Quinolizidine alkaloid status of Styphnolobium and Cladrastis (Leguminosae)

Biochemical Systematics and Ecology 31 (2003) 1409–1416 www.elsevier.com/locate/biochemsyseco

Quinolizidine alkaloid status of Styphnolobium and Cladrastis (Leguminosae) Geoffrey C. Kite a,∗, R. Toby Pennington b b

a Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK Royal Botanic Garden Edinburgh, 20a Inverleith Row, Edinburgh EH3 5LR, UK

Received 20 October 2002; accepted 26 February 2003

Abstract Reports of quinolizidine alkaloids in Styphnolobium Schott and Cladrastis Raf. (Leguminosae) conflict with their position in recent molecular phylogenies because they are not members of a major clade of quinolizidine alkaloid-accumulating taxa. The alkaloid status of these two genera was therefore re-investigated using gas chromatography–mass spectrometry. Quinolizidine alkaloids could not be detected in extracts of leaves, flowers or seeds of S. japonicum (L.) Schott, nor in leaves of S. affine (Torrey & A. Gray) Walp., C. delavayi (Franch.) Prain, C. kentukea (Dum.-Cours.) Rudd or C. platycarpa Mak. In contrast, Calia secundiflora (Ortega) Yakovlev, also currently placed outside the major clade of quinolizidine alkaloid-producing genera in molecular phylogenies, was confirmed to accumulate a range of quinolizidine alkaloids.  2003 Elsevier Ltd. All rights reserved. Keywords: Styphnolobium; Cladrastis; Calia; Sophora; Leguminosae; Quinolizidine alkaloids; Chemosystematics; Gas chromatography–mass spectrometry

1. Introduction Quinolizidine alkaloids have been reported to occur in about 65 genera of Leguminosae (Mears and Mabry, 1971; Wink, 1993; Ohmiya et al., 1995), all in subfamily Papilionoideae. Recent phylogenetic analyses of DNA sequences of legumes, when compared with data on other character types, have indicated that the presence

∗

Corresponding author. Tel.: +44-20-8332-5368; Fax +44-20-8332-5310. E-mail address: [email protected] (G.C. Kite).

0305-1978/$ - see front matter  2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0305-1978(03)00118-2

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of quinolizidine alkaloids may be of systematic significance at a relatively high taxonomic level in the family (Crisp et al., 2000; Pennington et al., 2001). All taxa with quinolizidine alkaloids except Styphnolobium, Cladrastis and Calia are indicated to be members of a single monophyletic group, the “genistoids”, by chloroplast trnL intron sequences (Pennington et al., 2001), a result confirmed by chloroplast matK sequences (Wojciechowski et al., unpublished data). Analyses of nuclear ribosomal internal transcribed spacer (ITS) sequences (Crisp et al., 2000) and chloroplast rbcL sequences (Kajita et al., 2001) differ slightly in that they place some quinolizidine containing taxa (Brongniarteae plus Poecilanthe and Cyclolobium of Millettieae) outside the genistoid clade, although as close relatives. Reports of quinolizidine alkaloids in Styphnolobium and Cladrastis conflict strongly with their position in all current molecular phylogenies (Crisp et al., 2000; Pennington et al., 2001; Kajita et al., 2001), since these genera are not placed close to other quinolizidine alkaloidproducing taxa. Calia is generally placed closer to quinolizidine containing taxa, but in no case is it a member of the same monophyletic group. The listing of Cladrastis as a quinolizidine alkaloid-producing genus in review papers (e.g. Mears and Mabry, 1971; Kinghorn and Balandrin, 1984; Wink, 1993) appears to originate from the abstract (Chemical Abstracts 51, 5369) of a paper by Nikonov (1956) reporting cytisine and other alkaloids in C. amurensis (no authority given). The original paper, however, quotes the taxon studied as Maackia amurensis Rupr. & Maxim., which is the name currently accepted for this species; Cladrastis amurensis (Rupr. & Maxim.) Benth. is a synonym. Several quinolizidine alkaloids have since been reported from M. amurensis (e.g., Kinghorn et al., 1982; Wang et al., 2000) as well as other species of Maackia Rupr. (e.g. Wang et al., 1999), but no reports exist for currently recognised species of Cladrastis. Data on the quinolizidine alkaloid status of the genus Styphnolobium, as circumscribed by Sousa and Rudd (1993), come from analyses of S. japonicum and S. affine under their former classification as species of Sophora L. Several quinolizidine alkaloids were identified from seeds of S. japonicum (as Sophora japonica L.) by Abdusalamov et al. (1972) but only low levels were found in the leaves. The species was also scored positive for quinolizidine alkaloids in a phylogenetic analysis of legumes using molecular and chemical characters (Ka¨ ss and Wink, 1995). However and Izaddost (1975) could not detect any alkaloids in seeds of either S. japonicum (as Sophora japonica) or S. affine (as Sophora affinis Torrey & A. Gray). Thus, given the potential importance of this chemical character in legume systematics, further study of Cladrastis and Styphnolobium was merited to clarify their quinolizidine alkaloid status.

2. Materials and methods 2.1. Plant material Most leaf material for analysis was obtained from plants growing at the Royal Botanic Gardens, Kew. Specimen and voucher details are given in Table 1. For

Leaf Leaf Leaf Leaf Leaf Leaf, Flower, Pod, Seed Seed

1969-16092 (F) RMP

1982-5415 (F) LR 1983-3116 (F) LR

1931-18102 1908-35901 1973-11942 1877-18312 1897-62202 1972-10834

–

S. microphylla (9425)

S. tomentosa (9417) S. velutina var. zimbabweensis (9418) Styphnolobium affine (9424) S. japonicum (9450) S. japonicum (10661) S. japonicum (9415) S. japonicum (9414) S. japonicum (10527)

S. japonicum (10536)

Leaf

31/5/01 5/6/01 13/8/02 31/5/01 31/5/01 10/8/01 10/8/01 24/10/01 16/11/01 24/9/69

31/5/01 31/5/01

31/5/01

6/6/01 2/2/92 6/8/02 11/6/01 11/6/01 11/6/01 6/8/02 31/5/01

Date collected

S.L.Kelsey 382. Mt Cuba Botanical Park, USA

R.T. Pennington s.n. (E) R.T. Pennington s.n. (E) 1973-11942: ref. G.C. Kite, BI 10661 (K) – 1897-62202: ref. G.C. Kite, BI 9414 (K) 1972-10834: ref.G.C. Kite, BI 10527 (K)

– C.E. Hughes 1579 1920-10301: ref: G.C. Kite, BI 10652 (K) – 1973-15671: ref. G.C. Kite, BI 9464 (K) 1910-65043: ref. G.C. Kite, BI 9463 (K) 1969-16287: ref. G.C. Kite, BI 10651 (K). R.T. Pennington s.n. (E); 1988-8714: ref. G.C. Kite, BI 9416 (K) Sophora tetraptera var. microphylla (Aiton) Hook.f.: ref. wall of Melon Yard, det. R. Polhill 1971 (K) – –

Herbarium voucher (flowering)

a F = Fully verified; P = Partially verified; LR = L. Rico; GPL = G.P. Lewis; SA = S. Andrews; RMP = R.M. Polhill; RTP = R.T. Pennington; JB = J. Barham.

(F) RMP (F) RMP

(P) RTP (F) SA

Leaf Leaf

1990-899 (F) LR – 1920-10301 (F) GPL 1973-18516 (F) SA 1973-15671 (P) SA 1910-65043 (F) SA 1969-16287. (F) RMP 1988-8714 (F) JB

Calia secundiflora (9451) C. secundiflora (10537) Cladrastis kentukea (10652) C. kentukea (9462) C. platycarpa (9464) C. delavayi (9463) C. delavayi (10651) Sophora davidii (9416)

Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf

Kew living collections Organ accession no. (verification) identifiera

Species (Sample No.)

Table 1 Plant specimens analysed for quinolizidine alkaloids. G.C. Kite, R.T. Pennington / Biochemical Systematics and Ecology 31 (2003) 1409–1416 1411

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S. japonicum, collections were also made of flowers, fruits and seeds, as well as leaf collections at different times during the year. Additionally, a mature seed of S. japonicum from a herbarium specimen was also analysed. Some species of Sophora s.l. that were predicted to contain quinolizidine alkaloids from data in the literature were also included in the study. These were S. davidii (Franch.) Pavol. cv. ‘Hans Fliegner’, S. tomentosa L., S. velutina var. zimbabweensis J.B. Gillett & Brummitt, S. microphylla Sol. ex Aiton and S. secundiflora (Ortega) Lag. ex DC. [now classified as Calia secundiflora (Ortega) Yakovlev]. 2.2. Sample preparation Extracts were prepared from fresh material immediately after collection. Between 0.5–2 g of material was macerated in 0.5 M aqueous HCl. After 2 h at room temperature the residue was removed by centrifugation and the supernatant was shaken with dichloromethane. The phases were separated by centrifugation and the aqueous phase was removed and made alkaline (to pH 10) by addition of ammonium hydroxide then shaken again with dichloromethane. Following centrifugation, the organic phase was removed and evaporated to dryness. The residue was dissolved in 100 µl dichloromethane for every 100 mg (fresh weight) of material originally extracted; some residues of S. japonicum were dissolved at ten times this concentration. Dry herbarium material was processed in the same manner, except the compounds extracted from 100 mg were finally dissolved in 1 ml for analysis. 2.3. GC–MS analysis Samples were analysed using a quadrupole GC–MS system (Perkin-Elmer TurboMass). 1 µl injections (split 1:10) were vaporised at 250 °C onto a 30 m x 0.25 mm (i.d.) x 0.25 µm DB1-MS (J & W Scientific) capillary column and chromatography was undertaken using a temperature gradient of 120–320 °C at 6 °C/min with 1 ml/min helium carrier gas. Mass spectra following electron ionisation (70 eV; source temperature 180 °C) were recorded at 0.75 s/scan in the range m/z 38–600. To effect identification of quinolizidine alkaloids, the mass spectra and retention indices were compared with published data (Wink, 1993; Wink et al., 1995).

3. Results and discussion GC–MS analysis revealed a range of quinolizidine alkaloids in the leaf extracts of Sophora davidii, S. tomentosa, S. velutina var. zimbabweensis, S. microphylla and Calia secundiflora (Table 2). However, quinolizidine alkaloids could not be detected in leaf material of the species of Styphnolobium and Cladrastis studied, even in the more concentrated extracts of S. japonicum. Neither could quinolizidine alkaloids be detected in the samples of flowers, pods and seeds of S. japonicum. In contrast, even minor alkaloid components in C. secundiflora and the Sophora species studied produced easily observed peaks in the total ion chromatograms; for example,

1424 1701 1773 1801 1833 1856 1954 1992 2144 2169 2177 2178 2196 2249 2262 2274 2281 2286 2406 2749

169 234 234 232 232 208 204 190 246 244 264 248 248 230 230 244 246 248 244 302

Lupinine α-Isosparteine Sparteine Unknown A 11,12-Dehydrosparteine Ammodendrine N-Methylcytisine Cytisine 5,6-Dehydrolupanine Rhombifoline Lamprolobine Lupanine Aphylline Unknown B 11-Allylcytisine Sophoramine Sophocarpine Matrine Anagyrine 13β-Acetoxyanagyrine

Assignment

2.3 0.8

2.4 1.6

0.6 0.5 48.5 23.6 0.4 6.2

21.7 7.9

5.6

9.5 31.7 8.1

6.5

10537

9451 0.1 0.4 12.6

C. sec

C. sec.

9.2 2.5

1.0

16.5 1.5

4.3 0.5 5.2 10.9 4.0 4.6 24.0 8.7

9425

S. mic.

Relative amount (% total alkaloids)

2.1 52.0 13.6

19.7 0.9

9416

S. dav.

86.5

2.0

8.9

9417

S. tom.

0.9

96.6

0.6

0.7

0.5

9418

S. vel.

EIMS 70 eV; m/z (rel. int.): Unknown A: 232 (M+, 47), 175 (22), 148 (29), 134 (95), 98 (100), Unknown B: 230 (M+, 27), 189 (21), 160 (24), 147 (100), 134 (33), 68 (43)

(2240) (–) (–) (2265) (2390) (2700)

(1840) (1865) (1955) (1990) (2133) (2155) (2165) (2165) (2180)

(1422) (1710) (1785)

(Publ. RI)

m/z (%)

(36) (25) (18) (47) (32) (42) (18) (47) (22) (2) (15) (32) (20) (27) (8) (51) (68) (100) (23) (14)

Expt. RI

M+ (rel. int.)

Table 2. Quinolizidine alkaloids detected in Calia secundiflora and species of Sophora (percent total alkaloids by peak area in total ion chromatograms). Unknowns constituting less that 1% of quinolizidine alkaloids not listed. Experimental retention indices (RIs) are for DB1 phase. Published RIs are from Wink (1993) and Wink et al. (1995).

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α-isosparteine constituting 0.4% of the volatile quinolizidine alkaloids in C. secundiflora gave a peak in the total ion chromatogram with a signal to noise ratio of 22:1. This indicated that the sample preparation and analysis methods were effective at detecting accumulated quinolizidine alkaloids. Wink and co-workers have also recently re-evaluated S. japonicum and they were also unable to detect quinolizidine alkaloids (Prof. M. Wink, personal communication). The failure to detect quinolizidine alkaloids in Styphnolobium and Cladrastis is in accordance with recent data on the phylogenetic position of these taxa. In a cladistic analysis of basal papilionoid legumes using sequences of the chloroplast intron trnL (Pennington et al., 2001), all the genera in a major monophyletic group, the ‘genistoids’, were ones reported to accumulate quinolizidine alkaloids (Wink, 1993; Kite, unpublished data for quinolizidine alkaloids in Cyclolobium). These included Sophora velutina and S. davidii studied here and confirmed to accumulate quinolizidine alkaloids. These species are representative of Sophora sensu stricto (as defined by Sousa and Rudd, 1993), and we anticipate that all species of this genus are likely to contain quinolizidine alkaloids, which is supported by reports from the literature (Ohmiya et al., 1995). The only genera occurring outside of the genistoid clade in the analysis of Pennington et al. (2001) for which reports of quinolizidine alkaloids exist are Styphnolobium, Cladrastis and Calia. Styphnolobium (represented by S. japonicum and S. affine) and Cladrastis (represented by C. kentukea and C. delavayi) formed a clade well removed from the genistoids. A similar position for Styphnolobium and Cladrastis outside the genistoids was also reported in analyses of rbcL and ITS sequences (Ka¨ ss and Wink, 1997; Crisp et al., 2000; Kajita et al., 2001). It should be noted that in the analysis of Pennington et al. (2001) a DNA sequence from Styphnolobium japonicum was located in the clade containing Styphnolobium and Cladrastis and was incorrectly assigned to Sophora pachycarpa C. Meyer, a species is known to produce quinolizidine alkaloids (see Sadykov and Kushmuradov, 1962, and references therein). It is difficult to prove conclusively an absence of a chemical trait, but the failure to detect quinolizidine alkaloids in Styphnolobium and Cladrastis agrees better with their phylogenetic position in the molecular analysis of Pennington et al. (2001) than past reports of the presence of alkaloids in these genera. However, this statement must be tempered by the results of other molecular analyses such as that of Kajita et al. (2001) which place some quinolizidine alkaloid-containing genera, those in Brongniartieae plus Poecilanthe (Millettieae), outside the genistoid clade, albeit with poor bootstrap support. In this context, it is notable that preliminary cladistic analyses of chloroplast matK sequences (Wojciechowski et al., unpublished data) are entirely congruent with the trnL results. The matK results also indicate Pickeringia (Thermopsideae) to be a member of the Styphnolobium–Cladrastis clade, and this monospecific genus is also reported not to accumulate quinolizidine alkaloids (Turner, 1981). The analysis of Pennington et al. (2001) placed Calia secundiflora as one of several unresolved elements which potentially might be sister to the genistoid clade. A similar position for C. secundiflora (as Sophora secundiflora) was found by Crisp et al. (2000) but in the analysis of Kajita et al., (2001) the taxon was placed further

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from the genistoids. Several previous studies report on the quinolizidine alkaloids in C. secundiflora (Chavez and Sullivan, 1984; Murakoshi et al., 1986; new data in Ohmiya et al., 1995) and the presence of alkaloids is confirmed here (Table 1), although the two samples examined showed some differences in the alkaloids they contained. Given the non-congruence of current DNA sequence analyses with respect to C. secundiflora, it will be of interest to monitor the position of this taxon in future and more robust molecular phylogenies of Leguminosae to ascertain whether the presence of quinolizidine alkaloids is a character that defines a single major clade of legumes. References Abdusalamov, B.A., Aslanov, H.A., Sadykov, A.C., Horoshkova, O.A., 1972. Investigation of the alkaloid content of Sophora japonica. Khimiia Prirodnykh Soedinenii 5, 658. Chavez, P.I., Sullivan, G., 1984. A qualitative and quantitative comparison of the quinolizidine alkaloids of the fasciated and normal stems of Sophora secundiflora. J. Nat. Prod. 47, 735–736. Crisp, M.D., Gilmore, S., Van Wyk, B.-E., 2000. Molecular phylogeny of the genistoid tribes of papilionoid legumes. In: Herendeen, P.S., Bruneau, A. (Eds.), Advances in Legume Systematics, Part 9. Royal Botanic Gardens, Kew, pp. 249–276. Izaddost, M., 1975. Alkaloid chemotaxonomy of the genus Sophora. Phytochemistry 14, 203–204. Kajita, T., Ohashi, H., Tateishi, Y., Bailey, C.D., Doyle, J.J., 2001. RbcL and legume phylogeny, with particular reference to Phaseolae, Millettieae and Allies. Syst. Bot. 26, 515–536. Ka¨ ss, E., Wink, M., 1995. Molecular phylogeny of the Papilionoideae (Family Leguminosae): rbcL gene sequences versus chemical taxonomy. Bot. Acta 108, 149–162. Ka¨ ss, E., Wink, M., 1997. Phylogenetic relationships in the Papilionoieae (family Leguminosae) based on nucleotide sequences of cpDNA (rbcL) and ncDNA (ITS1 and 2). Molecular Phylogenetics and Evolution 8; 1, 65–88. Kinghorn, A.D., Balandrin, M.F., 1984. Quinolizidine alkaloids of the Leguminosae: structural types, analysis, chemotaxonomy, and biological activities. In: Pelletier, S.W. (Ed.), Alkaloids, Vol. 2:. Chemical and Biological Perspectives, Wiley, New York, pp. 105–148. Kinghorn, A.D., Balandrin, M.F., Lin, L.-J, 1982. Alkaloid distribution in some species of the papilionaceous tribes Sophoreae, Dalbergieae, Loteae, Brongniartieae and Bossiaeeae. Phytochemistry 21, 2269–2275. Mears, J.A., Mabry, T.J., 1971. Alkaloids in the Leguminosae. In: Harborne, J.B., Boulter, D, Turner, B.L. (Eds.), Chemotaxonomy of the Leguminosae. Academic Press, London, pp. 73–178. Murakoshi, I., Kubo, H., Ikram, M., Isar, M., Shafi, N., Ohmiya, S., Otomasu, H., 1986. (+)-11-Oxocytisine, a lupine alkaloid form leaves of Sophora secundiflora. Phytochemistry 25, 2000–2002. Nikonov, G.K., 1956. Maackia amurensis—a new source of cytisine. Aptechnoe Delo 5, 30–35. Ohmiya, S., Saito, K., Murakoshi, I., 1995. Lupine alkaloids. In: Cordell, G.A. (Ed.), The Alkaloids, Vol. 47: Chemistry and Pharmacology. Academic Press, San Diego, pp. 1–114. Pennington, R., Lavin, M., Ireland, H., Klitgaard, B.B., Preston, J., Hu, J.-M, 2001. Phylogenetic relationships of basal papilionoid legumes based upon sequences of the chloroplast trnL intron. Syst. Bot. 26, 537–556. Sadykov, A.S., Kushmuradov, Y.K., 1962. Studies on alkaloids of the C15 series. VIII. A separation of the alkaloids of Sophora pachycarpa. J. Gen. Chem. USSR 32, 1322–1325. Sousa, S.M., Rudd, V.E., 1993. Revision del genero Styphnolobium (Leguminosae: Papilionoideae: Sophoreae). Ann. Missouri Bot. Gard. 80, 270–283. Turner, B.L., 1981. Thermopsideae. In: Polhill, R.M., Raven, P.H. (Eds.), Advances in Legume Systematics, Part 1. Royal Botanic Gardens, Kew, pp. 403–407. Wang, Y.H., Li, J.S., Jiang, Z.R., Kubo, H., Higashiyama, K., Ohmiya, S., 2000. Lupin alkaloids from Chinese Maackia amurensis. Chem. Pharm. Bull. 48, 641–645.

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