Cuticular Wax Profiles of Leaves of Some Traditionally Used African

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Cuticular Wax Profiles of Leaves of Some Traditionally Used African Bignoniaceae Rainer Gormanna, Lukas Schreiberb, and Herbert Kolodzieja,* a Institut für Pharmazie, Pharmazeutische Biologie, Freie Universität Berlin, Königin-Luise-Str.2+4,D-14195 Berlin, Germany. Fax: +30-838-53729. E-mail: [email protected] b Institut für Botanik, Abteilung Ökophysiologie, Rheinische-Friedrich-Wilhelms- Universität Bonn, Kirschallee 1, D-53115 Bonn, Germany * Author for correspondence and reprint requests Z. Naturforsch. 59c, 631Ð635 (2004); received May 24/June 15, 2004 The cuticular waxes, obtained by chloroform extraction from the leaves of four African Bignoniaceae, Newbouldia laevis, Markhamia acuminata, Spathodea campanulata and Kigelia africana were analysed by GC-MS. The principal constituents were represented by a homologous series of n-alkanes (C23ÐC33), n-alcohols (C18ÐC30) and related carboxylic acids (C16Ð C36). For N. laevis and M. acuminata, ursolic and oleanolic acid were the most abundant wax components (52 and 60%, respectively), followed by the C29, the C31 and the C33 n-alkanes. The predominant components of S. campanulata were n-alcohols (35%), with octacosanol and triacontanol as the most abundant ones, while K. africana is distinguished from these three members by the conspicuous absence of triterpenoic acids and the predominance of n-alkanes (70%) with hentriacontane and tritriacontane as the main representatives. Other notable constituents were sterols, albeit present in trace amounts. The wax profiles are dis- cussed in terms of taxonomic characters. Key words: Bignoniaceae, Cuticular Wax, Chemotaxonomic Characters

Introduction (Schoonhoven et al., 1998), with long chain hydrocarbons and derivatives thereof as principle Members of the Bignoniaceae are common in constituents (Baker, 1982). Terpenoids and flavo- the tropical areas of the New World, while only a noids also occur, the latter being reported for cu- few occur in the rain forests and savannah zones ticular waxes of some species and used as taxo- of Africa. Although many species are cropped nomic features (Stermitz et al., 1992; Wollenweber (Janzen, 1975; Shepherd et al., 2000; Nyberg et al., et al., 1996; Blatt et al., 1998; Alcerito et al., 2002). 2002) and employed in traditional medical systems This paper deals with compositional studies of for the treatment of various diseases (Burkill, the cuticular waxes of N. laevis., M. acuminata., 1985), little attention has been paid to the chemi- S. campanulata and K. africana and their possible cal constituents of plants of the Bignoniaceae. The taxonomic significance. systematic application of morphological characters of, e.g., fruits and seeds to the establishment of subfamilial boundaries is limited and has produced Materials and Methods seven tribes, with at least for the Tecomeae tribe Plant material considerable heterogeneity (Gentry, 1980; Arm- strong, 1985). The present study was therefore ini- Adult leaves of N. laevis were provided by the tiated to re-examine the taxonomic grouping of Royal Botanical Garden of Belgium, adult leaves the medicinally used plants Newbouldia laevis of M. acuminata, S. campanulata and K. africana Seem., Markhamia acuminata (Klotzsch) K. were obtained from the Botanical Garden of Ber- Schum., and Spathodea campanulata Beauv. (tribe lin (Germany). All species were grown in a tropi- Tecomeae) and Kigelia africana (Lam.) Benth. cal greenhouse under similar conditions. Voucher (tribe Coleeae) on the basis of their leaf wax com- specimens are deposited at the Institut für Phar- positions as chemical characters. The role of sur- mazie, Pharmazeutische Biologie, Freie Universi- face waxes in plant defence is well established tät Berlin, Berlin, Germany.

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Extraction S. campanulata, M. acuminata and N. laevis are Four to six leaves of each plant were extracted morphologically well characterized and can be dis- by immersion in 50 ml chloroform for 30 s. The tinguished by their spathaceous calyx splitted on wax extracts were filtered over defatted cotton adaxial (S. campanulata, M. acuminata) or abaxial and the solvent subsequently evaporated under re- side (N. laevis), respectively. Furthermore, the lat- duced pressure. After gravimetric determination, ter two members can be differentiated by their the extracts were dissolved in chloroform (2 mg/ ovules and fruits (Hutchinson and Dalziel, 1963; ml) with tetracosane as internal standard (100 µg/ Steentoft, 1988). These aspects have obviously ml) and analysed by GC-MS and GC-FID. Meas- been ignored in the past. urements of the surface area were performed by The average total wax load for the leaves of N. tracing the photocopied image of each leaf using laevis, M. acuminata, S. campanulata and K. afri- µ 2 standard calibration paper. cana were 18.5, 14.7, 1.6 and 0.6 g/cm , respectively. From the chloroform extracts about 62% (N. laevis), 67% (M. acuminata), 52% (S. campa- Qualitative and quantitative analysis of the nulata) and 79% (K. africana) of their constituents wax fractions were unambiguously identified by GC-MS analy- Aliquots (100 µl) of the wax fractions were dried ses and the results are presented in Table I. Unfor- and then silylated with 20 µl BSTFA (N,N-bistri- tunately, separate GC-MS analyses of the wax ex- methyl-silyltrifluoroacetamide; Macherey-Nagel) tracts on a Restek Rtx“-5Sil column using a in the presence of 10 µl pyridine at 70 ∞C for Finnigan MD 800 instrument and comparison of 30 min. Qualitative analyses were carried out by the mass spectra with those of Wiley and NIST GC-MS using a Hewlett-Packard HP 5890 II in- Library by means of AMDIS failed to identify fur- strument, coupled with a quadrupole mass selec- ther constituents. In each case, the lipophilic frac- tive detector (HP MSD 5971). A DB-1 fused-silica tions consisted of a complex mixture with hy- capillary column (30 m x 0.32 mm i.d., film thick- drocarbons and oxygenated triterpenoids as the µ ness 0.1 m; Fisons) was used with a temperature dominating constituents. Several homologous programme of 50Ð200 ∞C (40 ∞C minÐ1) and 200Ð series were identified, including n-alkanes (C23Ð 300 ∞C(3∞C minÐ1), 300 ∞C (20 min); pressure pro- C33), n-alcohols (C18ÐC30) and related carboxylic gramme: 10 kPa (40 min), 10Ð100 kPa (10 kPa acids (C ÐC ), which are typical constituents of Ð1 16 36 min ), 100 kPa (30 min); helium was the carrier cuticular waxes (Baker, 1982). Conspicuously, gas at a flow rate of 2 ml/min. For MS analysis, the large gaps were observed in these series (Table I), ionisation energy was 70 eV. The compounds were a finding that can not be satisfactorily explained. identified on the basis of their retention times and A range of sterols was also detected, although in mass-spectral fragmentation patterns compared trace amounts (< 0.01%). To our knowledge, the with those of reference compounds stored on our occurrence of stigmasterol (M. acuminata, S. cam- spectrometer database. panulata) and campesterol (S. campanulata) was Quantification of identified constituents was not previously described for leaf extracts of the µ performed by injecting 1 l of the samples (on-col- indicated members. umn injector; FID; hydrogen as carrier gas). The Compositional studies of the wax extracts of following temperature and pressure programmes N. laevis and M. acuminata revealed the presence were used: 50 ∞C (2 min), 50Ð200 ∞C (40 ∞C minÐ1), Ð1), of considerable amounts of oxygenated triterpe- 200Ð320 ∞C(3∞C min 320 ∞C (20 min); 40 kPa noids, accounting for 52% and 60% of the iden- Ð Ð1 (40 min), 40 150 kPa (10 kPa min ), 150 kPa tified constituents, respectively, followed by long- (30 min). chain n-alkanes (C26ÐC33) with ca. 9% and 7%, respectively (Table I). The triterpenoid compo- Results and Discussion nents in the waxes of N. laevis and M. acuminata The selection of plants was prompted by the were oleanolic acid (14.5% and 23%, respectively) synonym citation of some of the specimens, e.g. and ursolic acid (around 37%), with the latter as Markhamia acuminata syn. Spathodea acuminata the major component in each case. It should also and Newbouldia laevis syn. Spathodea laevis (Mis- be noted that hederagenin occurred in M. acumi- souri Botanical Garden), giving rise to some con- nata as a trace component, but was apparently fusion in their botanical classification. However, absent or only present below detection limits R. Gormann et al. · Cuticular Wax Profiles of Leaves of Some African Bignoniaceae 633

Table I. Cuticular wax profiles and distribution of hydrocarbons, alcohols, fatty acids, triterpenoids and sterols for N. laevis, M. acuminata, S. campanulata and K. africana.

Species/ N. laevis M. acuminata S. campanulata K. africana Substance class abababab n-Alkanes Tricosane C23 Ð ÐÐÐÐÐ* 0.5 Hexacosane C26 0.1 ð 0.006 0.4 ÐÐÐÐÐÐ ͷ Heptacosane C27 0.1 ð 0.006 0.2 0.1 ð 0.002 0.1 0.7 * 1.7 Octacosane C28 0.1 ð 0.004 0.4 ÐÐÐÐÐÐ ͷ Nonacosane C29 0.5 ð 0.11 2.9 0.1 ð 0.021 0.6 1.0 0.1 ð 0.012 7.0 Triacontane C30 0.1 ð 0.013 0.6 ÐÐÐÐÐÐ ͷ Hentriacontane C31 0.5 ð 0.07 3.0 0.4 ð 0.037 2.9 1.7 0.3 ð 0.04 42.5 Dotriacontane C32 0.1 ð 0.017 0.4 ÐÐÐÐÐÐ ͷ Tritriacontane C33 0.2 ð 0.026 1.4 0.5 ð 0.015 3.2 2.3 0.1 ð 0.02 18.2 Σ 1.7 9.3 1.1 6.8 0.1 5.7 0.5 69.9 n-Alcohols Octadecanol C18 ÐÐÐÐ* 0.6 Eicosanol C20 ÐÐ* 0.6 * 1.2 Hexacosaol C26 ÐÐ0.1 ð 0.006 2.0 ÐÐ Heptacosanol C27 ÐÐ* 0.5 ÐÐ Octacosanol C28 * 0.1 0.2 ð 0.064 14.8 ÐÐ Triacontanol C30 ÐÐ0.3 ð 0.129 17.1 ÐÐ Σ ÐÐ* 0.1 0.6 35.0 * 1.8 Fatty acids ͷ ͷ ͷ Hexadecanoic C16 ÐÐ 0.2 0.9 1.2 ͷ ͷ ͷ Octadecanoic C18 ÐÐ 0.1 0.5 1.5 ͷ ͷ Hexacosanoic C26 0.2 0.1 ÐÐÐÐ ͷ Dotriacontanoic C32 0.3 ÐÐÐÐÐÐ ͷ Hexatriacontanoic C36 ÐÐÐÐ 0.9 ÐÐ Σ ͷ 0.5 ͷ 0.4 ͷ 2.3 ͷ 2.7 Esters ͷ C44 ÐÐÐÐÐÐ 4.9 Triterpenoids Ursolic acid 7.0 ð 1.02 37.9 5.2 ð 0.320 36.6 ͷ 1.8 ÐÐ Oleanolic acid 2.7 ð 0.50 14.5 3.3 ð 0.165 23.1 0.1 ð 0.043 6.7 ÐÐ Hederagenin ÐÐ** Lupeol * * α-Amyrin * * Σ 9.7 52.4 8.5 59.7 0.1 8.5 ÐÐ Sterols -Sitosterol * * * * * * Stigmasterol * * * * Campesterol * * * * Cholesterol ** a Abundance on leaf surface [µg/cm2]. b Percent of total wax. * Trace amounts < 0.01%. ͷ Trace amounts < 0.1 µg/cm2. Each species was tested with n =4Ð6 replicates and results are expressed as mean values ð SD.

(< 1 ng/µl) in the remaining three species investi- The total amount of chloroform soluble surface gated. With regard to the identified n-alkanes, the wax constituents of the leaves of S. campanulata C29, the C31 and the C33 were the most abundant was markedly lower than those of N. laevis and compounds, consistent with the composition of M. acuminata (vide supra), placed within the same most waxes (Gülz, 1994). tribe Tecomeae. Also worthy of mention was the 634 R. Gormann et al. · Cuticular Wax Profiles of Leaves of Some African Bignoniaceae finding that the cuticular wax chemical profile of several individuals of each species at different de- S. campanulata differed significantly from those of velopmental stages and from plants of natural the latter tribal-associated members by the con- population will be needed to ascertain the stability siderably reduced levels of triterpenoic acids and the individual variability of the chemical char- (8.5%) and the increased level of primary alcohols acters. Since only single specimens maintained in (35%), with octacosanol and triacontanol as the greenhouse conditions were available, appropriate predominant ones. Other striking features ap- studies were excluded accordingly. In an alterna- peared to be the significantly higher percentage of tive approach, gene analysis is currently in pro- fatty acids and the diversity of sterols in S. cam- gress for extracting further taxonomic information panulata, suggesting less taxonomic affinity. from the sequence variation of ndhF and rbcL The picture that emerged from examining the genes. wax profile of K. africana disclosed differences, as Finally, with regard to their traditional uses, the compared to overall amounts and distribution presence of triterpenoic acids in considerable within the terpenoids, the hydrocarbons and their amounts is significant in that these compounds are oxygenated analogues. Conspicuously, triterpenoic well known to possess antimicrobial (Collins and acids were absent, while the diversity of sterols Charles, 1987) and potent antitumoural activity was somewhat reminiscent of that of S. campanu- (Liu, 1995). Accordingly, the use of leaves of lata. Here, the n-alkanes constituted the most N. laevis, M. acuminata and S. campanulata in abundant group (70%), with the C31 (42.5%) and traditional medicine for the treatment of skin can- C33 (18%) as the main components. cer may, at least in part, be rationalised in terms With regard to the identified wax profile, shared of the presence of these triterpenoic acids with key characters support the location of N. laevis, established antitumoural activities, while the utili- M. acuminata, S. campanulata in a common tribe, zation of K. africana should be based on the pres- Tecomeae, as originally delineated by morphologi- ence of naphthoquinones. However, the complex- cal features (Gentry, 1980). Although S. campanu- ity of extracts necessitates that traditional uses lata appears chemically distinct, it would be impru- have to be evaluated on a rational basis in each dent to exclude this member from this tribe on case. limited chemical grounds without various addi- tional non-chemical and chemical criteria. The re- markable absence of any triterpenoids and the Acknowledgements conspicuous abundance of n-alkanes in the cuticular wax of K. africana support its treatment as a Leaves of N. laevis were kindly provided by the separate member as reflected by the placement in Royal Botanical Garden of Belgium, and samples the Coleeae (Gentry, 1980). Although the results of M. acuminata, S. campanulata and K. africana are consistent with the present taxonomic group- were generously supplied by the Botanical Garden ing, analyses of fresh plant material collected from of Berlin (Germany). R. Gormann et al. · Cuticular Wax Profiles of Leaves of Some African Bignoniaceae 635

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