Biochemical Systematics and Ecology, Vol. 10, No. 1, pp. 49-53, 1982. 0305-1978/82/010049-05 S03.00/0 Printed in Great Britain. © 1982 Pergamon Press Ltd

Ecological Significance of the Chemistry of the Leaf Resin of Bytropappus rhinocerotis

PETER PROKSCH*, MARGARETA PROKSCH', PHILIP W. RUNDELt- and ELOY RODRIGUEZ* "Phytochemical Laboratory and

1- Department of Ecology and Evolutionary Biology, University of California, lrvine, CA 92717, USA

Key 11Nord Index - Elytropappusrhinocerotls; Inuleae; Asteraceae; leaf resins; methylated flavonoids; phenolic acids; biological activity; ecology.

Abstract - Elytropappus rhinocerotis (Asteraceae) is a dominant, fire-adapted species of renosterveld shrub communities in the Cape region of South Africa. The leaves are covered by a thick lipophilic resin comprising up to 20% of their dry weight. Approximately 80% of the leaf resin is composed of phenolic and acidic products with the methoxylated flavones cirsimaritin, hispidulin, eupafolin and the flavonol quercetin the main components. Seven benzoic and cinnamic acid derivatives are also present in minor amounts. About 20% of the resin consisted of aliphatic constituents. Bioassays indicated atlelopathic effects of the resin on the seed germination of Lacruca and Raphanus. The predominance of lipophilic flavonoids and phenolics suggests a dual role of repelling herbivores and reducing cuticular transpiration. Calorimetric measurements indicated no significant contribution of the non-volatile resin to the flammability of E. rhinocerotis.

Introduction The genus Elytropappus Cass. (Asteraceae - Tribe Inuleae) includes eight shrub species endemic to the Cape Province of South Africa [1,2]. Seven of these species are generally restricted within the boundaries of the Cape zone in the western part of the Cape Province, while E. rhinocerotis Less. extends from these winter rainfall areas eastward into summer rainfall areas. This gray, resinous shrub is the dominant element of the coastal and mountain renosterveld (dry shrub). These renosterveld communities show a number of parallels with those of the coastal sage and other shrubs in southern California.

Elytropappus rhinocerotis reproduces from seed in great numbers following natural fire outbreaks and then grows rapidly in dense pure stands which suppress the reproduction ot other species [3-5]. The growth pattern is such that it suggests the possibility of allelopathic interactions. The chief characteristic of chemical interest is the resinous nature of the leaves of the plant. It has been suggested that these resins are highly flammable and promote burning in stands of E. rhinocerotis [4] and thus favour its dominance.

In this paper we describe the secondary chemistry of the external leaf resin of E. rhino- cerotis and discuss its possible ecological roles and functions.

(Received 1 August 1981 )

49

Results and diecumdon The leaves of Elytropappus rhinocerotis secrete large amounts of ether-soluble resin (15-20% per dry wt) by glandular trichomes present on the surface. Up to 80% of this resin consists of phenolic and acidic material. The four major components of the phenolic fraction are the three methoxylated flavones: hispidulin (6-methoxy- apigenin); cirsimaritin (7-O-methylhispidulin); eupafolin (6-methoxyluteolin) and quercetin (Fig. 1 ). These compounds were identified by their UV, IH NMR and MS spectra and by comparison with published data [6-10]. Seven benzoic and cinnamic acid derivatives were shown to be present in minor amounts bv HPLC with p- hydroxybenzoic acid as the main component (Fig. 1).

Since E. rhinocerotis exhibits such a strong growth dominance in renosterveld communities, we conducted bioassay experiments to test the allelopathic potential of the phenolic resin from its leaves. Discs of filter paper, soaked with various amounts of resin, were moistened with water and used to study the germination of seeds of Lactuca sativa and Raphanus sativus. The seeds were analysed after 24 h and considered germinated with the first appearance of the radical (Fig. 2). The inhibitory effects of the resin were shown to be linear by calculating the regression slopes of resin concentrations germinated seeds (correlation coefficients: - 0.971 for Lactuca and - 0.949 for Raphanus seeds). Lactuca seeds were thus more

50 PETER PROKSCH, MARGARETAPROKSCH, PHILIPW RUNDELAND ELOY RODRIGUEZ

OH

EUPAFOLIN

I,-I

.,co-

HISPIDULIN

OH

y ' - ~ "OH OH OH

QUERCETIN

CH ,O'ICOOH I mm CH-CHCOOH Elytropoppus rhinocerotis

COOH 0CH~ OH ~COOH L ~ OH

COUMARIC ACID I~-')l y FERULIC ACID

OH BENZOIC p-HYDROXY-

ACID BENZOIC ACID COOH CH=CHCOOH

OH ~ ~ OCH3 OH ~ OH

PROTOCATECHUIC OCH3 SINAPIC ACID ACID OEH 3

VERATRIC ACID

FIG. 1. BRANCH WITH LEAVES (BLACK) OF ELYTROPAPPUS RHtNOCEROTIS AND STRUCTURES OF THE IDENTIFIED PHENOLIC CONSTITUENTS OF THE LEAF RESIN.

lOC

~ 40 E

'~ 2 o

I i I00 2~0 300

m~ Resin per Pefri Dish

FIG 2. INHIBITION OF SEED GERMINATION OF LACTUCA SALIVA AND RAPHANUS SArlVUS CAUSED BY INCREASING AMOUNTS OF LEAF RESIN FROM ELYI'ROPAPPUS RHINOCEROTIS

sensitive to the inhibitory effects of the resin than those of Raphanus. A concentration of 220 mg resin/8.5 cm petri dish with 20 seeds of Lactuca resulted in a 50% inhibition of germination whereas a similar inhibition of Raphanus seeds required approximately 310 mg resin. The slope of the regression grade for Lactuca was - 0.2233 and remarkably steeper than the slope of Raphanus at - 0.1578. The seeds of Lactuca and Raphanus that

germinated on filter discs with resin concentrations of approximately 80 mg and more were inhibited in their radicle development compared to the ones that were treated with smaller amounts of resin. Whereas the latter had a radicle length of about 1 cm and showed the development of root hairs, the radicle length of the inhibited seeds was about 0.5 cm and less and no root hairs were apparent. Such inhibition of radicle development through chemical effects might be highly significant ecologically, since fine root development is an important

CHEMISTRY OF THE LEAF RESIN OF ELYTROPAPPUS RHINOCEROTIS 51

aspect of adaptation in low phosphorus soils [11]. The high amounts of resin present on the leaf surface of E. rhinocerods indicate that inhibition of seed germination and/or radicle development is due to accumulation of resin compounds in the soil, beneath these plants, either by rainwash or by litter. Phenolic compounds have often been shown to exhibit allelopathic effects. The growth of Adenostoma fasciculatum H. 8 A. (Rosaceae), the dominant shrub of the Mediterranean-like Californian chaparral community, has been assigned to phenolic leaf constituents similar to those present in the resin of E. rhinocerotis [12]. The concentrations of phytotoxins from A. fascicula~um necessary to exhibit inhibitory effects on other species have, however, not been determined.

Despite the early suggestions that the resin of E. rhinocerotis might contribute to the well-known flammability of the species [4], our analysis suggests that there is little basis for this hypothesis. The dominant phenolic and acidic materials we have isolated from the resins have very low volatility and thus would do little to increase the ignitability of Elytropappus. The energy content of these compounds is also quite low. On a dry wt basis, the energy content of young branches overall is 21.81 k J/g, while the energy content of the resin alone is 2.78 kJ/g branch, resulting in an energy content of 13.89 kJ/g resin. Thus, we attribute the high flammability of those shrubs to the fine branch structure and dense growth rather than to the non-volatile resinous nature of any chemical compound studied.

The chemical composition and quantity of the resin of E. rhinocerotis closely resemble the lipophilic resin of the leaves of the desert shrub Larrea tridentata Sesse 8 Moc. ex DC Coville (Zygophyllaceae) [13]. R hoades and Cates showed in their studies that the phenolic constituents of the resin of Larrea are composed of methoxylated flavonoids and phenolics. The resin exhibits anti- herbivore properties by binding to proteins and thereby acting as tannin-like compounds [14-16].

It is very likely that the resin of E. rhinocerotis might also act in much the same manner as the defensive compounds of Larrea. Besides the well- known enzyme inhibition caused by many flavonoids [17], it has been suggested that increasing the lipophilic properties of such compounds which contain methoxyl groups should result in substances that are less likely to be detoxified by animals [18]. A range of methoxylated flavonoids, including cirsimaritin and eupafolin, have been shown to be cytotoxic against tumor cells [19].

It has also been suggested that flavonoids may act as photoprotective compounds against UV radiation [ 17, 20]. A UV spectrum of the crude resin of E. rhinocerods shows a strong absorption from approximately 370 nm to lower wavelength ranges and is probably due to the flavonoid aglycones and hydroxy-aromatic acids. Therefore, the possible role of the secreted flavonoids as a UV radiation shield seems plausible.

The significance of cuticular waxes coating the aerial parts of a plant for reduction of transpiration iswell established [21-25]. Since the resin is mainly composed of lipophilic material and comprises up to 20% of the dry wt, another possible role is the reduction of water toss by cuticular transpiration. Other adaptations (morphological) of E. rhinocerods to drought conditions are reduction of leaf area and presence of long non-glandular trichomes that cover the leaf surfaces.

From our studies, it is apparent that the resin of E. rhinocerotis exhibits a wide range of activities which may contribute to the ecological success of this species in the renosterveld communities of South Africa. Similar compounds are also present in related desert dominants such as Flourensia cemua, Encelia farinosa (Asteraceae) and Larrea tfidentata in North America and may play an important role in supporting the dominant nature of these taxa [26].

Experimental Isolation and identification of resin components. Elytro- pappus rhinocerotis Less. was collected in December 1980 in coastal renosterveld north of Cape Town, South Africa. A voucher specimen is on file at the UCI herbarium, No. MSB- 18732. The external resin was extracted by dipping leafy branches into Et20 for 1 min (yield 15-20% per dry wt). The resin was partitioned between Et20 and 5% NaOH to determine the amount of phenolic and acidic compounds (80% of the resin) vs the amount of apolar primarily aliphatic components (20% of the resin). UV spectroscopy and TLC of the resin indicated the presence of flavonoids and phenolic acids. The four major flavonoids were isolated by preparative TLC on polyamide plates (solvent system: benzene-methyl-ethyl ketone-methanol, 60:26:14), purified by column chromatography on Sephadex LH-20 (solvent methanol) and identified by spectroscopical means. Spectra were recorded on the following instruments: UV spectrophotometer Beckmann model 25; 1H-NMR Varian EM 390 (spectra were recorded at 90 MHz in CCI 4 with TMS as internal standard); Finnigan Quadrupole MS 4000 GC-MS with Tektronix 4010-1 data system and Data General Nova 3 (spectra are given by heated probe and 70 ev).

Cirsimaritin (7-O-methylhispidulin). R fO.72, color under UV, purple; color with NH3, yellow. UV ~'max MeOH nm: 276, 335; NaOMe-MeOH. 274, 389; AICI3-MeOH: 292,303, 364; AICI~- HCI-MeOH: 292, 303, 358; NaOAc-MeOH: 277, 390; NaOAc- H3BO4-MeOH: 277, 335. 1H NMR of trimethylsilated com- pound: 6 3.67 (3H, s, C6-OMe), 3.87 (3H, s, CT-OMe), 6.25

52 PETER PROKSCH, MARGARETA PROKSCH, PHILIP W. RUNDEL AND ELOY RODRIGUEZ

(1H, s, C3), 6.45 (1H, s, Ca), 6.77 (2H, d, J= 9Hz, C3,,5,), 7.67 (2H, d, J = 9Hz, C2, e.). The signal at d 3.87 shifted to 3.3 when the spectrum was recorded in CeD e. MS m/z (rel. int.): 314 (M+,100), 299 (M+-15, 90), 285 (22), 271 (M + -43, 40), 181 (A1- 15, 25), 153(m/z181 -28,32), 121 (B 2, 15), 119(25), 118(B1, 20).

Hisp/dulin (6-methoxyapigenin). Rf 0.5, color under UV, purple; color with NH 3, yellow: UV ~.rr~x MeOH nm: 276, 335; NaOMe-MeOH: 278, 325, 394; AICI3-MeOH: 290, 304, 365; AICI3-HCI-MeOH: 290, 304, 359; NaOAc-MeOH: 276, 310, 385; NaOAc-H3BO4-MeOH: 278, 340, IH NMR of trimethyl- silated compound: (J 3.70 (3H, s, Ce-OMe), 6.25 (1H, s, C3), 6.45 (1H, s, Cs), 6.77 (2H, d, J=9Hz, C3,.5,), 7.67 (2H, d, J=9Hz, C2,6,). MS m/z (rel. int.): 300 (M +, 100), 285 ( M + - 15, 75), 282 ( M + - 18, 62), 257 (M+-43 , 95), 167 (A 1 - 15, 30), 153 (22), 139 (m/z 167-28, 65), 129 (35), 124 (10), 121 (B 2, 32), 119 (65), 118 (B~, 42).

Eupafolin (6-methoxyluteolin). Rf 0.2, color under UV, purple; color with NH3, yellow. UV~max MeOH nm: 257, 275, 345; NaOMe-MeOH: 270, 330, 406; AICI3-MeOH: 275, 302, 422; AICI3-HCI-MeOH: 295, 360; NaOAc-MeOH: 273, 330, 392; NaOAc-H3BO4-MeOH: 268, 365; 428 sh tH NMR of trimethylsilated compound: (~ 3.70 (3H, s, Ce-OMe), 6.20 (1 H, s, C3), 6.45 (1H, s, C8), 6.80 (1H, d, J= 9Hz, C5,), 7.20 (1H, d, J=2Hz, C2,), 7.25 (1H, dd, J = 9 and 2Hz, C6,). MS m/z (rel. Int.): 316 (M +, 100), 301 (M + -15, 65), 300 (25), 298 (M + -18, 55), 288 (M + -28, 25), 273 (m/z301 -28, 75), 167 (A 1 - 15, 25), 166 (35), 153 (10), 139 (m/e 167-28, 55), 137 ( B 2, 30), 135 (67), 135 (52), 134 (B1,50), 129 (20), 124 (35), 123 (28), 111 (25).

Quercetin. RfO.06, color under UV, yellow; color with NH 3, yellow. The spectroscopical properties were identical with those of an authentic sample. Protocatechuic, p-hydroxy- benzoic, benzoic, p-coumaric, veratric, ferutic and sinapic acid were identified by HPLC. The external ether extract was taken to dryness, redissolved with absolute methanol, filtered to remove precipitating lipids and injected into a Waters HPLC machine. Identification was achieved by co-chromatography with known standards [27].

Bioassay. The crude resin was dissolved with methanol and filtered to remove precipitating lipids. Germination experi- ments were conducted with seeds of Lactuca sativa and Raphanus sativus on discs of filter paper placed in petri dishes first soaked with 2 ml methanol containing various concentra- tions of the soluble resin fraction and then dried. The applied concentrations of resin per petri dish were 330, 165, 82.5, 33 and 3.3 mg. The filter paper in control dishes was soaked with 2 ml methanol. After complete evaporation of the solvent, 20 seeds of Lactuca or 15 seeds of Raphanus respectively were placed in each petri dish. The filter papers were then moistened with water. The Lactuca seeds were given a light/dark rhythm of 10/14 h at room temperature and the Raphanus seeds were grown in the dark. After 24 h the germination of the seeds was recorded.

Calorimetric measurements. Replicate energy measure- ments were made for unextracted and extracted branch tips using a Paar semimicro bomb calorimeter. All values were calibrated to standard benzoic acid controls and expressed as per cent of oven dry wt.

Acknowledgement=- We thank NSF (DEB 7912204) and the Fulbright Commission for financial support, Eugene Moll

for providing us with ample collections of Elytropappus rhinocerotis for our studies and Gall Baker for laboratory help.

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