Ng, N.

Walker, J. R. L.

English

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MAURI ORA, 1975, 3: 3-10 A CHEMOTAXONOMIC STUDY OF THE NEW ZEALAND ARALIACEAE : PHENOLIC COMPOUNDS NANCY NG and J . R . L . WALKER D e p a r t m e n t o f B o t a n y , U n i v e r s i t y o f C a n t e r b u r y C h r i s t c h u r c h , New Z e a l a n d ABSTRACT A p r e l i m i n a r y Chroma tog raph i e s u r v e y of p h e n o l i c Compounds p r e s e n t i n t h e l e a v e s of New Zea l and s p e c i e s of A r a l i a c e a e was a t t e m p t e d . With t h e e x c e p t i o n of Pseudopanax anomalum, b o t h woody and h e r b a e e o u s s p e c i e s e x h i b i t e d s i m i l a r b a s i c p a t t e r n s of p h e n o l i c c o n s t i t u e n t s . C o n t r a r y t o p r e v i o u s r e p o r t s l e u e o a n t h o e y a n i d i n s were d e t e c t e d i n some, b u t n o t a l l , woody s p e c i e s of A r a l i a c e a e . I t was of s p e c i a l i n t e r e s t t o f i n d t h a t t h e j u v e n i l e l e a v e s of P . simplex showed a h i g h e r c o n c e n t r a t i o n of l e u c o a n t h o -c y a n i d i n t h a n d i d t h e a d u l t l e a v e s . INTRODUCTION H i g h e r p l a n t s e x h i b i t an e n o r m o u s V a r i a t i o n i n t h e c h e m i c a l m a n i f e s t a t i o n s o f t h e i r s e c o n d a r y m e t a b o l i s m a n d , d u r i n g t h e p a s t two d e c a d e s , s y s t e m a t i s t s h a v e made i n c r e a s i n g u s e of p h y t o c h e m i c a l s t u d i e s a s an a i d t o C l a s s i f i c a t i o n . The r a t i o n a l e o f b i o c h e m i c a l s y s t e m a t i c s h a s b e e n d i s c u s s e d by Swain ( 1 9 6 3 , 1966) and H a r b o r n e ( 1 9 6 7 , 1 9 7 3 ) , who h a v e shown t h a t p h e n o l i c and f l a v o n o i d Compounds f u l f i l m o s t o f t h e r e q u i r e m e n t s f o r t a x o n o m i c m a r k e r s . M o r e o v e r , t h e s e Compounds a r e r e a d i l y s e p a r a t e d and i d e n t i f i e d by r e l a t i v e l y s i m p l e t e c h n i q u e s of c h r o m a t o g r a p h y and UV s p e c t r o s c o p y ( H a r b o r n e 1 9 7 3 ) . To d a t e r e l a t i v e l y l i t t l e work h a s b e e n done on t h e c h e m o s y s t e m a t i c s of New Z e a l a n d p l a n t s and t h i s p a p e r r e p o r t s a p r e l i m i n a r y s t u d y o f t h e p h e n o l i c c o n s t i t u e n t s of t h e New Z e a l a n d A r a l i a c e a e . The A r a l i a c e a e a r e r e p r e s e n t e d i n New Z e a l a n d by t h r e e s m a l l g e n e r a : Meryta (1 s p . ) , Schefflera (1 s p . ) and Stilbocarpa (3 s p p . ) t o g e t h e r w i t h t h e l a r g e r g e n u s Pseudopanax w h i c h c o n t a i n s f o u r t e e n s p e c i e s . P h i l i p s o n (1971) s u b - d i v i d e d t h e l a t t e r g e n u s on a b a s i s o f s h o o t c h a r a c t e r i s t i c s i n t o t h r e e ma in g r o u p s a s f o l l o w s : arboreum ( " f i v e - f i n g e r " ) , P . colensoi , mackinty rei, P . laetum. gilliesii*, P. lessorii, P. discolor. crassifelium ( " l a n e e w o o d " ) , P . ferox, chathamicum, P . lineare. simplex, P . edgerleyi*. anomal urr.. *not available for this investigation. Group Group Group Group Group I: IIa: IIb: IIc: III : P P P P P P P 4 MAURI ORA, 1975, Vol. 3 The aim of the studies reported here was to attempt to examine the distribution of the phenolic constituents within these species as a possible support for the above Classification. MATERIALS AND METHODS PLANT MATERIAL Leaf material from twelve of the species of Pseudopanax listed above were investigated together with samples of Meryta sinclairii , Schefflera digitata, Stilbocarpa polaris and S. robusta. All plant material was preserved by freeze-drying in an Edwards (U.K.) 30P2 centrifugal freeze-dryer. EXTRACTION OF PHENOLIC COMPOUNDS Freeze-dried leaf material was homogenised in 70% (v/v) methanol containing 1% conc. HCl and extracted for two hours. The extract was filtered through Whatman No. 1 paper, and extracted with a small quantity of petroleum ether to remove Chlorophylls and carotenoids. It was then concentrated in a rotary vacuum evaporator. Most flavonoid and phenolic Compounds are present in plants as a multiplicity of glycosides and other derivatives so it is normal practice, in the initial stages of a phytochemical survey, to hydrolyse the crude extract so that only the parent phenolic aglycones are present. This was achieved by refluxing the concentrated extract with an equal volume of 2N-HC1 for one hour in a boiling water bath. CHROMATOGRAPHIC PROCEDURES The routine Separation and identification of plant phenolics is best achieved by two-dimensional chromatography on paper or cellulose thin-layers using Standard solvent Systems (Mabry and Markham 1970; Harborne 1973) to facilitate comparison of Rf values. In the present work the hydrolysed extracts were separated by this procedure on Whatman No. 1 paper using TBA solvent (tert-butanol/acetic acid/water, 3:1:1) for the first direction followed by 15% (v/v) aqetic acid as the second solvent. The dry chromatograms were viewed under long wavelength UV light (365 mm), before and after exposure to NH3 fumes, to locate fluorescent spots. Relative concentrations of each component were judged visually by the size and intensity of fluorescence of each spot. SPECTROSCOPY OF PHENOLICS Most phenolic Compounds exhibit characteristic UV absorption spectra and the position of the absorption maximum may be shifted by the addition of group specific reagents such as borate and AICI3, thus providing valuable data for their identification (Harborne 1973). Identification of phenolic Compounds present in high concen-tration was attempted by eluting the spots from the chromatogram with methanol and then recording their spectra on a Pye-Unicam SP1800 recording UV spectrophotometer. Additions of sodium acetate, sodium tetraborate, or AICI3 were made to test for vicinal phenolic hydroxyl groups and unsaturated side-chains. NG & WALKER - ARALIACEAE CHEMOTAXONOMY 5 LEUCOANTHOCYANIDIN ASSAY Leucoanthocyanidins (LA's) yield a red colour, the "phloba-phene reaction", when heated in the presence of strong mineral acid. Swain and Hillis (1959) adapted this reaction to give a quantitative assay for LA's and this procedure was used to determine the levels of LA in 20 mg samples of freeze-dried leaf material. RESULTS AND DISCUSSION Examination of the individual two-dimensional chromatograms under UV light revealed the presence of 28 different UV-fluores-cent or absorbing spots; these were most probably phenolic or flavonoid in nature. Figure 1 shows a master composite chroma-togram derived from all the individual chromatograms. Table 1 gives the semi-quantitative distribution of the different pheno-lic Compounds in the various species examined. Considering first Pseudopanax, Group I, it may be seen that this group possesses a richer variety of phenolic constituents than the other groups. Species within the group appear to be closely related except for P. laetum which lacks spot 2 but possesses additional spots 18, 19 and 23, absent from the remainder of Group I. Similarly the composition of the plants of Group II appears to correlate reasonably well with the Classification based on morphological and other characteristics. Of particular interest is the absence of spot 3 from Groups IIa and IIb and the restric-tion of spot 28 to the plants of Group IIb. One of the most interesting observations from the chemo-systematic point of view is the uniquely different array of Com-pounds present in P. anomalum. For example, spots 21, 22, 23, 24, 25 and 26 were observed only in this species whereas the commonly occurring spots 1 and 11 were absent. Spot 21 is also unique in that it was the only Compound to exhibit a piiik fluo-rescence. Thus both chemically and morphologically P. anomalum is anomalous! Moreover, greater chemical diversity was found within the Single genus Pseudopanax than between such unrelated genera as Stilbocarpa, Meryta and Schefflera. Attempts were made to identify some of the Compounds separated on the chromatograms by comparison of Rf values with those of authentic Compounds and from spectroscopic data. Com-pounds 1 and 2 appeared to be very similar and may be eis and trans isomers* the spectral data suggested ortho-dihydroxy groups and an unsaturated side-chain but the Compound(s) were not identical with caffeic acid. Spots 8 and 9 were of interest since their oecurrence appeared to be complimentary; spot 8 was normally more prevalent but absent when 9 was dominant, as in P. anomalum, P.chathamicum and P. laetum. Spots 8 and 9 both yielded similar spectra and the present data suggests that they might be the eis and trans isomers of caffeic acid since 15% (v/v) acetic solvent separates these isomers. It may also be significant that spot 8 was one of the few Compounds present in all the genera examined. Spots 12 and 16 were tentatively identified as quercetin derivatives and spot 11 was probably kaempferol. Bate-Smith (1958) has reported that flavonoids together with hydroxy- and methoxy-substituted phenolic acids are 6 MÄURI ORA, 1975, Vol. 3 solvent front solvent 1, TBA © solvent 2. HAc Fluorescence colour: UV light: | | blue \=j blue dark yellow-brown pink absorbing spot UV light plus NH3 vapour: blue green or yellow-green bright yellow-brown pink orange-brown Fig. 1. Composite two-dimensional chromatogram of phenolic Compounds extracted from leaves of New Zealand Araliaceae. NG & WALKER - ARALIACEÄE CHEMOTÄXONOMY t) t) 3^ ^ t)Q I" I' ^) a^ [a ^jo w *3 Q> » O ft ö O ^ C 8 MAURI ORA, 1975, Vol. 3 TABLE 2. RELATIVE LEVELS OF LEUCOANTHOCYANIDINS IN NEW ZEALAND ARALIADS Species Absorbance1 (550 nm filter) reading Araliaceae Stilbocarpa robusta Stilbocarpa polaris Meryta sinclairii Pseudopanax arboreum P. colensoi P. mackintyrei P. laetum P. lessonii P. discolor P. crassifolium P. ferox P. chathamicum P. lineare P. simplex (adult) P. simplex (juvenile) P. anomalum 2 Umbelllferae Daucus carota (carrot) Petroselinum crispum (parsley) Foeniculum vulgare (fennel) 1.5 1.2 1.4 1.5 6.6 1.1 1.4 1.0 5.0 1.9 1.3 1.0 3.2 1.2 3.1 1.3 1.9 1.1 2.0 (high) (high) (moderate) (moderate) Note 1. Leucoanthocyanidin Standards were not available therefore only relative levels are given. Note 2. Specimens of non-woody Umbelliferae are included to provide a base-line level. NG & WALKER - ARALIACEAE CHEMOTAXONOMY 9 commonly present in the Araliaceae. Comparison of Schefflera digitata and Meryta sinclairii with the genus Pseudopanax shows that all possess an essentially similar ränge of phenolic Compounds. The similarity of s. digitata to Pseudopanax Group I is in agreement with the observed similarities of shoot morphology. However, a greater difference might have been expected between Pseudopanax Group I and M. sinclairii in view of their differences in morphology. It seems therefore, that the family as a whole, with the notable exception of P. anomalum, exhibit a basic similarity in their phenolic constituents. When this investigation was begun it was hoped that chemical evidence might help resolve the problem of the Classification of the aberrant genus stilbocarpa which is herbaceous whereas most other araliads are woody in nature. It was considered that the LA levels in the various plants might shed light on this problem since Harborne (196 7) has suggested that there may be a correla-tion between the presence of LA's and woodiness. Many authors have considered the Araliaceae and ümbelliferae to have the Status of an order, the Umbellales or Umbelliflorae (Cronquist 1968; Hegnauer 1964) within which Rodriguex (1971) also included the family Cornales. LA's have been reported to be absent from the ümbelliferae (Bate-Smith 1958; Harborne 1967), but present in some Cornales (Hegnauer 1964) and absent from the Araliaceae (Bate-Smith 1958). The present results (Table 2) show that LA's are present in four of the sixteen species examined. However, if the presence of LA's is associated with woodiness than they would be expected to be present in the woody araliads but not in stilbocarpa which is herbaceous. One interesting point that needs further research is the finding of appreciable amounts of LA's in the juvenile form of P. simplex but only trace amounts in, the adult form. It is therefore necessary to determine if this loss of LA's with maturity is a common feature of all araliads and this may help clarify the question of the anomalous distribu-tion of LA's referred to above; in particular it could help resolve the problem of Stilbocarpa. This study is only a beginning; it yet remains to examine the distribution of other groups of Compounds such as proteins and allozymes, non-protein amino acids and other secondary meta-bolites. For example, Murray and Stanley (1952) have reported the occurrence of terpenoid Compounds in the essential oil from leaves of P. simplex and this should be a worthwhile area for future chemosystematic investigations. ACKNOWLEDGMENTS The authors wish to thank the several people who helped in collecting plant material, also the University of Canterbury and the U.G.C. for grants for equipment. LITERATURE CITED BATE-SMITH, E.C. 1958. Plant phenolics as taxonomic guides. Proceedings of the Linnean Society of London. 169th Session: 198-211. CRONQUIST, A. 1968. The evolution and Classification of flowering plants. Houghton Mifflin Co., Boston. 396 pp. 10 MÄURI ORA, 1975, Vol. 3 HARBORNE, J.B. 1967. Comparative biochemistry of the flavonoids. Academic Press, London and New York. 383 pp. 1973. Phytochemical methods. Chapman and Hall Ltd, London. 278 pp. HEGNAUER, R. 1964. Chemotaxonomie der Pflanzen. Band III. pp. 173-183. Birkhäuser Verlag, Berlin. 743 pp. MABRY, T.J. and MARKHAM, K.R. 1970. Tne systematic Identification of flavo-noids. Springer-Verlag, Berlin. 345 pp. MURRAY, J. and STANLEY, B.G. 1952. The essential oil of Nothopanax simplex. Journal of applied Chemistry 2\ 5. PHILIPSON, W.R. 1971. Shoot differentiation in the Araliaceae of New Zealand. Journal of the Indian Botanical Society 50A: 188-195. RODRIGUEZ, R.L. 1971. The relationships of the Uiribellales. In Heywood, V.H. (Ed.), The biology and chemistry of the Umbelliferaei 63-91. Academic Press, London and New York. 438 pp. SWAIN, T. (Ed.). 1963. Chemical plant taxonomy. Academic Press, London and New York. 543 pp. — 1966. Comparative phytochemistry. Academic Press, London and New York. 383 pp. and HILLIS, W.E. 1959. The phenolic constituents of Prunus domestica, I. - The quantitative analysis of phenolic constituents. Journal of the Science of Food and Agriculture I: 63-68. 

A chemotaxonomic study of the New Zealand Araliacea: phenolic compounds

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A chemotaxonomic study of the New Zealand Araliacea: phenolic compounds

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