Pacific Science (1994), vol. 48, no. 4: 458-463 © 1994 by University of Hawaii Press. All rights reserved Flavonoids and Condensed Tannins from Leaves of Hawaiian Vaccinium reticulatum and V. calycinum (Ericaceae)l 2 3 2 BRUCE A. BOHM • AND MOHAMMED R. KOUPAI-ABYAZANI ABSTRACT: The flavonoids and condensed tannins of Hawaiian Vaccinium reticulatum Smith and V calycinum Smith have been isolated and their struc­ tures determined. Flavonoids present in both species were quercetin, quercetin­ 3-0-glucoside, quercetin-3-0-galactoside, quercetin-3-0-methyl ether, isorham­ netin, and (-)-epicatechin. The condensed tannin contained procyanidin units with cis stereochemistry only. Extension and terminal units, and number­ average molecular weight of the polymer were determined. A large quantity of neocWorogenic acid (a caffeic acid derivative) was also detected. The phenolic compounds of V reticulatum from a population on Mauna Kea and two pop­ ulations near K11auea, both on the island of Hawai'i, and from one population of V calycinum on Kaua'i were qualitatively identical. The high degree of similarity supports the view that these species are closely related. It is suggested that the phenolic chemistry ofthe species may have been fixed in the progenitor of the Hawaiian Vaccinium. IN THE RECENTLY PUBLISHED flora of the Ha­ Vander Kloet (1990) stressed the need for waiian Islands, Vander Kloet (1990) sug­ detailed studies of the group so that the gested that the variation seen in Vaccinium significance of variation patterns may be on the Islands can best be accommodated assessed. As part of our ongoing study of by recognizing three species, V calycinum polyphenolic compounds as systematic mark­ Smith, V den tatum Smith, and V reticu­ ers of Island taxa, we undertook an ex­ latum Smith. Vander Kloet (1990) empha­ amination of the flavonoid chemistry of two sized calyx lobe development, leaf persis­ species of Hawaiian Vaccinium. The high de­ tence, and leaf blade size in his treatment and gree of chemical uniformity observed in the placed less emphasis on leaf margin, blade material studied to date is entirely in line color, and indumentum, characters that had with the suggestion that this group of species been used in earlier species delimitations represents a closely related phylogenetic unit. (Skottsberg 1927, 1937, 1944, Degener 1940). The results of that study are presented in this In a more recent paper, Vander Kloet (1993) paper. described seed and seedling characteristics, juvenile leaf features, and some aspects of the breeding biology of all three species. Homo­ MATERIALS AND METHODS geneous juvenile leaf features along with limited variation in seed size, shape, and Source ofPlant Material sculpture led to the suggestion that the group is "... rather homogeneous and of recent Leaf samples of Vaccinium reticulatum origin" (Vander Kloet 1993). amounting to about 10 g dry weight were re­ moved from individual plants from three sites on the island of Hawai'i: (I) ca. 100 m east of the Desolation Trail trailhead; (2) I Manuscript accepted 10 October 1993. 2Department of Botany, University of British Co­ west side of the road near the Volcano Ob­ lumbia, Vancouver, British Columbia V6T IZ4 Canada. servation Center; and (3) east flank ofMauna 3 Author for correspondence. Kea at ca. 3000 m elevation. Eleven plants 458 Flavonoids from Hawaiian Vaccinium-BoHM AND KOUPAI-ABYAZANI 459 were sampled at site 1, 10 at site 2, and four Lichrospher 100 RP-18 column (250 x 4 mm at site 3. Efforts were made to avoid collect­ i.d.) with monitoring at 280 nm. A linear ing nearest neighbors. Two specimens of V gradient of 1% aqueous acetic acid and calycinum Smith were collected on the north methanol was used for elution (Koupai­ shore of Kaua'i by K. Marr. Vouchers have Abyazani et al. 1993a,b). been deposited in UBC. 13C-NMR spectra were recorded in water: d6 -acetone (1 : 1 vol/vol) on a Varian XL Isolation and Identification ofFlavonoids (300 MHz) spectrometer. 1H-NMR analysis was carried out in d4-methanol on a Bruker Air-dried leaf samples were extracted WH-400 (400 MHz) spectrometer. Optical repeatedly with 80% aqueous methanol at rotations of polymers dissolved in methanol­ room temperature. The combined extract of water (1: 1 vol/vol) were measured in a each sample was then evaporated to dryness 10 mm path length cell at 20°C in a lasco and subjected to a combination of column 1710 spectrophotometer. and preparative thin-layer chromatography (TLC) according to described procedures (Wilkins and Bohm 1976, Gornall and Bohm RESULTS 1980). Flavonoid structures were established using standard ultraviolet (UV) (Mabry et al. Flavonoids 1970) and mass spectral (MS) methods (Mabry and Markham 1975, Markham Twenty-five individuals of V reticulatum, 1982). Sugar analysis was carried out using representing three populations, and two of V methods described by Ceska and Styles calycinum were subjected to 2D-TLC ana­ (1984) and Kartnig and Wegschaider (1971). lysis. The chromatograms exhibited identical It should be noted that detailed listings of patterns of flavonoids with only slight varia­ UV spectral maxima, MS peaks, and IH and tion in concentration (by visual inspection). 13C proton magnetic resonances (PMR) for Five flavonoids (UV absorbing) and a phe­ the compounds described have been omitted nylpropanoid (blue fluorescense) were ob­ to save space. In all cases, observed values served as the major spots on the chromato­ agreed with published data. grams. These compounds were isolated by column chromatography and purified by Isolation, Purification, and Analysis of means of preparative TLC. Initial compar­ Condensed Tannins isons of these compounds with standards us­ ing TLC indicated that two were flavonoid Dried leaves were blended with 75% aque­ monoglycosides and the other three were fla­ ous acetone containing 0.1 % ascorbic acid, vonoid aglycones. UV analysis showed that and the mixture was filtered. Acetone was the monoglycosides were 3-0-substituted removed by evaporation under reduced pres­ flavonols (Markham 1982). The glycosidic sure. The aqueous residue was then subjected components were shown to be glucose and to column chromatography using Sephadex galactose. The MS spectra of these com­ LH-20 according to the procedure described pounds showed an ion with m/z = 302, by Koupai-Abyazani and coworkers (1992). which is characteristic of quercetin, and ions with m/z = 153 and m/z = 137, which rep­ Characterization ofthe Polymer resent the A-ring and B-ring fragments of quercetin, respectively (Mabry and Mark­ The structure of the purified tannin was ham 1975). The two glycosides were, there­ determined using a hydrolytic procedure that fore, quercetin-3-0-glucoside (1) and querce­ employed phloroglucinol as the nucleophilic tin-3-0-galactoside (2). The UV data of the scavenger of the freed monomeric units. Re­ third compound revealed that it was also a 3­ sultant adducts were analyzed by high pres­ O-substituted flavonol (Markham 1982), but sure liquid chromatography (HPLC) using a acid hydrolysis resulted in unchanged com- 460 PACIFIC SCIENCE, Volume 48, October 1994 pound and no sugar. The color reaction of ular weight (MW) (Mn) of the polymer was the compound with diphenylboric acid etha­ estimated to be in the range of ca. 1160 nolamine complex was the same as for 3-0­ (assuming an average unit MW of 290) by substituted quercetin derivatives. The com­ integrating the signals near 73 ppm (C-3 of pound ran faster than quercetin in nonpolar PC units) and 67 ppm (C-3 ofterminal flavan­ solvent systems. The MS spectrum of this 3-01 units). This was in total agreement with compound showed an ion at m/z = 316, the results of HPLC analysis of the phloro­ which is consistent with a flavone having four glucinol adducts of the polymer (Koupai­ OH groups and one O-methyl group. Ions at Abyazani et al. 1993a,b). Acid hydrolysis of m/z = 301, which arises by loss ofthe methyl the polymer in the presence ofphloroglucinol group; m/z = 153, which arises from the yielded the extension units as flavan-3-01­ A-ring; and m/z = 137, which arises from phloroglucinol adducts and terminal units as the B-ring, are consistent with the behavior flavan-3-01s. Analysis of the hydrolysis prod­ of quercetin 3-0-methyl ether (3). The other ucts by RP-HPLC indicated the presence of two aglycones were identified as quercetin (4) (-)-epicatechin-4-phloroglucinol (7) and (-)­ and isorhamnetin (5) by comparison of their epicatechin (8). TLC, UV, and MS data with those of stan­ The ethyl acetate-soluble fraction, ob­ dards. No isorhamnetin glycosides were tained during the tannin extraction proce­ detected. dure, was examined by TLC on cellulose (6% The lH-NMR spectrum of the isolated aqueous acetic acid). Development of a deep phenylpropanoid indicated that it was a caf­ red spot after spraying the chromatogram feic acid derivative (Ibrahim and Barron with vanillin/HCl reagent indicated the 1989). Comparison of the MS data with presence of flavan-3-01s. Further analysis of those reported by Sakushima et al. (1985) this fraction by 2D-TLC and HPLC showed suggested that the compound is neo­ compound 8, (-)-epicatechin, to be present. chlorogenic acid (6). Identical results were obtained for all collec­ tions of Vaccinium. Condensed Tannins An aqueous acetone extraction of Vac­ cinium leaves yielded the purified polymer DISCUSSION after a combination of liquid partitioning The flavonoid profile of Vaccinium calyci­ and chromatography on Sephadex LH-20 col­ num and V reticulatum consists of quercetin, umn. The polymer had an E 1% value of 134 its 3-0-g1ucoside, 3-0-galactoside, 3-0-methyl at its absorption maximum near 278 urn, ether, 3'-O-methyl ether (isorhamnetin), and which is typical of a procyanidin (PC) poly­ (-)-epicatechin.