onderstepoort J. Vet. Sci. Anim. Ind. 20, 75. Wall, M.E., Wani, M.C., Manikumar, G., Taylor, H., McGivney, R., 1989. J. Nat. Prod. 52, 774.