The composition of aristolochic acids from different populations of four Aristolochia species was analysed by HPLC; quantitative data were submitted to discriminant and statistical analyses. The interspecific variability was confirmed, and significant differences were detected by canonical discriminant functions. The aristolochic acid II and the ratio AAII/AAI are significant in the discrimination of the examined species

Cateni, Francesca

Coassini Lokar, Laura

Martini, Fabrizio

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Studia Geobotanica. Vol. 16: 45-49 (1997) CHEMOSYSTEMA TIC VALUE OF ARISTOLOCIDC ACIDS IN THE GENUS ARISTOLOCHIA L. IN FRIULI-VENEZIA GIULIA (NEITALY) Francesca CATENI 1), Laura COASSINI LOKAR2), and Fabrizio MARTINI2) l) Dipartimento di Scienze Farmaceutiche, Università di Trieste, P.le Europa 1, 1-34 127 Trieste - 2) Dipartimento di Biologia, Università di Trieste, Via L. Giorgieri 10, 1-34127 Trieste Keywords: Aristolochia L., aristolochie acids, chemotaxonomy, disc1iminant analysis. Abstract: The composition of aristolochie acids from different populations of four Aristolochia species was analysed by HPLC; quantitative data were submitted to discriminant and statistica[ analyses. The interspecific variability was confirmed, and significant differences were detected by canonica! discriminant functions. The aristolochie acid II and the ratio AAII/ AAI are significant in the discriminati on of the examined species. Introduction Aristolochia L. (Aristolochiaceae) is a well-known genus since the ancient times for its officinal properties. In ltaly, several Aristolochia species have been widely used as medicinal plants since Plinius up to the last century (Savi 1805, Targioni­Tozzetti 1824, Cassone 1850). Ten species occur in ltaly (Nardi 1984), four of which are present in the Friuli-Venezia Giulia Region: A. clematitis L., A. rotunda L., A. pallida Willd. and A. lutea Desf. (Gortani 1906, Poldini 1980, 1991, Martini 1990). Characters related to the hypogean apparatus and the flowers have been considered as the most discrirninant taxonornically (Nardi 1984). Nardi's revision was mainly based both on the frequently disregarded flower morphological characters and on chromosome numbers. Some species, i.e. A. pallida and A. lutea, are morphologically sirnilar, and their deterrnination on morphological evidence alone is difficult. A previous study (Cateni et al. 1993), reported the chernical composition of various populations of A. clematitis, and the distribution of aristolochieacids in different parts of the plants, also withregard to the seasonal stages.In this paper we exarnine the aristolochie acid composition of the underground apparatus of the Aristolochia species occurring in the Friuli-Venezia Giulia Region, which were not yet phytochernically investigated. The distinct properties of aristolochie 45 acids among the species, and the evidence they provide on their mutuai relationship were also considered. The four species differ in their ecology. A.clematitis is the most autoapophytic, and grows in waste grounds along streams, ditches and sandy places near seashores, edges of cultivated fields, vineyards, hedges, rubble, margins of woods, coppices, on damp and aerated soils, rich in nitrates, up to 400 m. Aristolochia rotunda prefers damp grassy places along field margins, near ditches and streams, hedges, woods, shrubs, up to 400 m. Aristolochia pallida grows in woods, hedges, grassy places, mainly on plains and hills (up to 600 m). Aristolochia lutea is the taxon of the A. pallida complex with the widest distribution, and is also the most euriterrnic, growing mainly in Ostrya carpinifolia-woods (but also in beech woods), up to 1000 m. The distribution area of A. pallida includes the western part of the Region, while A. lutea is distributed in the eastern part; the northern populations of A. lutea co-exist with those of A. pallida and are often hardly distinguishable by morphological characters (Martini 1990). The purpose of this study is to ascertain whether the four species can be distinguished on the basis of their aristolochie acids content, and to contribute further to the knowledge of aristolochie acids variation within the genus. F. CATENI, L. COASSINI LOKAR, F. MARTINI Materials and methods Sampling concerned four populations of each species of Aristolochia in the following localities: Aristolochia clematitis: 1) Gorizia, loc. Mainizza, 40 m; 2) Sgonico (TS), 255 m; 3) Polcenigo (PN), loc. S. Giovanni, 60 m; 4) Monfalcone (GO), loc. Alberoni, 2 m. Aristolochia rotunda: 5) Muggia (TS), loc. Noghe­re, 5 m; 6) Muzzana del Turgnano (UD), loc. Bosco Coda di Manin, 6 m; 7) Latisana, Precenicco (UD), 3 m; 8) Monfalcone (GO), loc. Alberoni, 2 m. Aristolochia pallida: 9) Polcenigo (PN), loc. Rui Bragam, 70 m; lO)Tramonti di Sotto (PN), loc. Stalle Spinespes, 590 m; 11) Peonis (UD), loc. Rio Sech, 400 m; 12) Jouf di Maniago (PN), 450 m. Aristolochia lutea: 13) M. S. Leonardo (TS), 350 m; 14) Fernetti (TS), 360 m; 15) Ajdovsèina (Slo­venia), Trnovski Gozd, M. Kucelj, 650 m; 16) Maniago (PN), 400 m. The plants were collected at full anthesis to exarnine the quantitative variability in the hypogean apparatus. In each population, five samples of ten wild plants were randomly collected and voucher specimens were deposited in the Herbarium Universitatis Tergestinae (TSB). The hypogean parts were treated with liquid nitrogen, finely grounded (60 mesh) and weighed (1 g dry weight estimated on another sample). The extraction was can-ied out with 150 ml of methanol in Soxhlet to exhaustion. The extracts were evaporated in vacuum to small volume. The residue was redissolved in 10 ml methanol. The extracts were analysed by high performance liquid chromatography (HPLC) as described in a previous paper (Cateni et al. 1992). HPLC analyses were performed using a Perkin Elmer ODS RP-column filled with bonded octadecylsilane on silica (10 µm), flow rate 1,8 ml/rnin, detection 310 nm (detector sensitivity 0,01 a.u.f.s.) and methanol/water/acetic acid (80:20: 1) as mobile phase. All the determinations were pe1formed at 22° C and at isocratic condition of elution. The analytical data were subrnitted to elaboration with statistica! methods (Zar 1984, Lagonegro & Feoli 1985a, 1985b). Results and discussion The study and the experiment were considered a good test for application of aristolochie acid data to 46 Tab. 1 - V ariation and discriminant analysis in aristolochie acids I and II (% dry weight) in the examined populations of Aristolochia. Spccies Populations Aristolochie acids Ratio [(%) 11(%) AAII/AAI A. clematitis I 0.54 0.58 I I 2 0.25 0.24 I.O 3 0.26 0.28 I.I 4 0.-12 0.54 l.3 Average 0.37 0.41 I.I St. dev. 0.14 0.17 0.13 A. rotunda 5 0.09 0.09 I.O 6 0.05 0.07 1.4 7 0.07 0.09 1.2 8 0.07 0.09 1.2 Average 0.07 0.08 1.2 St. dev. 0.02 0.01 0.17 A. pallida 9 0.05 0.16 3.1 IO 0.05 0.17 3.5 Il 0.06 0.19 3.4 12 0.06 0.18 3.2 Average 0.05 0.18 3.3 St. dev. 0.003 0.02 0.2 A. lutea 13 0.04 O.I 2.4 14 0.05 O.I 2.0 15 0.04 O.I 2.2 16 0.08 O. 18 2.2 Average 0.05 0.12 2.2 St. dev. 0.017 0.04 0.16 Variab\e Wilkslambda F Significance AA[ 0.!766 I 18.1 o AAII 0.2697 68.6 o taxonornic problems in the genus Aristolochia which is cun-ently under rev1s10n. All the populations of the four exarnined species contain in the underground apparatus the same aristolochie acids (AAI and AAII) in different quantities. The quantitative composition in AAI and AAII and their ratio value in the various populations and species are reported in Tab. 1. The quantitative acid composition of A. clematitis L. is clearly distinct from those of the other investigated species, and all its populations are more productive, about two to ten times higher; furthermore, in all the populations (except n. 2) AAII prevails. The quantity of AAII in the population n. 16 (A. lutea) is sirnilar to that of A. pallida. These samples were derived from an isolated locality inside the distribution area of A. pallida. The ratio between the two aristolochie acids in the four species is very different for A. pallida, A. lutea and the remaining species: for A. clematitis the mean value is 1.1, for A. rotunda 1.2, for A. pallida 3.3 and for A. lutea 2.2. The analysis of variance was used to display Studia Geobot. 16 (1998) - Chemosystematic value of aristolochie acidsTab. 2. - Analysis of variance of aristolochie acid I (left) and II (right) based on o/o dry weight in the populations of the four species of Aristolochia L. (Ac = Aristolochia clematitis; Ar = A. rotunda; Ap = A. pallida; Al = A. lutea).(F0_05_(3_16) =3.24; DF=degree of freedom; F= variance ratio test; Q= statisti es for Cohrans's Q test). AAl AAII Species Source of Sum of DF Mean F Q Species Source of Sum of DF Mean F Q variarìon squares squarcs variation squares squarcs Treatment 0.293 3 0.098 285.691 Treatment 0.453 3 0.151 487.210 Error 0.005 16 o Errar 0.005 16 o Tocai 0.299 19 Total 0.458 19 Ac-Ar 208.0 I 5 24.981 Ac-Ar 308.661 30.430 A. clemmiris Ac-Ap 192.511 24.032 A. clema1ilis Ac-Ap 242.963 26.998 Ac-AI 35.083 10.259 Ac-AI 4.956 3.856 Ar-.-lp 0.300 0.949 Ar-Ap 3.926 3.432 Ar-AI 72.244 14.722 Ar-Al 235.395 26.574 Ap-AI 63.230 IJ.773 Ap-AI 178.519 23.142 Treatment 0.004 3 0.001 326.077 Treatmem 0.002 3 0.001 23.966 Errar 0.001 16 o Errar o 16 o Total 0.005 19 Tota! 0.002 19 Ac-Ar 325.167 31.233 Ac-Ar 20.363 7.816 A.rotunda Ac-Ap 75.288 15.023 A.rotunda A c -Ap 1.006 l.738 Ac-AI 67.132 14. 191 Ac-AI 0.698 1.447 Ar-Ap 87.591 16.210 Ar-Ap 12.315 6.078 Ar-AI 96.805 17.024 Ar-Al 13.521 6.369 Ap-AI 0.230 0.831 Ap-AI 0.028 0.291 Treacment o 3 o 18.368 Treatment o 3 0.001 68.335 Errar o 16 o Errar o 16 o Tarai o 19 Total o 19 Ac-Ar 0.462 1.177 Ac·Ar 12.782 6.193 A.pallida Ac-Ap 8.297 4.989 A.pallida Ac-Ap 67.065 14.184 Ac-AI 5.678 4.127 Ac-AI 22.086 8.140 Ar -Ap 12.673 6. 166 Ar-Ap :i 1.290 7.992 Ar-Al 9.377 5.304 Ar-Al 1.264 1.947 Ap-AI 0.248 0.862 Ap-AI 12.179 6.045 Treatment 0.004 3 0.001 342.908 Treatment o 3 0.008 920.049 Errar o 16 o Errar o 16 o Tocai 0.004 19 Tota! o 19 Ac-Ar 11.837 5.959 Ac-Ar 0.043 0.360 A.lutea r!c-Ap 1.606 2.195 A.lutea Ac-Ap 0.043 0.360 Ac-AI 269.599 28.439 Ac-A! 6 I 3.250 42.892 Ar-Ap 4.724 3.746 Ar·Ap 0.173 0.721 Ar-Al 168.453 22.480 Ar-AI 602.990 42.532 Ap-AI 229.595 26.245 Ap-AI 623.598 42.253 Tab. 3.- T Student's results for regression lines: centroids slopes and intercepts values. Df= 5, a< 0.05. (Ac= Aristolochia clematitis;Ar= A.. rotunda; Ap= A. pallida; Al= A. lutea). Species Centroids Slopes lntercepts I i T Student Ì Prob (%) T Studenti Prob (%) T Student Prob (%) : Ac-Ar 6.49 I 0.17 2.29 8.37 12.29 0.004 Ac-Ap 6.79 0.25 4.92 0.8 13.39 0.004 Ac-Al 7.09 0.21 2.78 4.97 13.36 0.004 Ar-Ap 11.51 0.03 13.66 0.02 9.24 0.02 Ar-Al 2.71 5.38 9.33 0.08 9.47 0.02 Ap -Al 3.28 3.04 6.36 0.31 11.20 0.001 47 F. CATENI, L. COASSINILOKAR, F. MARTIN! .2 .6 .1 5 .4 ...... .1 ...... ...... ...... :-9 .2 :-9 (.) (.) c:<:S c:<:S .:2 .:2 ..e: -8 (.) o o 2 C/) .;:'.'.; 'i:: .... -c:i:: o o <i:: o .O 5 .1 .2 .3 .4 .5 .6 Aristolochie acid I Fig. 1. - Ellipses of egual concentration, regression lines and centroids for the examinated Aristolochia species at the 5% leve! of confidence. The slopes and intercepts of the reg:ression lines are significantly different (p< 0.05). 1: A. clematitis; 2: A. rotunda; 3: A pallida; 4: A. lutea. The quantities are expressed as % dry weight. the source of noticed variation among the four species. Statistically significant variation occurred for AAI and AAII. The most significant is AAI (F == 118.1) (Tab. 1). The results of the analysis of variance among populations are reported in Table 2. Because the critica! value F is 3.14 at the 0.05 level of significance, all the values, which are smaller of 3.14, are not rejected. Therefore, A clematitis populations are significatively distinct from those of the other species, in terms of both AAI and AAII. Statistically significant variations in the quantities of AAI occur among all the other populations of the various species. The populations of A lutea significantly differ from those of A. pallida, although the two species are hardly distinguishable morphologically. The four species can be discriminated by regression lines and centroid values of the aristolochie acids (Fig. 1). The values of centroid, slope and intercept are reported in Tab. 3. Most discriminant are the intercept and centroid values. The greatest disjunction is between A. clematitis and the other species. 48 Conclusions The results demonstrate that aristolochie acids may be used for chemotaxonomical purposes in Aristolochia. The co-occurrence of structurally closely related compounds points to the chemical uniformity of the investigated species, differences being only quantitative. Aristolochie acid I and the ratio AAII/ AAI are the more significant taxonomically: chemical and morphological data are highly congruent. Severa! questions regarding systematic relationships can be addressed using chemical data. The four Aristolochia species may be distinguishable from one another, based on aristolochie acid II data. Quantitative differences were compared: (a) among populations of different species, and (b) by combining the populations of each species to increase the sample size. The two closely related species, A. pallida and A. lutea, can be also distinguished on a chemical basis. It is confirmed that, chemically, A. clematitis is more distant one from the other species. Studia Geobot. 16 (1998) - Chemosystematic value of aristolochie acids -These results have implications both for basic research on plant populations and for applied studies on plant secondary products as defensive or attractive agents. The different concentrations of the same compounds in the populations and in the species suggest that they may have a specific biologica! role, and could be related both to their possible function as allelopathic agents (Nishida & Fukarni 1989) and to their ecologica! role, since the plant toxins can be sequestred from the diet and stored by insects for defence. Aristolochie acids are actually used for this purpose by the butterfly Battus archidamas Boisd. (larval stage, Harborne 1987, 1988). lt is interesting to note that in Friuli-Venezia Giulia, another butterfly, Zerynthia polyxena Schiff. (= Z. hypsipyle Schulz., Papilionidae) also feeds on Aristolochia (Morandini, ex verbis). Riassunto VALORE CHEMOSISTEMATICO DEGLI ACIDI ARISTOLOCHICI NEL GEN. ARISTOLOCHIA L. NEL FRIULI-VENEZIA GIULIA. La composizione in acidi aristolochici di differenti popolazioni appartenenti alle quattro specie di Aristolochia L. presenti nella regione Friuli - Venezia Giulia è stata analizzata tramite HPLC, analisi discriminanti e statistiche. La variabilità interspecifica è stata confermata e differenze significative sono state evidenziate tramite le funzioni canoniche discriminanti. Gli acidi aristolochici I e II e in particolare il rapporto AAII/AAI hanno valore di marcatori chemotassonomici data la congruenza fra dati chimici e morfologici. Acknowledgments The Authors wonld like to thank the Director of the Museo Friulano di Storia Naturale (Udine) Dr. C. Morandini, for information on the butterfly Zerinthia polyxena. This project was carried out thanks to the financial support of the Italian Ministry of Public Instruction (M.P.I. 40%). 49 References Cassone F., 1850. Flora medico farmaceutica. 5. Torino. Cateni F., Mamolo M.G. & Coassini Lokar L., 1992. Rapid HPLC analysis for quantitative determination of aristolochie acids. Il Farmaco, 47 (10): 1335-1342. Cateni F., Coassini Lokar L., Martini F. & Vrech E., 1993. Aristolochie acids variation and distribution in some Aristolochia clematitis L. populations. Studia Geobot.,13: 299-312.Gortani L. & M., 1906. Flora friulana con speciale riguardo alla Carnia. 2. Udine. Harborne J.B., 1987. Chemical signals in the ecosystem. In: Dodge J.E. (ed.), New Perspectives in Plani Science. London, New York Harborne J.B., 1988. Introduction to Ecologica/ Biochemistry. New York. Lagonegro M. & Feoli E., 1985a. The use of ellypses of equa! concen.tration to analyse ordination vegetation patterns. Studia Geobot., 5: 143-165. Lagonegro M. & Feoli E., 1985b. Analisi multivariata di dati. Manuale d'uso di programmi BASIC per persona/ computer. Libreria Goliardica, Trieste. Martini F., 1990. Il gruppo Aristolochia pallida nell'Italia nordorientale. Giorn. Bot. Ital., 124: 731-743. Nardi E., 1984. The genus "Aristolochia" L. (Aristolochiaceae) in Italy. Webbia, 38: 221-300. Nishida R. & Fukami H., 1989. Ecologica/ adaptation of an Aristolochiaceae-feeding swallowtail butterfly, Atrophaneu­ra alcinous to aristolochie acid. J Chem. Eco!., 15: 2549-2563. Poldini L., 1980. Catalogo floristico del Friuli-Venezia Giulia e dei territori adiacenti. Studia Geobot., 1: 313-474. Poldini L., 1991. Atlante corologico delle piante vascolari nel Friuli-Venezia Giulia. Regione Autonoma Friuli-Venezia Giulia, Direzione Regionale Foreste e Parchi, Udine, & Dipartimento di Biologia, Università di Trieste .. Savi G., 1805. Materia medica vegetabile toscana. Firenze. Targioni-Tozzetti A., 1824 Scelta di piante officinali più necessarie a conoscersi. Firenze. Zar J.H., 1984. Biostatistical analysis, 2nd ed. Prentice- Hall Inc., New Jersey. Received November 11, 1996 Accepted November 10, 1997 

Chemosystematic value of aristolochic acids in the genus aristolochia l. in Friuli-Venezia Giulia (NE Italy)

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Chemosystematic value of aristolochic acids in the genus aristolochia l. in Friuli-Venezia Giulia (NE Italy)

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