XANTHOGALENOL AND 4'-O-METHYLXANTHOHUMOL CONTENT IN SOME AMERICAN HUMULUS

The resins produced by either lupulin or leaf glands of more than 41 genotypes of botanical varieties of Humulus lupulus L. collected from the wild in 9 mid-western and western American states were analyzed for the presence of prenylated flavonoids. Xanthogalenol and other 4′-O-methylchalcones are fixed in H. lupulus var. lupuloides E. Small and in H. l. var. pubescens E. Small. Most of the H. l. var. neomexicanus Nelson and Cockerell collected east of the Rocky Mountains contained 4′-O-methylchalcones, while most specimens of H. l. var. neomexicanus from west of the Rocky Mountains did not. In 2 localities, the Kaibab Plateau, Arizona, and Phantom Canyon, Colorado, individuals of H. l. var. neomexicanus containing 4′-O-methylchalcones and those without, were sympatric. We have expanded the results of Stevens et al. (2000) who reported that European hops (Humulus lupulus var. lupulus L.), like most H. l. var. neomexicanus from west of the Rocky Mountains, lack 4′-O-methylchalcones, whereas Japanese hops (H. l. var. cordifolius (Miquel) Maximowicz) contain 4′-O-methylchalcones, like the majority of North American hop samples east of the Rocky Mountains. This geographic disjunction and polymorphism in the occurrence of 4′-O-methylchalcones suggests a complex evolutionary history in Humulus lupulus. Further morphological and molecular evaluations of this wild collected germplasm are in progress. INTRODUCTION The species Humulus lupulus L. in North America currently has 3 recognized varieties (Small 1978, 1997). Stevens et al. (2000) recognized a geographic dichotomy of phenotypes in Native American hops regarding their prenylflavonoid composition. They observed that, while all Humulus lupulus varieties contain xanthohumol, the presence of xanthogalenol (3′-prenyl-4′-O-methylchalconaringenin), 4′-O-methylxanthohumol, and 4′, 6′-di-O-methylchalconaringenin was limited to North American hops from the Missouri and Mississippi River drainages (H. l. var. lupuloides E. Small, H. l. var. neomexicanus Nelson and Cockerell east of the Rocky Mountains, and H. l. var. pubescens E. Small) and to the Japanese hops (H. l. var. cordifolius (Miquel) Maximowicz). Neither Southwestern American hops (H. l. var. neomexicanus west of the Rocky Mountains) nor European hops (Humulus lupulus var. lupulus L.) produced 4′-O-methylchalcones (Stevens et al., 2000). Small (1978) considered the Native American hop H. l. var. lupuloides to be more closely related to European hop (H. l. var. lupulus) than to the other two North American botanical varieties. Hampton et al. (2002), however, found that Proc. I IS on Humulus Eds. K.E. Hummer and J.A. Henning Acta Hort. 668, ISHS 2005 230 unlike European hops, all 109 H. l. var. lupuloides cone samples they analyzed produced 4′-O-methylchalcones, in agreement with findings of Stevens et al. (2000). The objectives of our research were to analyze additional samples of Native American hops, including H. l. var. lupuloides, H. l. var. pubescens, and H. l. var. neomexicanus, particularly those collected in watersheds on either side of the Rocky Mountains, and from isolated mountains in New Mexico and Arizona; and to examine the pattern of geographic distribution of 4′-O-methylchalcones in American sub-specific taxa. MATERIALS AND METHODS Hop cones were obtained from hop plants collected from the wild in 9 midwestern and western American states. One hop cone, or about 0.1 g hops, was immersed in 100 ml of MeOH-HCOOH (99:1 by vol.). The cone was extracted for 2 hours, with occasional agitation during the first hour. For the leaf gland extracts, about 30 glands were removed from the abaxial side of young leaves with a solid needle at 7x (stereomicroscope), transferred to a 0.25 ml sample vial, and extracted with MeOHHCOOH (99:1, 0.1 ml) at room temp for 2 h with occasional agitation during the first hour. Hop extracts were analyzed for prenyl flavonoids using liquid chromatographytandem mass spectrometry (LC-MS) as described by Stevens et al. (1999) and Stevens et al. (2000). In brief, an aliquot of each hop extract was separated on a 250 x 4.6 mm Synergi Hydro-RP column (Phenomenex, Torrance, CA) using a gradient of 40 – 100% acetonitrile in 0.5% formic acid (aq) over 15 minutes, followed by 7 min at 100% MeCN, at a flow of 0.8 ml/min. The effluent from the HPLC was introduced to a PE-Sciex API III+ triple quadrupole mass spectrometer via an atmospheric pressure chemical ionization interface. Prenylflavonoids were detected in the multiple reaction monitoring mode: (1) xanthohumol and xanthogalenol, m/z 355.2 to 179.0; (2) desmethylxanthohumol, m/z 341.2 to 165.0; (3) 4’-O-methylxanthohumol, m/z 369.2 to 193.0; and (4) 4’, 6’-di-Omethylchalconaringenin, m/z 301.2 to 181.0. Taxonomy and species identification followed Small (1997). RESULTS AND DISCUSSION The presence and absence of 4′-O-methylchalcones of 41 genotypes of North American H. lupulus (Tables 1 and 2) and the geographical distribution of these samples are reported. Xanthogalenol and other 4′-O-methylchalcones were fixed in H. l. var. lupuloides and in H. l. var. pubescens. H. l. var. neomexicanus was polymorphic for the presence of the 4′-O-methylchalcones. The majority of specimens east of the Rocky Mountains, including H. l. var. neomexicanus, had xanthogalenol and 4′-Omethylxanthohumol while most of the specimens west of the Rocky Mountains did not. In 2 localities, the Kaibab Plateau, Arizona, and Phantom Canyon, Colorado, individuals of H. l. var. neomexicanus containing 4′-O-methylchalcones and those without, were sympatric. The degree of polymorphism and heritability for 4′-O-methyl substitutions in populations of H. l. var. neomexicanus is unknown. Further sampling within populations and controlled crosses are planned. Most individuals of H. l. var. neomexicanus from the western United States and all native European hops (Stevens et al., 2000) lack any 4′-Omethyl substitutions. H. l. var. lupuloides and H. l. var. pubescens, share the presence of 4′-O-methyl chalcones with H. l. var. cordifolius from Japan (Stevens et al., 2000). This geographic disjunction and polymorphism in the occurrence of 4′-O-methylchalcones suggests a complex evolutionary history in Humulus lupulus and possible introgression among North American taxa. Molecular markers will be applied to further examine the relationships of this species complex. Based on material of H. l. var. neomexicanus that we collected throughout the western United States, much variability was observed for other traits besides the chemical polymorphism mentioned above. While Small (1978, 1997) describes this variety as usually having well defined, 5-lobed leaves, our observations suggest this variety exhibits a wide range of leaf and cone morphology (data not shown). This suggests that H. l. var.