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c 2001) Journal of Chemical Ecology, Vol. 27, No. 12, December 2001 (°
ETHYL m-DIGALLATE FROM RED MAPLE, Acer rubrum L., AS THE MAJOR RESISTANCE FACTOR TO FOREST TENT CATERPILLAR, Malacosoma disstria Hbn.
MAMDOUH M. ABOU-ZAID,1, * BLAIR V. HELSON,1 CONSTANCE NOZZOLILLO,2 and J. THOR ARNASON2 1 Natural
Resources Canada, Canadian Forest Service Great Lakes Forestry Centre 1219 Queen St. East, Sault Ste. Marie, Ontario, Canada P6A 2E5 2 Ottawa-Carleton Institute of Biology University of Ottawa Ottawa, Ontario, Canada K1N 6N5 (Received March 10, 2001; accepted August 1, 2001)
Abstract—An ethanolic extract of red maple (Acer rubrum L.) leaves (RME) applied to trembling aspen (Populus tremuloides Michx.) leaves reduced feeding in choice test assays with forest tent caterpillar larvae (Malacosoma disstria Hbn.) (FTC), whereas a trembling aspen foliage extract, similarly applied, stimulated feeding. Compounds isolated from the RME were gallic acid, methyl gallate, ethyl gallate, m-digallate, ethyl m-digallate, 1-O-galloyl-β-D-glucose, 1-O-galloyl-α-L-rhamnose, kaempferol 3-O-β-D-glucoside, kaempferol 3-O-βD-galactoside, kaempferol 3-O-β-L-rhamnoside, kaempferol-3-O-rhamnoglucoside, quercetin 3-O-β-D-glucoside, quercetin 3-O-β-L-rhamnoside and quercetin 3-O-rhamnoglucoside, (−)-epicatechin, (+)-catechin and ellagic acid. All of the gallates, (−)-epicatechin, and kaempferol 3-O-β-L-rhamnoside deterred feeding on trembling aspen leaf disks when applied at 0.28 mg/cm2 . The two digallates deterred feeding by 90% and were the most effective. HPLC analysis indicated that ethyl m-digallate is present in amounts 10–100 × higher in RME (∼2.5– 250 mg/g) than any other compound. Thus, ethyl m-digallate appears to be the major compound protecting red maple from feeding by FTC, with a minor contribution from other gallates. Key Words—Acer rubrum L., red maple, A. saccharum L., sugar maple, Aceraceae, Populus tremuloides Michx., trembling aspen, Salicaceae, Malacosoma disstria Hubner, forest tent caterpillar, Lepidoptera, Lasiocampidae, feeding deterrence, phenolics, gallates, flavonols.
∗ To
whom correspondence should be addressed. E-mail:
[email protected]
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ABOU-ZAID, HELSON, NOZZOLILLO, AND ARNASON INTRODUCTION
Hardwood forests are endangered by severe defoliation caused by insects. The forest tent caterpillar (FTC) (Malacosoma disstria Hubner) is a major pest of Canadian deciduous forests feeding mainly on poplar (Populus spp.), particularly trembling aspen (P. tremuloides Michx.). It also defoliates other species, including sugar maple (Acer saccharum Marsh) (Howse, 1995; Nicol et al., 1997). Red maple (A. rubrum L.), another prominent maple occupying a similar range (Farrar, 1995), is seldom attacked by FTC. Beavers (Castor canadensis) also have been reported to avoid red maple trees (M¨uller-Schwarze et al., 1994). Except for a 13% higher content of polyphenols in red maple (Ricklefs and Matthews, 1982), leaves do not exhibit major differences in characteristics, e.g., toughness, sugar content, water content, or nitrogen content, from those of sugar maple. Among polyphenols, tannins are considered as the major defense compounds in tree leaves, but since red and sugar maple leaves contain similar amounts of these complex molecules (Bate-Smith, 1978), some other constituent(s) must provide red maple leaves with a deterrent to feeding by FTC. In the present study, the constituents of an aqueous ethanolic extract of red maple leaves were isolated, identified, and then tested in leaf disk feeding assays in order to determine the phytochemical basis of insect resistance in red maple leaves.
METHODS AND MATERIALS
Plant Material. Red maple leaves were collected in June 1992 from 10 mature trees in Sault Ste. Marie, Ontario, Canada (lat. 46.34N, long. 84.17W). Leaves of trembling aspen were collected locally (Sault Ste. Marie) or grown in the greenhouse as needed for feeding tests. Pressed voucher specimens are deposited in the Canadian Forest Service-Sault Ste. Marie herbarium as Acer rubrum L. (CFSSSM #s 1001–1010), family Aceraceae and trembling aspen (Populus tremuloides Michx.) (CFS-SSM #s 1255–1265), family Salicaceae. Extraction. Fresh red maple leaves (2 kg fresh wt) were extracted at room temperature. First, leaves were steeped for 24 hr in 100% EtOH (1 g fresh wt/10 ml solvent), followed by chopping in a commercial Waring blender, and decanting the solvent. Next, the chopped residue was steeped for an additional 24 hr in 50% aqueous EtOH (10 g fresh wt/100 ml solvent). The combined ethanolic extracts (RME) were evaporated under reduced pressure until most or all of the EtOH had been removed. The residue was freeze-dried to obtain 242 g of crude extract (RME). Thus, from each gram fresh weight of leaves, 121 mg of RME was obtained. Fresh aspen leaf material (2 kg fresh wt) was similarly extracted to yield 204 g residue. Fractionation. RME (100 g) was adsorbed onto 200 g of polyvinylpolypyrrolidone (PVPP) powder (Sigma Chem. Co., St. Louis, Missouri) preconditioned by
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soaking in distilled water for 24 hr and packed in a Buchner funnel (4 liters). Elution was carried out at a slow rate using 5 liters of water followed by 5 liters aliquots of increasing concentrations (20, 50, 70, 100%) of ethanol. All five fractions were tested for antifeedant activity (data not shown) with maximum activity found in the fraction eluting with 50% ethanol, henceforth referred to as the phenolic fraction (RMP). The active fraction was concentrated under vacuum and chromatographed on Whatman No.1 chromatography paper (PC) using either BAW (n-butanol–acetic acid–water, 4:1:5, upper phase), water, or acetic acid–water (15:85) to isolate the pure compounds. Further fractionation was carried out on a PVPP column (7 × 120 cm ID.) using the following solvent systems: (i) CH2 Cl2 – EtOH–MeCOEt–Me2 CO (1:1:1:1), (ii) EtOH–MeCOEt–Me2 CO–H2 O (1:1:1:1), and (iii) EtOH–H2 O (1:1). Purification was achieved on a Chemco low-prep pump (model 9 1-M-8R), and fractions were tested for purity by HPLC. HPLC. A Waters Delta Prep 4000 liquid chromatograph was used equipped with a computer and Millennium 2010 software, an autoscan photodiode array detector (Waters 996), and a Waters Nova-Pak C18 reverse-phase analytical column ˚ 3.9 × 150 mm ID). A modified gradient chromatographic technique (4 µm, 60 A, (van Sumere et al., 1993) was used at room temperature: solvent A = MeOH; B = 5% aq. HCOOH; and a flow rate of 0.9 ml/min. Two fixed detection wavelengths were used, 280 nm and 350 nm, and resolved peaks were scanned from 250 to 400 nm by the photodiode array detector. Identification of Isolated Compounds. UV spectra were recorded on a UV-Vis Beckman DU series 640 spectrophotometer. 1 H NMR and 13 C NMR spectra were recorded on a Bruker AMX-500 spectrometer, at 500 MHz and 125 MHz, respectively; samples were dissolved in DMSO-d6 with TMS as an internal standard. Structures of purified compounds were determined by comparison with authentic samples where available using standard methods (Harborne, 1994; Markham, 1982): acid hydrolysis in 2 M and 0.1 M HCl (mild hydrolysis) at 100◦ C for 60 min, enzymatic hydrolysis with β-glucosidase (Sigma) using an acetate buffer (pH 5), hydrogen peroxide oxidation, UV spectroscopy, 1 H NMR, 13 C NMR, and FABmass spectroscopy (positive and negative). The glycosides and aglycones obtained from hydrolysis of isolated compounds were identified by cochromatography with authentic samples (Aapin and Extrasynthese) using PC, TLC, and HPLC. Sugars released by hydrolysis were identified by PC and TLC using standards. Standard gallic acid and gallate derivatives were kindly provided by Profs. G. Gross, Universt¨at Ulm, Ulm, Germany, and T. Yoshida, Okayama University, Okayama, Japan. Quantitative and Qualitative Analysis. A dilute solution (10 mg/ml) of RME or RMP was filtered and 10 µl was injected onto an HPLC column (van Sumere et al., 1993) with and without spiking with standards. Peaks were identified on the basis of retention times and UV spectra. Peak heights, measured at 280 nm, were converted to milligrams per milliliter using conversion factors obtained
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for standards (Slacanin et al., 1991). Such HPLC analyses were performed in triplicate. Insect Material. Egg bands of forest tent caterpillar, Malacosoma disstria Hubner, Lepidoptera, Lasiocampidae (FTC) were collected from trembling aspen trees from Cochrane and Thunder Bay, Ontario, Canada. Eggs were stored at 2◦ C for at least 13 weeks. Larvae were reared to the required stage on artificial diet (Addy, 1969; Singh and Moore, 1985) in environmental chambers (21◦ C, 80% relative humidity, and 16L:8D). Leaf Disk Assays. Leaf disks (2-cm diam.) were cut with a #13 cork borer from freshly collected leaves and placed into a 9-cm-diam. plastic Petri plate (two disks of each species in alternating positions) to compare feeding on red maple and trembling aspen leaves. The disks were pinned in place through small holes in the bottom of the plate. Five newly molted fourth-instar larvae were placed into each of seven replicate plates. After 24 hr, the leaf disks were photocopied and the percentage of each disk remaining was determined with an Image Analyser (Artek, Image Editor and Video Memory, model 940). The antifeedant index (AI) for each plate was calculated as %AI = (C − T )/(C + T ) × 100 where C is the percentage of two control disks eaten, and T is the percentage of two treated disks eaten (Blaney et al., 1994). A second choice leaf disk assay method was used to determine the feeding deterrency of red maple and aspen leaf extracts applied to aspen leaf disks in side by side comparisons. Four, 1.5-cm-diam. disks from greenhouse-grown trembling aspen leaves were placed into a 9-cm-diam. plastic Petri plate and pinned in place. The upper surfaces of two opposite disks were each treated with four applications of 12.5 µl of 5% solutions (acetone–water, 80:20) of the extracts for a total of 2.5 mg of each crude extract. The other two disks were treated with solvent only. Five fourth-instar larvae were placed in each of 12 replicate plates for each extract. The percentage of foliage eaten was estimated visually until 35–65% of the disks in a dish were consumed. The AI was then calculated as above. The RMP extract (13 replicate plates) was also tested separately in the same fashion. Feeding deterrency of individual compounds found in the RME extract, either available commercially or purified from the extract, was determined with the second leaf disk assay method. An equal mass (0.5 mg) of each compound was similarly applied to individual aspen leaf disks. Six to 29 replicate plates in at least duplicate tests were used for each compound. Methyl gallate was tested in 116 replicate plates as a regular standard. Statistical analysis using appropriate parametric or nonparametric procedures was undertaken with GraphPAD InSTAT software (GraphPad, San Diego, California) or SigmaStat Statistical Software, Version 2 (SPSS Inc., Chicago, Illinois).
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TABLE 1. HPLC ANALYSIS OF CRUDE RED MAPLE LEAF EXTRACT (RME) AND PHENOLIC FRACTION FROM RED MAPLE LEAF EXTRACT (RMP) ELUTED FROM PVPP WITH 50% ETHANOL–WATER mg/g dried extract Compound 1-O-galloyl-β-D-glucosea Gallic acida 1-O-galloyl-α-L-rhamnosea Methyl gallatea Ethyl gallatea m-Digallatea Ethyl m-digallatea Quercetin 3-O-β-D-glucosidea Quercetin 3-O-rutinosidea Kaempferol 3-O-β-D-galactosidea Quercetin 3-O-β-L-rhamnosidea Kaempferol 3-O-β-D-glucosidea Kaempferol-3-O-rutinosidea Kaempferol 3-O-β-L-rhamnosidea Kaempferol (galloyl?)rutinosideb Kaempferol (galloyl?)rhamnnosideb
RME
RMP
10.3 24.5 30.2 2.5 2.0 24.5 248.5 6.1 5.3 6.2 15.0 4.1 4.1 4.1 28.0 3.0
5.4 22.9 16.1 2.5 3.0 58.6 302.5 12.2 5.3 4.1 28.0 6.2 6.2 8.3 32.0 3.0
a Compounds isolated from red maple leaf extract and used in Table 3. b The tentatively identified acylated kaempferol rutinoside and rham-
noside were most probably hydrolysed to the free glycosides during the prolonged separation and isolation steps.
RESULTS AND DISCUSSION
Identification of Isolated Compounds. Seventeen compounds were isolated and identified from Acer rubrum L. including the 14 listed in Table 1 and an additional three, (−)-epicatechin, (+)-catechin and ellagic acid (listed in Table 3 below). Structures (Figure 1) were established using standard procedures as described in Methods and Materials (Harborne, 1994; Markham, 1982). UV spectra, 1 H NMR, 13 C NMR, and MS were identical with standards. Quantitative and Qualitative Analysis. The depside ethyl m-digallate, present at 248.5 mg/g is the predominant compound in both RME and RMP extracts (Table 1). Gallic acid is an important constituent of red maple leaf extracts (24.5 mg/g) as are 1-O-galloyl-β-D-glucose (10.3 mg/g), 1-O-galloyl-α-Lrhamnose (30.2 mg/g) and m-digallate (24.5 mg/g). By contrast, methyl gallate and ethyl gallate are present in RME in relatively low amounts (2.5 mg/g and 2 mg/g; respectively).
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2523 FIG. 1. Structures of some of the phenolic compounds that have been isolated from ethanolic extracts of red maple (Acer rubrum L.) leaves.
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The presence in maple leaves of gallic acid, its esters, and glycosides was previously examined by Haslam (1965), but the identification of 1-O-galloyl-αL-rhamnose is a recent discovery (Abou-Zaid and Nozzolillo, 1999). Bailey et al. (1986) isolated methyl gallate from methanolic extracts of silver maple leaves (A. saccharinum L.), but this constitutes the first report of its presence in red maple leaves (Table 1). Haddock et al. (1982) pointed out the significance of the depside as a hydrolyzable tannin component of red maple and related species, but this is the first report of its presence as a free compound. Ellagic acid, (+)-catechin, and (−)-epicatechin were not detectable in the HPLC traces, but small amounts of each were present in red maple extract. Several flavonol peaks also were present, of which seven were isolated and identified. Delendick (1990) identified the three quercetin glycosides, but only one of the four kaempferol glycosides. Only quercetin 3-O-β-L-rhamnoside was present in excess of 10 mg/g of extract (Table 1). However, there was an estimated 28 mg/g of the putative kaempferol galloyl rhamnoglucoside in leaf extracts that was most probably hydrolyzed during the lengthy isolation procedure and purified as kaempferol 3-O-rhamnoglucoside. Antifeedant Tests. Red maple leaf disks strongly deterred feeding by FTC larvae. In the choice test employed, 100% of aspen leaf disks were eaten in contrast to only 3.3 ± 6.9% of red maple leaf disks after 24 hr, giving an AI of 94.3% (N = 7). This result confirms the field observation by Nicol et al. (1997) that red maple is not a preferred host of FTC. RME applied to aspen leaf disks at 1.4 mg/cm2 deterred feeding, yielding an AI of 56.2% (Table 2). In contrast, feeding was stimulated by crude aspen leaf extract (Table 2). These findings suggest that the resistance of red maple to feeding by FTC larvae is due to specific phytochemicals present in the leaves of this species. Since the RMP fraction contains higher levels of phenolic compounds and had an TABLE 2. CHOICE BIOASSAYS SHOWING FEEDING RESPONSES CATERPILLAR LARVAE TO RED MAPLE LEAF EXTRACT (RME), ASPEN LEAF EXTRACT (AE), OR PHENOLIC FRACTION OF RED MAPLE EXTRACT (RMP)
OF FOREST TENT
Test Experiment 1 RME AE Experiment 2 RMP a+
mg/cm2 of extract applied
Replicates (N )
Antifeedant index (%)a
1.4 1.4
12 12
+56.2 ± 32.9s −54.4 ± 23.5s
1.4
13
+88.1 ± 16.3s
= antifeedant; − = phagostimulant. Wilcoxon’s one-tailed test (H1 ; µ > 0), differences from untreated (control) aspen leaf disks indicated as s = significant (P < 0.05) or ns = not significant, (P > 0.05).
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AI of 88.1% (Table 2), we suggest that these compounds might be responsible for the antifeedant activity of this fraction. Previously reported choice tests with six commercially available phenolic compounds present in red maple also demonstrated that gallic acid and methyl gallate were feeding deterrents at 0.28 mg/cm2 (Abou-Zaid et al., 2000b). When the full range of 14 phenolic compounds identified in red maple leaf extract was tested at 0.28 mg/cm2 in the present study, all the gallates (gallic acid, methyl gallate, ethyl gallate, m-digallate, ethyl m-digallate, 1-O-galloyl-β-D-glucose, and 1-O-galloyl-α-L-rhamnose) showed antifeedant activity (Table 3). However, ethyl m-digallate and m-digallate were clearly the most inhibitory to feeding, with AIs of about 90% (Table 3). Only ethyl m-digallate was the predominant compound in both RME and RMP. Neither compound is present in sugar maple leaves (AbouZaid et al., 2000a). The amount of ethyl m-digallate in RME and RMP applied to the leaf disks in Table 2 was 0.36 and 0.42 mg/cm2 , respectively, as compared to 0.28 mg/cm2 of the pure compound in Table 3. Only RMP inhibited feeding to the same extent as the digallate. Other compounds in RME may act as stimulants to reduce insect feeding deterrence since this extract inhibited feeding by only TABLE 3. CHOICE BIOASSAYS TO TEST FEEDING RESPONSES OF FOREST TENT CATERPILLAR LARVAE TO ASPEN LEAF DISKS TREATED WITH PHENOLIC COMPOUNDS ISOLATED FROM RED MAPLE (0.28 mg/cm2 OF LEAF DISK) Compound
µM
Antifeedant index (mean % ± SD)a
N
m-Digallate Ethyl m-digallate Methyl gallate 1-O-galloyl-α-L-rhamnose 1-O-galloyl-β-D-glucose (−)-Epicatechin Gallic acid Ethyl gallate Kaempferol 3-O-β-L-rhamnoside Quercetin 3-O-β-L-rhamnoside Ellagic acid Quercetin 3-O-rhamnoglucoside Quercetin 3-O-β-D-glucoside (+)-Catechin Kaempferol 3-O-β-D-glucoside Kaempferol-3-O-rhamnoglucoside
0.9 0.8 1.6 0.9 0.9 1.0 1.7 1.4 0.7 0.6 0.9 0.5 0.6 1.0 0.6 0.5
+90.4 ± 12.5s +89.2 ± 9.1s +52.1 ± 29.5s +43.7 ± 33.8s +40.5 ± 36.5s +35.7 ± 31.8s +33.7 ± 33.8s +33.4 ± 32.9s +29.2 ± 38.1s +13.6 ± 28.9ns +5.2 ± 23.3ns + 2.8 ± 23.2ns −6.8 ± 32.3ns −6.9 ± 29.0ns −15.8 ± 54.0ns −24.2 ± 29.1s
9 12 116b 8 10 14 20 9 10 11 18 29 18 10 6 10
a+
= antifeedant; − = phagostimulant. Wilcoxon’s one-tailed test (H1 ; µ > 0), differences from untreated (control) aspen leaf disks indicated as s = significant, (P < 0.05) or ns = not significant, (P > 0.05). b Used as a standard for all tests.
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56%. These include the flavonol glycosides that stimulated feeding in tests with the pure compounds (Table 3). A crude extract such as RME also contains free sugars, amino acids, and other soluble metabolites that are not present in RMP and may stimulate feeding. Further evidence of the potential importance of such metabolites to feeding activity is provided by the test with aspen leaf extract (Table 2), which stimulated feeding in contrast to the inhibitory effect of a similar amount of RME. Of the two building blocks of proanthocyanidins, (+)-catechin and (−)epicatechin, only the latter elicited inhibitory activity similar to gallic acid. Only kaempferol 3-O-β-L-rhamnoside showed an antifeedant effect of the six flavonols tested (Table 3). Expression of the concentrations of the compounds in molar amounts (Table 3) makes it possible for direct comparison of biological activity. For gallic acid and its esters, biological activity would be lower than that recorded on a milligram basis. By the same token, kaempferol 3-O-β-L-rhamnoside is a more effective antifeedant than the AI value of 29.2 would indicate. In conclusion, FTC are deterred by gallates. The five most active compounds in red maple all possess the galloyl moiety, suggesting that this structure may constitute the basis for the feeding deterrence of these compounds and that the functional groups attached to this basic structure may influence the level of deterrency. Of all the gallates, ethyl m-digallate has both the highest antifeedant activity and concentration in red maple, suggesting it is the major resistance factor. Acknowledgments—We acknowledge the financial support of the Integrated Forest Pest Management Green Plan Initiative and the Canadian Biotechnology Strategy Fund.
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BLANEY, W. M., SIMMONDS, M. S. J., LEY, S. V., ANDERSON, J. C., SMITH, S. C., and WOOD, A. 1994. Effect of azadirachtin-derived decalin (perhydronaphthalene) and dihydrofuranacetal (furo[2,3b]pyran) fragments on the feeding behavior of Spodoptera littoralis. Pest. Sci. 40:169–173. DELENDICK, T. J. 1990. A survey of foliar flavonoids in the Aceraceae. Mem. NY Bot. Gard. 54:1–129. FARRAR, J. L. 1995. Trees in Canada. Canadian Forest Service & Fitzhenry and Whiteside Ltd., Markham, Ontario, Canada. HADDOCK, E. A., GUPTA, R. K., AL-SHAFI, S. M. K., LAYDEN, K., HASLAM., E., and MAGNOLATO, D. 1982. The metabolism of gallic acid and hexahydroxydiphenic acid in plants A: Biogenetic and molecular taxonomic considerations. Phytochemistry 21:1049–1062. HARBORNE, J. B. 1994. The Flavonoids: Advances in Research Since 1986. Chapman and Hall, London. HASLAM, E. 1965. Galloyl esters in the Aceraceae. Phytochemistry 4:495–498. HOWSE, G. M. 1995. Forest Insect Pests in the Ontario Region. In J. A. Armstrong and W. G. H. Ives (eds.). Forest Insect Pests in Canada. Natural Resources Canada, Ottawa, Ontario, Canada. MARKHAM, K. R. 1982. Techniques of Flavonoid Identification, Academic Press, London. ¨ ¨ ¨ -SCHWARZE, D., SCHULTE, B. A., SUN, L., MULLER -SCHWARZE, A., and MULLER -SCHWARZE, MULLER C. 1994. Red maple (Acer rubrum) inhibits feeding by beaver (Castor canadensis). J. Chem. Ecol. 20:2021–2034. NICOL, R. W., ARNASON, J. T., HELSON, B. V., and ABOU-ZAID, M. M. 1997. Effect of host and non-host trees on the growth and development of the forest tent caterpillar, Malacosoma disstria Hubner (Lepidoptera: Lasiocampidae). Can. Entomol. 129:995–1003. RICKLEFS, R. E. and MATTHEWS, K. 1982. Chemical characteristics of the foliage of some deciduous trees in southeastern Ontario. Can. J. Bot. 60:2037–2045. SINGH, P. and MOORE, R. F. 1985. Handbook of Insect Rearing, Vol II. Elsevier Science Publishers, Amsterdam, The Netherlands, 369 pp. SLACANIN, I., MARSTON, A., HOSTETTMANN, K., DELABAYS, N., and DARABELLAY, C. 1991. Isolation and determination of flavonol glycosides from Epilobium species. J. Chromatogr. 557:391–394. VAN SUMERE, C., FACHE, P., CASTEELE, K. V., DE COOMAN, L., EVERAERT, E., DE LOOSE, R., and HUTSEBAUT, W. 1993. Improved extraction and reversed phase high performance liquid chromatographic separation of flavonoids and identification of Rosa cultivars. Phytochem. Anal. 4:279–292.