Humulus lupulus Cannabaceae

The resins derived from female flowers of Humulus lupulus L., known commonly as hops, provide the bitter and aromatic components so essential for the brewing of beer. According to Mabberley (1997), the genus consists of two species, H. lupulus of northern temperate regions and H. japonicus Sieb. & Zucc. of temperate eastern Asia. Owing to intense selection for flavor and yield characteristics, many cultivated varieties of H. lupulus have been developed. Although there is opinion to the contrary, J. F. Stevens et al. (2000), whose chemical study is discussed below, prefer the infraspecific taxa formally described by Small (1978). Thus, wild hops of Europe are considered to be H. lupulus var. lupulus, while North American hops are segregated into three varieties primarily on the basis of geography: H. lupulus var. lupuloides E. Small from central and eastern North America, H. lupulus var. pubescens E. Small from midwestern United States, and H. lupulus var. neomexi-canus Nelson and Cockerell of western North America. These are often referred to generally as "wild American hops." According to Stevens et al. (2000), the wild hops of "Japan and perhaps eastern mainland Asia" can be referred to as H. lupulus var. cordifolius (Miquel) Maximowicz.

In addition to the bitter acids and essential oils, the flowers of hops offer a rich array of polyphenolic compounds, primarily chalcones and their accompanying flavanones, many of which are prenylated derivatives (Stevens et al., 1997, 1999a, b). The most prominent flavonoid in all plants studied was xanthohumol [342] (3'-prenyl-6'-0-methylchalconaringenin; chalconaringenin is 2',4',6',4-tetrahydroxychalcone) (see Fig. 4.11 for structures 342-346). Several additional chalcones—variously adorned with 0-methyl and/or C-prenyl functions—were also encountered, along with their respective flavanones. Three new compounds were described in the Stevens et al.

Fig. 4.11 Compounds 342-346, polyphenolic compounds from Humulus lupulus, hops

Fig. 4.11 Compounds 342-346, polyphenolic compounds from Humulus lupulus, hops

(2000) paper: xanthogalenol [343] (the common name derives from the cultivar "Galena" used in the study), xanthohumol D [344]; and xanthohumol E [345]. Of chemotaxonomic significance was the finding that the three 4'-O-methylchalcones observed in the survey (>120 plants), xanthogalenol, 4'-O-methylxanthohumol [346], and 4',6'-di-O-methylchalconaringenin, occurred only in wild H. lupulus var. cordifolius plants collected in the Missouri-Mississippi River Basin and in their descendants (cultivars derived from var. cordifolius) and in Japanese wild plants. The absence of these compounds from European and southeastern North American members of the species suggests, as pointed out by Stevens et al. (2000), that at least two separate lineages of H. lupulus exist. The North American members are considered to resemble the ancestral form, which means that 4'-O-methylation is an ancestral feature that was subsequently lost by European hops (or by the ancestor from which the European line arose). Although there were points of difference, the results obtained in the flavonoid survey are in overall agreement with those obtained in a study of restriction fragment-length polymorphisms of ribosomal DNA (Pillay and Kenny, 1996).

4.4 South Pacific

4.4.1 Eucryphia (Eucryphiaceae)

Eucryphia, the sole genus in Eucryphiaceae, consists of six (Mabberley, 1997, p. 270) or seven species (Wollenweber et al., 2000) and can be found occurring on the Australian mainland, in Tasmania, and in southern South America. Specifically, E. cordifolia Cav. and E. glutinosa Cav. occur in Chile, E. lucida (Labill.)

Baillon. and E. milliganii Hook. f. occur in Tasmania, and E. moorei F. Muell., E. jinksii P. I. Forst., and E. wilkiei B. Hyland occur in Australia. The first two named Australian species are temperate rainforest species; E. wilkiei is an outlier known from higher elevations in tropical northeastern Queensland.

The genus has attracted a good deal more chemical attention than might have been expected for a group its size, including a study of the principal aroma constituent of leatherwood (E. lucida) honey, which was shown to be 3,7-dimethyl-1,5,7-octatrien-3-ol [347] (Rowland et al., 1995) (see Fig. 4.12 for structures 347-356). Our concern with the genus, however, lies with the patterns of distribution of flavonoids, including several structurally uncommon ones. The earliest interest in flavonoids of Eucryphia appears to have been that of E. C. Bate-Smith (1962), who observed an unusual yellow-fluorescent spot in an extract of E. glutinosa examined as part of his chemotaxonomic survey of the dicots. Subsequent work established the structure of the compound responsible as quercetin 5-methyl ether (azaleatin) [348] (Bate-Smith et al., 1966). In the following year, these workers (Bate-Smith et al., 1967) described a survey of the two Chilean species and three Australian species. (Note: the earlier workers considered the genus to consist of five species.) Reference to Table 4.5 illustrates the striking differences among the species. The capacity to synthesize both azaleatin and caryotin (quercetin 3,5-dimethyl ether) [349], the rare 5-methyl ethers of quercetin, as well as very similar arrays of quercetin glycosides, plus the two unidentified flavonoids, by both Chilean species speaks clearly for their close

CH3O O

CH3O O

oh o

Fig. 4.12 Compounds 347-356, a terpene alcohol and flavonoids from Eucryphia

Table 4.5 Occurrence of flavonoids in Eucryphia (from Bate-Smith, 1967)

Flavonoidb

Chilean

speciesa

Australian speciesa

COR

GLU

MOO MIL LUC

Caryatin

+

+

- - -

Azaleatin 3-Gal

-

+

- - -

Azaleatin 3-DiGlc

+

+

- - -

Azaleatin 3-GalAra

+

-

- - -

Unidentified flavanone

+

+

- - -

Quercetin 3-Gal

+

+

- + -

Quercetin 3-Rhm

+

+

- - -

Quercetin 3-DiGly

+

+

- - -

Quercetin 3-TriGly

-

-

+ - -

Dihydroquercetin 3-Glyc

-

-

- + -

Kaempferol 3,7-DiMe

-

-

- - +

Unidentified flavan

+

+

- - -

a COR=E. cordifolia; GLU=E. glutinosa; MOO = E. moorei; MIL=E. milliganii; LUC=E. lucida. b Gal = galactose; Glc = glucose; Ara=arabinose; Rhm = rhamnose; DiMe = dimethyl (ether). c Dihydrokaempferol and dihydroquercetin were subsequently reported from E. cordifolia (Tschesche et al., 1979).

a COR=E. cordifolia; GLU=E. glutinosa; MOO = E. moorei; MIL=E. milliganii; LUC=E. lucida. b Gal = galactose; Glc = glucose; Ara=arabinose; Rhm = rhamnose; DiMe = dimethyl (ether). c Dihydrokaempferol and dihydroquercetin were subsequently reported from E. cordifolia (Tschesche et al., 1979).

relationship. By contrast, the two Tasmanian and single Australian species studied exhibited extremely simple flavonoid profiles showing none of the "specialized" compounds present in the Chilean species. This conclusion has had to be tempered somewhat by more recent research involving the Chilean species. Tschesche et al. (1979) identified the 3-0-rhamnosides of dihydroquercetin and dihydrokaempferol from E. cordifolia. In a more recent paper, dihydroquercetin 3-0-xyloside [350], caryatin 7-0-glucoside [351], and jaceidin 5-0-glucoside (jaceidin is 5,7,4'-trihydroxy-3,6,3'-trimethoxyflavone) [352] were obtained from twigs of E. glutinosa (Sepulveda-Boza et al., 1993).

The most recent contribution to the chemistry of Eucryphia came from the work of Wollenweber et al. (2000) who, not surprisingly, examined the flavonoid components of glandular exudates of all members of genus, which in the present view comprises seven species. The tables appear to be completely turned with this subset of Eucryphia flavonoids. In contrast to the richness of profiles exhibited by the Chilean taxa with regard to their polar components, their exudate chemistries are by far the simplest seen in the genus; E. cordifolia afforded a single compound, apigenin-7,4'-dimethyl ether [353], and only in trace amounts. The other South American species, E. glutinosa, yielded only two compounds, apigenin-7,4'-dimethyl ether and luteolin-7,4'-dimethyl ether [354]. Apigenin-7,4'-dimethyl ether was also detected in the two Tasmanian species and in the two mainland species, such as E. jinksii and E. moorei. The richest arrays of aglycones came from the two Tasmanian species with a profile based on O-methylated derivatives of api-genin, luteolin, kaempferol, and quercetin, 17 in E. lucida, and 13 in E. milliganii. Eucryphia jinksii exhibited a somewhat simpler profile, based as well on both fla-vones and flavonols, but clearly distinguished from all other species in the genus by the capacity to make the 8-oxygenated flavones isoscutellarein-8,4'-dimethyl ether [355] and 7,8,4'-trimethyl ether [356]. Eucryphia moorei exhibited a profile somewhat simpler than that of E. jinksii but, as mentioned, lacked flavones with the isoscutellarein oxygenation pattern. Eucryphia wilkiei exhibited a profile consisting of a single, unidentified aglycone, seen as well in E. lucida (Tasmania) and E. jinksii (mainland Australia). It was also unique in the genus in having flavonol glycosides as exudate components.

Numerical treatments of the flavonoid data were performed using nonmetric multidimensional scaling (NMDS), and unweighted pair group method with arithmetic mean (UPGMA) or average linkage clustering of Bray-Curtis dissimilarities. The NMDS treatment resulted in clear separation of E. wilkiei, but the other six taxa appeared scattered, although geographically close pairs, for the most part, appeared closer to each other than to other species. The UPGMA treatment of Bray-Curtis values resulted in clear separation of E. wilkiei from all other species and the following pairings, E. cordifolia-E. glutinosa (Chilean species); E. jinksii-E. moorei (mainland Australia); and E. lucida-E. millinganii (Tasmania). Cladistic analysis of the flavonoid data failed to resolve any relationships. A recent cladistic analysis using morphological data (Taylor and Hill, 1996) suggested that E. lucida and E. milliganii are sister taxa, which is supported by the phytochemical data. Neither the relationship of E. wilkiei to E. lucida and E. milliganii, as suggested by the cladistic analysis of Taylor and Hill (1996), nor to E. jinksii, as viewed by Forster and Hyland (1997), is supported by the flavonoid data. Because of incongruities between relationships suggested by the phytochemical data and those that arose from cladistic analysis of morphological data, one is once again inclined to suggest that gene-sequence studies might be of value in this system. In addition to the obvious phylo-genetic insights that might be gained from such a study, some ideas of what gains and losses have occurred in the flavonoid biosynthetic pathway that resulted in the significantly different profiles of compounds observed within the genus.

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