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Investigations on Hoya species. IV. Leaf Phenolics and Leaf-Wax Triterpenes of Hoya australis R. BR. ex TRAILL in Relation to Leaf Age
Botanisch Laboratorium, Rijksuniversiteit Utrecht, Nederland
Investigations on Hoya species. IV/) Leaf Phenolics and Leaf-Wax Triterpenes of Hoya australis R. BR. ex TRAILL in Relation to Leaf Age GERARD J. NIEMANN, THOMAS K. F. SCHULZ, HENDRIK H. VAN GENDEREN and WIM J. BAAS With 5 figures Received September 7, 1979 . Accepted October 3, 1979
Summary Roya australis leaves contain large amounts of chlorogenic acid, smaller amounts of iso-chlorogenic acid and other phenolic depsides such as p-coumaric acid ester and small concentrations of the apigenin-type flavones. Anthocyanins were found only in very young leaves. With increasing leaf age the concentrations of chlorogenic- and isochlorogenic acid decreased; a more or less stable level was reached at the moment when anthocyanins could no longer be detected and when the concentration of the triterpene lupeol in the leaf wax began to increase. Key words: Roya australis, Asclepiadaceae, jlavonoids, phenolics, triterpenes.
Introduction In the course of our study of H oya australis in relation to ageing (BAAS and FIGDOR, 1978 a; BAAS and NIEMANN, 1979 a) two sites of triterpene synthesis were investigated. It turned out that the triterpene composition found in the latex was totally independent of the age of the plant part investigated. In the cuticular wax, on the other hand, there were large variations in the triterpene composition with leaf age. Some of these variations seemed to correlate with colour changes of the leaf which are probably brought about by changes in the anthocyanin content. There is no direct connection between the biosynthesis of phenols such as the anthocyanins and triterpenes, but an overlap in their physiological significance cannot be excluded. Little is known about the overall physiological function of triterpenes and phenolics, although their involvement in vital biochemical processes has been reported. Flavonoids may regulate plant growth through their involvement 1) Part III: G. 1125-1128 (1979).
J.
NIEMANN', W.
J.
BAAS, E. BESSON and
J.
CHOPIN, Z. Naturforsch. 34 c,
Z. Pjlanzenphysiol. Bd. 97. S. 241-248. 1980.
242
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J. NIEMANN, T. K. F. SCHULZ, H. H. VAN GENDEREN and W. ]. BAAS
in the IAA metabolism or in the phosphorylation (MCCLURE, 1975), and phenolic acids may play a major role in seed and bud dormancy (RICE, 1977) and in the inhibition of flowering (PRYCE, 1972). It has been shown that some triterpenoids possess weak growth-stimulating or growth-inhibiting properties (BOITEAU et al., 1964). In some cases treatment of seedlings with triterpenoids has also led to an increase in the anthocyanin level (BOITEAU et al., 1964, p. 992). The main ecological significance of both groups of compounds, and of some other secondary plant products is that they probably have a potential protective function (FRAENKEL, 1959; JANZEN, 1972; SWAIN, 1977; SEIGLER, 1977). Some of these compounds are known to protect plants against fungal or bacterial disease. They may also be involved in allelopathy, help to reduce insect and/or herbivore predation and act as a protection against photodestruction. As in the case of triterpenoids (BOITEAU et al., 1964, p. 753), the variation in phenol content with leaf age has often been investigated with regard to seasonal variation. Active flavonoid metabolism and/or flavonoid accumulation was found mainly during periods of intensive growth; a more steady situation followed when leaves were fully grown. As far as anthocyanins are concerned this can often be observed directly in plant species through the reddish colour of the fast-growing parts. With other flavonoids such a correlation was found in Avena (EFFERTZ and WEISSENBOCK, 1976), Cucurbita (STRACK and REZNIK, 1976), Corylus (STAUDE and REZNIK, 1973), Larix (NIEMANN, 1976) and in several other plants.
In studies on the Asclepiadaceae MELIN (1964) compared very young and fully developed leaves of Periploca graeca L. In fullygrown leaves he found two kaempferol glycosides, which did not occur in the young ones, and he also observed that ageing brougt about an increase in the concentration of an aesculetin derivative and a decrease in the concentration of chlorogenic acid and isochlorogenic acid. Not much is known about leaf phenolics in H oya species. Chlorogenic acid has been reported in leaves of H. bandanensis (GORTER, 1909) and H. australis (BAAS and NIEMANN, 1979 a). Ferulic acid and acylated flavonol glycosides have been found in H. bella (BAAS and NIEMANN, 1979 b) and the glucose ester of ferulic acid and some C-glycosyl flavones in H. lacunosa leaves (NIEMANN et al., 1979). H. camosa leaves possibly contain leucocyanidin, kaempferol and quercetin (KOZJEK et al., 1973). The aim of the present investigations was to obtain some insight into the behaviour of phenolics and triterpenes in H. australis leaf during leaf development.
Material and Methods Plant material
We used cuttings of Hoya australis R. BR. ex TRAILL about 6 months old, which had been cultivated in the greenhouses. All leaf pairs of one plant were collected at the same time. Z. Pflanzenphysiol. Bd. 97. S. 241-248. 1980.
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ExtraCtion and separation A leaf-wax extract was obtained by dipping the leaves for 20 seconds in a mixture of chloroform-methanol 2 : 1, the extract was further separated by Al 20 a column chromatography and analysed by GLC (BAAS and FIGDOR, 1978 a). To obtain flavonoids and other phenolics the chloroform-washed leaves were homo genised in methanol-1 % formic acid and then extracted with acetone. The combined extract was filtered, lipophilic substances were removed by extraction with ligroin and, after concentration, the residue was extracted with butanol. Analysis of the phenols
For the quantitative determination of the phenolic compounds a Dupont 830 high-performance liquid chromatograph was used, coupled to a Dupont 837 spectrophotometer and an Infotronics 304 integrator. Conditions: The fractions were dissolved in acidified methanol and eluted at 50°C and at 1100 psi (7500 kPa, flow around 0.7 ml/min) from a 20rbax ODS column (precolumn with Co Pell ODS) with a gradient (concave 2, 45-100010) of water-methanol with 0.1 010 of phosphoric acid. The compounds were detected at 360 (flavonoids and phenolic depsides) or 530 nm (anthocyanins) and the peak surfaces were measured. For identification a butanol extract was prepared from a large quantity of leaves. This extract was separated by preparative HPLC on a Jobin-Yvon Chromatospec-prep 100 chromatograph, with a silica column (100 g of silica) 80 mm ID, eluted with chloroformacetic acid-methanol 75 : 1 : 25, without detection, at a pressure of 1000 kPa and at room temperature. The fractions obtained were further purified by repeated paper chromatography. Jdentification The compounds were obtained in solution. They were identified by UV spectral data (inclusive shifts), R f , colour under UV, ibid. with ammonia, and by different spray reagents, using the original products and their acid- (NIEMANN, 1972) and lor alkaline hydrolysis/degradation products. HPLC t:s were compared against referent compounds and with the original butanol extract by co-chromatography.
Results and Discussion
As previously mentioned, Raya australis leaves contain large amounts of chlorogenic acid (BAAS and NIEMANN, 1979 a). In addition to this compound two other phenolic depsides were identified: isochlorogenic acid and the glucose ester of p-coumaric acid. Flavonoids occurred mainly in comparatively low concentration; all compounds showed an apigenin- or luteolin-type UV spectrum. We identified apigenin-7-glucoside, -7-rutinoside and -7-(ferulylglucoside) and chrysoeriol7-rutinoside. For other flavonoids present the quantities were too low for complete identification. The anthocyanins, which occurred only in very young leaves, appeared to be cyanidin glucosides. From our studies on Raya phenolics, it would seem that the few species hitherto investigated have a rather specific flavonoid chemistry. For the investigation of the influence of leaf age on the leaf phenol and wax triterpene composition, we took nodal cuttings from plants about 6 months old and at the same time collected the leaves from the cuttings. Their relative age is
z. PJlanzenphysiol. Rd. 97. S. 241-248. 1980.
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determined by counting the nodes, starting with the one under the apex. A growth diagram of a typical plant is shown in Fig. 1. In addition to the apex this plant had eleven nodes; leaves on nodes 1-3 were too small for measurement. Visible anthocyanin colouration was observed in leaves at nodes 4-7 (for 7 traces only). weight surface area (cm 2)
5.0 50.0
0--<>---0.;:::--"'_ b
a
• surface area
4
5
6
7 8 9 node from apex
10
11
Fig. 1: Growth diagram of Boya australis: a gives the wet weight of a leaf pair in grammes and b the surface area in cm 2 •
Analysis of the alcohol fraction of the leaf wax is given in Fig. 2. This figure shows the behaviour of four of the main components: fJ-amyrin, lupeol, unknown Xl and the partly identified seco compound X 2 (BAAS and FIGDOR, 1978 a and b) in relation to leaf age. Basically, the behaviour of the compounds shown in the graphs resembles that found previously for much older plants (BAAS and FIGDOR, 1978 a), although growth conditions were different. A typical HPLC chromatogram of the phenolics in leaf extracts of a young (3 A) and somewhat older (3 B) leaf is shown in Fig. 3; in this figure the relatively high
ug/cm2
5
.Iupeol 0 component X, • component X 2 Cl B-amyrin
4
5
6
7 8 9 node from apex
10
11
Fig. 2: Amounts of the individual components of the leaf wax-alcohol fraction per cm 2 leaf area in relation to leaf age. Z. P/lanzenphysiol. Ed. 97. S. 241-248. 1980.
Investigations on Raya species. IV.
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1.5.
I
3
9
8
2
0
5
11
I
4
12
1fl13
I
10
15
20 minutes
0
-12
2 -3 1.5.
I
9
o
5
10
15
20 minutes
Fig. 3: HPLC analysis of Raya australis leaf extracts on Zorbax ODS eluted with a gradient of water-methanol with 0.1 % phosphoric acid. A: young leaf, B: older leaf. I.S. = internal standard vitexin, 3 = chlorogenic acid, 6 = p-coumarylglucose, 7 = isochlorogenic acid, 11 = apigenin-7-glucoside, 12 = apigenin-7-rutinoside + an unknown flavonoid, 13 = chrysoeriol-7-rutinoside.
concentrations of chlorogenic acid (compound 3) are apparent. In relation to leaf age (Fig. 4) the comparatively high cOntent of chlorogenic acid and of isochlorogenic acid (compound 7) in very young leaves shows a sharp decrease on leaf expansion (leaves at node 5 to 7). It is only peak 12, a mixture of apigenin-7-rutinoside and a yet unknown flavonoid, which increases slightly with increasing leaf age. With anthocyanins (Fig. 5) leaf cell extension seems to be a critical period. All four anthocyan ins are found in the small leaves at nodes 4 to 6. The cyanidin diglucosides Z. PJlanzenphysial. Bd. 97. S. 241-248. 1980.
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rei. abs.l cm 2 • chlorogenic acid (3) o isochlorogenic acid (?) • component 12
4
5
6 7 8 9 node from apex
10
11
Fig. 4: Amount of leaf phenolics per cm 2 leaf area in relation to leaf age. Component 12 = a mixture of apigenin-7-rutinoside and an unknown flavonoid.
5
rei. abs.l cm 2
4
5 6 node from apex
7
Fig. 5: Amount of anthocyan ins per cm 2 leaf area in relation to leaf age. Compounds 1-4 all are cyanidin derivatives.
(1 and 2) are the first to disappear and only traces of compounds 3 and 4 were found in the expanding leaves at node no. 7. Anthocyanins could not be detected in fully expanded leaves (from node 8 onwards). From our findings for all compounds investigated we can conclude that the period of leaf expansion seen at nodes no 7 to 8 is rather a critical stage. By this time anthocyanins have disappeared and concentration of phenolic depsides has fallen to a comparatively low level, but on the other hand a sudden increase occurs in the leaf wax compound lupeol. Information about the physiological significance of phenolics and triterpenes is scarce. If, however it is assumed that they have a general protective function, the simultaneous drop in concentration of the phenolic compounds and the rise in that of lupeol would seem to suggest some physiological interaction. A similar interdependence of triterpenes with anthocyanins has been found before. In Andromeda polilolia leaves, for instance, the parasite Exobasidium vaccinii causes a Z. Pflanzenphysiol. Bd. 97. S. 241-248.1980.
Investigations on Baya species. IV.
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decrease in the triterpene level and this is accompanied by the synthesis of two new anthocyanins (TAMAS et a!., 1975). In the latter case it should be noticed that injury may also lead to increased anthocyanin synthesis (Bopp, 1959). More evidence is certainly needed before firm conclusions can be drawn. Acknowledgement We are indebted to Mrs. JUDITH KOERSELMAN-KooY and Miss JANNY VAN SCHAlK for their assistance and to the RMP company for use of the Chromatospac prep 100. Miss S. McNAB is thanked for helping to revise the English of this text.
References BAAS, W. ]. and C. G. FIGDOR: Triterpene composition of Baya australis cuticular wax in relation to leaf age. Z. Pflanzenphysiol. 87, 243-253 (1978 a). - - Isolation of a new seco-nor-triterpenol from Baya australis leaf wax. Z. Naturforsch. 33 c, 337-339 (1978 b). BAAS, W. ]. and G. ]. NIEMANN: Investigations on Baya species. I. Latex lipids of Baya australis R. BR. ex TRAILL. and the effects of the age of the plant part and of environmental factors thereupon. Plant a med. 35, 348-353 (1979 a). - - Investigations on Baya species, II. Latex lipids and leaf phenolics of Baya bella HOOK. Z. Naturforsch. 34 c, 5-8 (1979 b). BOITEAU, P., B. PASISCH, and A. RAKOTO RATSIMAMANGA: Les Triterpenoi'des en physiologie vegetale et animale. Gauthier-Villars, Paris, 1964. Bopp, M.: aber die Bildung von Anthocyan und Leucoanthocyan an Wundrandern. Z. Botanik 47,197-217 (1959). EFFERTZ, B. und G. WEISSENBOCK: Dynamik der Flavonoid-Akkumulation in Primarblattgeweben von Avena sativa L. Ber. Deutsch. Bot. Ges. 89, 473-481 (1976). FRAENKEL, G. S.: The raison d'~tre of secondary plant substances. Science 129, 1466-1470 (1959). GORTER, K.: aber die Verbreitung der Chlorogensaure in der Natur. Arch. Pharm. 247, 184-196 (1909). JANZEN, D. H.: Community structure of secondary compounds in plants. Pure App!. Chern. 34, 529-538 (1973). KOZJEK, F., M. JAY, and G. NETIEN: Recherches chimiotaxonomiques sur les plantes vasculaires. XXIX. Contribution biochimique l'etude des flavonoides des Asclepiadacees. Candollea 28, 237-247 (1973). MCCLURE, ]. W.: Physiology and functions of flavonoids. In: ]. B. HARBORNE, T. ]. MABRY and H. MABRY (Eds.): The flavonoids, p. 970-1055. Chapman and Hall, London, 1975. MELIN, D.: Etude par chromatographie sur papier du contenu en flavonoi'des des feuilles de Periplaca graeca L. Ann. sci. Univ. Besan~on, ser. 2, 20, 87-93 (1964). NIEMANN, G. ].: Acid degradation of flavonoids as an aid in their identification. ]. Chl'Omatog. 74, 155-156 (1972). - Phenolics from Larix needles. XII. Seasonal variation of main flavonoids in leaves of L.leptalepis. Acta Bot. Neerl. 25, 349-359 (1976). NIEMANN, G. ]., w. J. BAAS, E. BESSON, and]. COPIN: Investigations on Boya species. III. Leaf phenolics and latex lipids of Baya lacunosa BL. Z. Naturforsch. 34 c, 1125-1128 (1979).
a
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VAN GENDEREN and W.
J. BAAS
PRYCE, R. J.: Gallic acid as a natural inhibitor of flowering in Kalanchoe blosfeldiana. Phytochemistry 11, 1911-1918 (1972). RICE, E. 1.: Some roles of allelopathic compounds in plant communities. Biochem. Syst. EcoI. 5, 201-206 (1977). SEIGLER, D. S.: Primary roles for secondary compounds. Biochem. Syst. EcoI. 5, 195-199 (1977). STAUDE, M. and H. REZNIK: Das Flavonoidmuster der Winterknospen und LaubbIatter von Corylus avellana 1. Z. PflanzenphysioI. 68, 346-356 (1973). STRACK, D. und H. REZNIK: Die Dynamik von Flavonolglykosiden wahrend der Keimlingsentwicklung von Cucurbita maxima DUCHESNE. Z. PflanzenphysioI. 79, 95-108 (1976). SWAIN, T.: Secondary compounds as protective agents. Ann. Rev. Plant PhysioI. 28, 479-501 (1977). TAMAS, M., G. LAZAR-KEUL, and V. SORAN: Variation of the flavonoids, anthocyan ins and triterpenes in Andromeda polifolia 1. leaves under the action of parasitism caused by Exobasidium vaccinii (FUCK) WORON. Rev. Roum. BioI. 20, 65-69 (1975). TISSUT, M. and K. EGGER: Les glycosides flavoniques foliaires de quelques arbres, au cours du cycle vegthatif. Phytochemistry 11, 631-634 (1972). Dr. GERARD ]. NIEMANN, Botanisch Laboratorium, Rijksuniversiteit Utrecht, Lange Nieuwstraat 106, Utrecht.
Z. Pflanzenphysiol. Bd. 97. S. 241-248. 1980.
Investigations on Hoya species. IV/) Leaf Phenolics and Leaf-Wax Triterpenes of Hoya australis R. BR. ex TRAILL in Relation to Leaf Age GERARD J. NIEMANN, THOMAS K. F. SCHULZ, HENDRIK H. VAN GENDEREN and WIM J. BAAS With 5 figures Received September 7, 1979 . Accepted October 3, 1979
Summary Roya australis leaves contain large amounts of chlorogenic acid, smaller amounts of iso-chlorogenic acid and other phenolic depsides such as p-coumaric acid ester and small concentrations of the apigenin-type flavones. Anthocyanins were found only in very young leaves. With increasing leaf age the concentrations of chlorogenic- and isochlorogenic acid decreased; a more or less stable level was reached at the moment when anthocyanins could no longer be detected and when the concentration of the triterpene lupeol in the leaf wax began to increase. Key words: Roya australis, Asclepiadaceae, jlavonoids, phenolics, triterpenes.
Introduction In the course of our study of H oya australis in relation to ageing (BAAS and FIGDOR, 1978 a; BAAS and NIEMANN, 1979 a) two sites of triterpene synthesis were investigated. It turned out that the triterpene composition found in the latex was totally independent of the age of the plant part investigated. In the cuticular wax, on the other hand, there were large variations in the triterpene composition with leaf age. Some of these variations seemed to correlate with colour changes of the leaf which are probably brought about by changes in the anthocyanin content. There is no direct connection between the biosynthesis of phenols such as the anthocyanins and triterpenes, but an overlap in their physiological significance cannot be excluded. Little is known about the overall physiological function of triterpenes and phenolics, although their involvement in vital biochemical processes has been reported. Flavonoids may regulate plant growth through their involvement 1) Part III: G. 1125-1128 (1979).
J.
NIEMANN', W.
J.
BAAS, E. BESSON and
J.
CHOPIN, Z. Naturforsch. 34 c,
Z. Pjlanzenphysiol. Bd. 97. S. 241-248. 1980.
242
G.
J. NIEMANN, T. K. F. SCHULZ, H. H. VAN GENDEREN and W. ]. BAAS
in the IAA metabolism or in the phosphorylation (MCCLURE, 1975), and phenolic acids may play a major role in seed and bud dormancy (RICE, 1977) and in the inhibition of flowering (PRYCE, 1972). It has been shown that some triterpenoids possess weak growth-stimulating or growth-inhibiting properties (BOITEAU et al., 1964). In some cases treatment of seedlings with triterpenoids has also led to an increase in the anthocyanin level (BOITEAU et al., 1964, p. 992). The main ecological significance of both groups of compounds, and of some other secondary plant products is that they probably have a potential protective function (FRAENKEL, 1959; JANZEN, 1972; SWAIN, 1977; SEIGLER, 1977). Some of these compounds are known to protect plants against fungal or bacterial disease. They may also be involved in allelopathy, help to reduce insect and/or herbivore predation and act as a protection against photodestruction. As in the case of triterpenoids (BOITEAU et al., 1964, p. 753), the variation in phenol content with leaf age has often been investigated with regard to seasonal variation. Active flavonoid metabolism and/or flavonoid accumulation was found mainly during periods of intensive growth; a more steady situation followed when leaves were fully grown. As far as anthocyanins are concerned this can often be observed directly in plant species through the reddish colour of the fast-growing parts. With other flavonoids such a correlation was found in Avena (EFFERTZ and WEISSENBOCK, 1976), Cucurbita (STRACK and REZNIK, 1976), Corylus (STAUDE and REZNIK, 1973), Larix (NIEMANN, 1976) and in several other plants.
In studies on the Asclepiadaceae MELIN (1964) compared very young and fully developed leaves of Periploca graeca L. In fullygrown leaves he found two kaempferol glycosides, which did not occur in the young ones, and he also observed that ageing brougt about an increase in the concentration of an aesculetin derivative and a decrease in the concentration of chlorogenic acid and isochlorogenic acid. Not much is known about leaf phenolics in H oya species. Chlorogenic acid has been reported in leaves of H. bandanensis (GORTER, 1909) and H. australis (BAAS and NIEMANN, 1979 a). Ferulic acid and acylated flavonol glycosides have been found in H. bella (BAAS and NIEMANN, 1979 b) and the glucose ester of ferulic acid and some C-glycosyl flavones in H. lacunosa leaves (NIEMANN et al., 1979). H. camosa leaves possibly contain leucocyanidin, kaempferol and quercetin (KOZJEK et al., 1973). The aim of the present investigations was to obtain some insight into the behaviour of phenolics and triterpenes in H. australis leaf during leaf development.
Material and Methods Plant material
We used cuttings of Hoya australis R. BR. ex TRAILL about 6 months old, which had been cultivated in the greenhouses. All leaf pairs of one plant were collected at the same time. Z. Pflanzenphysiol. Bd. 97. S. 241-248. 1980.
Investigations on Roya species. IV.
243
ExtraCtion and separation A leaf-wax extract was obtained by dipping the leaves for 20 seconds in a mixture of chloroform-methanol 2 : 1, the extract was further separated by Al 20 a column chromatography and analysed by GLC (BAAS and FIGDOR, 1978 a). To obtain flavonoids and other phenolics the chloroform-washed leaves were homo genised in methanol-1 % formic acid and then extracted with acetone. The combined extract was filtered, lipophilic substances were removed by extraction with ligroin and, after concentration, the residue was extracted with butanol. Analysis of the phenols
For the quantitative determination of the phenolic compounds a Dupont 830 high-performance liquid chromatograph was used, coupled to a Dupont 837 spectrophotometer and an Infotronics 304 integrator. Conditions: The fractions were dissolved in acidified methanol and eluted at 50°C and at 1100 psi (7500 kPa, flow around 0.7 ml/min) from a 20rbax ODS column (precolumn with Co Pell ODS) with a gradient (concave 2, 45-100010) of water-methanol with 0.1 010 of phosphoric acid. The compounds were detected at 360 (flavonoids and phenolic depsides) or 530 nm (anthocyanins) and the peak surfaces were measured. For identification a butanol extract was prepared from a large quantity of leaves. This extract was separated by preparative HPLC on a Jobin-Yvon Chromatospec-prep 100 chromatograph, with a silica column (100 g of silica) 80 mm ID, eluted with chloroformacetic acid-methanol 75 : 1 : 25, without detection, at a pressure of 1000 kPa and at room temperature. The fractions obtained were further purified by repeated paper chromatography. Jdentification The compounds were obtained in solution. They were identified by UV spectral data (inclusive shifts), R f , colour under UV, ibid. with ammonia, and by different spray reagents, using the original products and their acid- (NIEMANN, 1972) and lor alkaline hydrolysis/degradation products. HPLC t:s were compared against referent compounds and with the original butanol extract by co-chromatography.
Results and Discussion
As previously mentioned, Raya australis leaves contain large amounts of chlorogenic acid (BAAS and NIEMANN, 1979 a). In addition to this compound two other phenolic depsides were identified: isochlorogenic acid and the glucose ester of p-coumaric acid. Flavonoids occurred mainly in comparatively low concentration; all compounds showed an apigenin- or luteolin-type UV spectrum. We identified apigenin-7-glucoside, -7-rutinoside and -7-(ferulylglucoside) and chrysoeriol7-rutinoside. For other flavonoids present the quantities were too low for complete identification. The anthocyanins, which occurred only in very young leaves, appeared to be cyanidin glucosides. From our studies on Raya phenolics, it would seem that the few species hitherto investigated have a rather specific flavonoid chemistry. For the investigation of the influence of leaf age on the leaf phenol and wax triterpene composition, we took nodal cuttings from plants about 6 months old and at the same time collected the leaves from the cuttings. Their relative age is
z. PJlanzenphysiol. Rd. 97. S. 241-248. 1980.
244
G.
J. NIEMANN, T. K. F. SCHULZ, H. H. VAN GENDEREN and W. J. BAAS
determined by counting the nodes, starting with the one under the apex. A growth diagram of a typical plant is shown in Fig. 1. In addition to the apex this plant had eleven nodes; leaves on nodes 1-3 were too small for measurement. Visible anthocyanin colouration was observed in leaves at nodes 4-7 (for 7 traces only). weight surface area (cm 2)
5.0 50.0
0--<>---0.;:::--"'_ b
a
• surface area
4
5
6
7 8 9 node from apex
10
11
Fig. 1: Growth diagram of Boya australis: a gives the wet weight of a leaf pair in grammes and b the surface area in cm 2 •
Analysis of the alcohol fraction of the leaf wax is given in Fig. 2. This figure shows the behaviour of four of the main components: fJ-amyrin, lupeol, unknown Xl and the partly identified seco compound X 2 (BAAS and FIGDOR, 1978 a and b) in relation to leaf age. Basically, the behaviour of the compounds shown in the graphs resembles that found previously for much older plants (BAAS and FIGDOR, 1978 a), although growth conditions were different. A typical HPLC chromatogram of the phenolics in leaf extracts of a young (3 A) and somewhat older (3 B) leaf is shown in Fig. 3; in this figure the relatively high
ug/cm2
5
.Iupeol 0 component X, • component X 2 Cl B-amyrin
4
5
6
7 8 9 node from apex
10
11
Fig. 2: Amounts of the individual components of the leaf wax-alcohol fraction per cm 2 leaf area in relation to leaf age. Z. P/lanzenphysiol. Ed. 97. S. 241-248. 1980.
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1.5.
I
3
9
8
2
0
5
11
I
4
12
1fl13
I
10
15
20 minutes
0
-12
2 -3 1.5.
I
9
o
5
10
15
20 minutes
Fig. 3: HPLC analysis of Raya australis leaf extracts on Zorbax ODS eluted with a gradient of water-methanol with 0.1 % phosphoric acid. A: young leaf, B: older leaf. I.S. = internal standard vitexin, 3 = chlorogenic acid, 6 = p-coumarylglucose, 7 = isochlorogenic acid, 11 = apigenin-7-glucoside, 12 = apigenin-7-rutinoside + an unknown flavonoid, 13 = chrysoeriol-7-rutinoside.
concentrations of chlorogenic acid (compound 3) are apparent. In relation to leaf age (Fig. 4) the comparatively high cOntent of chlorogenic acid and of isochlorogenic acid (compound 7) in very young leaves shows a sharp decrease on leaf expansion (leaves at node 5 to 7). It is only peak 12, a mixture of apigenin-7-rutinoside and a yet unknown flavonoid, which increases slightly with increasing leaf age. With anthocyanins (Fig. 5) leaf cell extension seems to be a critical period. All four anthocyan ins are found in the small leaves at nodes 4 to 6. The cyanidin diglucosides Z. PJlanzenphysial. Bd. 97. S. 241-248. 1980.
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rei. abs.l cm 2 • chlorogenic acid (3) o isochlorogenic acid (?) • component 12
4
5
6 7 8 9 node from apex
10
11
Fig. 4: Amount of leaf phenolics per cm 2 leaf area in relation to leaf age. Component 12 = a mixture of apigenin-7-rutinoside and an unknown flavonoid.
5
rei. abs.l cm 2
4
5 6 node from apex
7
Fig. 5: Amount of anthocyan ins per cm 2 leaf area in relation to leaf age. Compounds 1-4 all are cyanidin derivatives.
(1 and 2) are the first to disappear and only traces of compounds 3 and 4 were found in the expanding leaves at node no. 7. Anthocyanins could not be detected in fully expanded leaves (from node 8 onwards). From our findings for all compounds investigated we can conclude that the period of leaf expansion seen at nodes no 7 to 8 is rather a critical stage. By this time anthocyanins have disappeared and concentration of phenolic depsides has fallen to a comparatively low level, but on the other hand a sudden increase occurs in the leaf wax compound lupeol. Information about the physiological significance of phenolics and triterpenes is scarce. If, however it is assumed that they have a general protective function, the simultaneous drop in concentration of the phenolic compounds and the rise in that of lupeol would seem to suggest some physiological interaction. A similar interdependence of triterpenes with anthocyanins has been found before. In Andromeda polilolia leaves, for instance, the parasite Exobasidium vaccinii causes a Z. Pflanzenphysiol. Bd. 97. S. 241-248.1980.
Investigations on Baya species. IV.
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decrease in the triterpene level and this is accompanied by the synthesis of two new anthocyanins (TAMAS et a!., 1975). In the latter case it should be noticed that injury may also lead to increased anthocyanin synthesis (Bopp, 1959). More evidence is certainly needed before firm conclusions can be drawn. Acknowledgement We are indebted to Mrs. JUDITH KOERSELMAN-KooY and Miss JANNY VAN SCHAlK for their assistance and to the RMP company for use of the Chromatospac prep 100. Miss S. McNAB is thanked for helping to revise the English of this text.
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