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The flavonoids of Umbellularia californica (lauraceae) Neville, Heather A. 1993

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THE FLAVONOIDS OF UMBELLULARIA CALIFORNICA (LAURACEAE)byHEATHER ANNE NEVILLEB.Sc. Chemistry (Co-op), University of Victoria, 1983A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Department of Botany)We accept this thesis as conforming to therequired standardsTHE UNIVERSITY OF BRITISH COLUMBIAJune 1993© Heather A. Neville, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of  A77-7/1 (7(The University of British ColumbiaVancouver, CanadaDate DE-6 (2/88)ABSTRACTAn analysis of the flavonoids of Umbellularia californica was performed on 23 samples collected fromthroughout the range of the species in Oregon andCalifornia. The goals of the study were to identify themajor leaf flavonoids and to establish the presence orabsence of flavonoid profile differences between the twogrowth forms that exist in this monotypic genus. Usingstandard isolation techniques nine major flavonoids wereidentified representing a relatively simple profile offlavonol glycosides. Virtually no differences in profilewere seen between the two forms. It was concluded that theflavonoid data do not provide any help in defininginfraspecific taxa. The flavonoid profile identified in U.californica is consistent with profiles reported from othermembers of the Lauraceae.iiTABLE OF CONTENTS^ABSTRACT     iiLIST OF TABLES ^  ivLIST OF FIGURES ACKNOWLEDGEMENTS ^  viI. INTRODUCTION  1CHEMOSYSTEMATICS ^  5FLAVONOIDS DEFINED  12II. MATERIALS AND METHODS ^  18SOURCE OF PLANT MATERIAL  18EXTRACTION PROCEDURES ^  19TLC METHODS ^  21ISOLATION OF VACUOLAR FLAVONOIDS ^  23SUGAR ANALYSIS ^  24SPECTROSCOPIC METHODS ^  25III. RESULTS ^  28FLOWER FLAVONOIDS ^  37LEAF SURFACE COMPOUNDS  37WOOD COMPONENTS ^  38IV. DISCUSSION ^  41FLAVONOIDS OF UMBELLULARIA AND OTHERLAURACEAE COMPARED ^  44GENERAL CONCLUSIONS  55FUTURE STUDIES ^  56V. LITERATURE  58iiiLIST OF TABLES1. Classes of Flavonoids and Possible Functions .^.^.^. 142. Collection Sites ^ 183. Taxonomic Placement of Lauraceae Showing Genera fromwhich Flavonoids have been Identified ^ 464. Distribution of Flavonoids in the Lauraceae usingthe Taxonomic System of Kostermans ^ 485. Flavonoid Occurrences in Lauraceous Genera ^ 52ivLIST OF FIGURES1. A flavonoid study of three diploid Asplenium speciesand their alloploid progeny ^  112. Flavonoid Structure Guide  133. Collection Sites of Umbellularia ^ 174. U.V. fluorescence of one dimensional chromatogram ofmain flavonoids of Umbellularia californica versusglycoside standards ^  265. One dimensional chromatogram of cleaved sugars fromisorhamnetin triglycoside ^  276. Two dimensional chromatogram of Umbellulariacalifornica leaf extract ^  297. U.V. fluorescence of two one dimensional chromatogramsof Umbellularia californica ^  328. Major Flavonoids of Umbellularia californica . . . ^ 369.^U.V. fluorescence of one dimensional chromatogramof shrub wood fractions ^  40ACKNOWLEDGEMENTSHeartfelt gratitude goes to Dr. B. A. Bohm who gaveexemplary supervision and encouragement. My appreciationalso goes to Drs. Edith Camm and Wilf Schofield, the othermembers of my supervisory committee, for their support andassistance. Thanks are also due Dr. Schofield, Dr. BruceTiffney and Ms. Ardis Hyde for helping to provide samplesused in this study. I also wish to thank Dr. MohammedKoupai-Abyazani for mass spectroscopic data and an anonymousartist for valuable help. And thanks to my family andfriends (especially Mum) for ears and shoulders. Finally,many thanks to Joe for making my graduate educationunforgettable.viI. INTRODUCTIONThe goal of this thesis was to identify the flavonoidsof Umbellularia californica and to apply the data totaxonomic concerns within the species as well as toquestions of relationships within the Lauraceae. There aretwo different growth forms of U. californica and if anydifferences in flavonoid profile exist between the two formsthis would constitute further evidence for possibleinfraspecific taxonomic recognition. To attain thissystematic goal both leaf and wood flavonoids wereinvestigated. The thesis questions were:(1) What are the flavonoids of U. californica?(2) Do the flavonoid profiles indicate a differencebetween the two forms?(3) How does the flavonoid profile of Umbellularia compare with flavonoids identified from othermembers of the Lauraceae and related families?The thesis begins with information on U. californica.Background on the use of flavonoids in chemosystematics ispresented to put the work in context. The body of thethesis comprises a description of the flavonoids of U.californica and their application to delineating groupswithin the species. Next, the present flavonoid findingsare compared with those for other genera of the Lauraceae.1Finally the flavonoids of the Laurales are briefly discussedin relation to the known flavonoid chemistry of theMagnoliidae.Umbellularia (Nees) Nuttall is a monotypic genus whichconsists of U. californica (Hook. & Arn.) Nutt. The speciesranges from southern California to southwestern Oregon. InCalifornia the range is divided between (1) coastal andinner coast ranges and (2) foothills of the Sierra Nevada.Umbellularia californica, commonly known as California Bay,Bay tree, pepperwood, Bay laurel or Oregon myrtle, is oftenused for decorative furniture because of its hardness,attractive grain and capacity to take a high polish. Theleaves are entire, oblong-lanceolate, alternate and shiny.Flowers are small, yellow-white and bisexual; the fruit is adrupe (Munz 1959). The leaves and wood are highly fragrant.The principal odour component is umbellulone, a monoterpeneketone known to irritate membranes and cause headache insome individuals (Drake & Stuhr 1935). Use of the leaves ofU. californica as a condiment has also been reported(Lawrence et al. 1974).A quantitative analysis of volatile constituents fromU. californica was done by Lawrence and coworkers (1974).Umbellulone was shown to be the major component (39.0%) ofthe steam-volatile fraction from leaves. The next majorcomponent was 1,8-cineol (eucalyptol) at 23.4% followed by a2number of lesser components: terpinen-4-ol (5.6%), sabinene(4.8%), alpha-pinene (3.6%), thymol (3.3%), beta-pinene2.3%), linalool (2.0%), gamma-terpinene (1.8%), myrcene(1.3%), and p-cymene (1.0%). Minor components (less than1.0% each) included the following: trans-beta-farnesene,alpha-terpinyl acetate, alpha-terpinene, alpha-p-dimethylstyrene, benzaldehyde, limonene, terpinolene, methyleugenol, selina-4,11-diene, alpha-phellandrene, guaia-6,9-diene, ethyl benzoate, delta-terpineol, beta-phellandrene,alpha-santalene, pinocarvone, alpha-selinene, beta-bisabolene, ethyl cinnamate, eugenol, verbenone andcitronellol.Pech and Bruneton (1983) reported three aporphinealkaloids from U. californica, domesticine, nor-domesticineand isoboldine, and bufotenine, which is 5-hydroxy-N,N-dimethyltryptamine.The only report of flavonoids from U. californica appears in a study done in 1962 by Bate-Smith. This work,which only involved acid-hydrolyzed leaf extracts, showedthe presence of kaempferol and quercetin. Bate-Smith'sstudy included representatives of the following genera aswell: Apollonias, Cinnamomum, Cryptocarya, Laurus, Linderaand Persea.3Umbellularia californica consists of two growth forms,a full-size tree (up to 45 meters) with broad crown andsingle trunk that can be up a meter in diameter (tree form),and a shrubby form with several smaller trunks (up to 15 cm.diameter) that may attain a height of about six meters(shrub form). The tree form occurs mainly in lower coastalwoodlands while the shrub form occurs in drier, inlandcanyons and chaparral. In several of these drier areas U.californica occurs on serpentine-derived soils whichpresents an edaphic challenge to many plants. Umbellularia californica is one of several taxa in the Californian florathat have evolved to inhabit serpentine soil (Kruckeberg1985).^The only infraspecific taxon described, however, isU. californica var. fresnensis Eastw. whose inflorescenceand lower leaf surface were finely tomentulose. It was notspecifically stated which growth form was involved (Munz1959).Rohwer (1986) pointed out that the name SciadiodaphneReichenbach had been proposed in 1841 for what we nowrecognize as Umbellularia (Nees) Nuttall (first appearancein 1842), but that it had never occurred with a specificepithet. Neither had it appeared in the Index NominumGenericorum to that date. Rohwer proposed that Umbellulariabe conserved since it refers to a well known decorativetree. The proposal to conserve was subsequently accepted(Nicholson 1991).4The genus can be regarded as relictual andpalaeoendemic (Raven and Axelrod 1978). The origin ofUmbellularia is unknown. Raven (1977) pointed out thatthere are many families in the Californian flora, Lauraceaeincluded, some members of which appear to have originated inSouth America and others from Eurasia.When the new genus arose is unknown, but it was shownto have existed between 15 and 20 million years ago basedupon fossils found near Bridge Creek in north-centralOregon. The fossil study, done by Chaney (cited by Ornduff1974), also showed that the frequency of occurrence of U.californica at the present time in the Muir Woods in MarinCounty (13%) is similar to what was found in the Oregon site(9%). If unchanged morphology implies evolutionary fitnessfor a particular niche, U. californica may be consideredmaximally suited to its environment.ChemosystematicsA systematic analysis involves study of certaincharacteristics for the purpose of understandingrelationships between organisms or groups of organisms. Athorough systematic analysis is behind every taxonomicdelineation and is also useful for evolutionaryconsiderations. Chemosystematics involves use of various5chemical types, usually stable secondary metabolites, insuch an analysis. For example, if certain groups within afamily contain a particular compound or compound type, whichthe others lack, some taxonomic delineation may beindicated. Acceptance of such delineations depends upon theextent to which such information agrees with data frommorphological, anatomical, cytological and other studies.Commonly used secondary metabolites include terpenoids,alkaloids and flavonoids. Of all the compounds used inchemosystematics, flavonoids are arguably the most usefulfor the following reasons:(1) The flavonoid structure is inherently stable althoughcertain types are comparatively less so. For example,anthocyanins are sensitive to changes in pH and chalconescan isomerize spontaneously to flavanones. However,herbarium specimens decades old have yielded excellentresults, and in the extreme, flavonoids have beensuccessfully extracted from certain fossilized plants(Giannasi and Niklas 1977, 1981). In the case of the fossilstudies comparisons were possible between 20 million yearold extinct and extant species.(2) The extraction, purification and structuraldetermination of flavonoids is relatively simple and doesnot require unusual or expensive equipment. A crudemethanolic extract can be resolved into its components usingcolumn (CC) or thin layer chromatography (TLC). Useful6structural information can often be obtained from acompound's chromatographic behaviour, its behaviour in UVlight, and its reaction with chromogenic reagents.Purification can readily be done using TLC. Essentialstructural information can then be obtained using standardultraviolet spectroscopic methods (Mabry et al. 1970;Markham 1982).(3) Flavonoids occur in virtually all vascular plantsincluding the angiosperms, gymnosperms and ferns.Flavonoids have also been found in some mosses and greenalgae. As very widespread secondary metabolites flavonoidsare potentially useful at every taxonomic level.(4) Flavonoids occur in all plant organs. The distincttissue distribution of different types of flavonoids maycoincide with their function. For example, the vividlycoloured anthocyanins are usually concentrated in flowerswhere they serve to attract pollinators.(5) Because there are several skeletal differences amongflavonoids and because oxidation levels and substitutionpatterns can vary, the range of structures is extensive.This makes the flavonoid group ideal for systematic study;the molecular diversity rivals that of the plant kingdom,providing enough variation for meaningful comparisons.(6) The use of flavonoids as systematic markers wasrecognized in the 1960's (Bate-Smith 1962). Since then asubstantial data base has been amassed by plant systematistsaround the world. The size of the data bases facilitates7application of these compounds to a variety of problems inthe plant kingdom.(7) The biosynthesis of flavonoids and genetic control ofthe various steps are well understood.It should also be mentioned that flavonoids are usefulin studies of hybridization because pigment profiles areusually inherited codominantly.Examples of the use of Flavonoids in Systematic StudiesFamily - On the basis of similarities in morphology theEricaceae and Empetraceae are set apart from the relatedfamilies Epacridaceae, Clethraceae and Diapensiaceae.Gossypetin (3,5,7,8,3 1 ,4f-hexahydroxyflavone), acomparatively uncommon flavonoid marker, occurs in membersof Ericaceae and Empetraceae but not in the other families.As a character, gossypetin strengthens the apparent linkbetween these two families (Moore et al. 1970).Subfamily - The Gesneriaceae consists of two subfamilies,the Gesnerioideae and Cyrtandroideae. Two flavonoiddistinctions between these subfamilies have been discussedby Harborne (1966). Members of the Gesnerioideae accumulate3-deoxyanthocyanins as well as normal anthocyanins in bothleaf and floral tissue whereas members of the Cyrtandroideaeappear to make only normal anthocyanins. The otherdifference involves chalcones and aurones which have been8reported from members of the Cyrtandroideae but appear to belacking in Gesnerioideae.Tribe - Within the tribe Vicieae of the Fabaceae there arefour genera that show apparent segregation patternsreflecting flavonoid variation. Lathyrus and Pisum arelinked because they form the phytoalexin pisatin (a 6a-hydroxypterocarpan). Vicia and Lens, however, producefuranoacetylenes as phytoalexins (traces of pterocarpanshave been found). The chemical distinction between Lathyrus and Vicia is striking, since the two genera aremorphologically so similar (Robeson & Harborne 1977).Genus - Apples and pears were once thought to belong to thesame genus (Pyrus L.) because of many shared characteristicsand the capacity to hybridize. Separate genera are nowrecognized, Malus for apple and Pyrus for pear, based ondifferences in flower and fruit morphology. Phenolicdifferences between the two groups support this view; Malus accumulates dihydrochalcones while the major phenoliccompound in Pyrus is the non-flavonoid arbutin (hydroquinoneglucoside) (Challice 1973).Species - Of eleven species of Chondropetalum (Restionaceae)tested for flavonoids, seven exhibit one profile (myricetin,larycitrin and syringetin) while four exhibit a second(kaempferol, quercetin, gossypetin, gossypetin 7-methyl9ether and herbacetin 4'-methyl ether) (Harborne et al.1985).Hybrids - Flavonoid patterns were very useful in helping tounravel relationships between species of the fern Asplenium in the Appalachian Mountains (Smith and Levin 1963; Harborneet al. 1973). Plants of putative hybrid origin showedchromatographic patterns that were the sum of parentalspecies; see Figure 1 below. Flavonoids were also found tobe useful in a study of hybridization and subsequentbackcrossing within the genus Dubautia on the Island ofHawai'i (Crins et al. 1988).Race - The fern Notholaena standleyi consists of three formscharacterized by plant size, geographic distribution and thenature of their leaf exudate chemistry. The 'gold' raceplants exude mainly kaempferol 7-methyl ether and kaempferol4'-methyl ether. 'Yellow' race plants produce kaempferoland its 3,7- and 7,4'-dimethyl ethers. The 'yellow-green'race accumulates the same kinds of kaempferol derivativesbut the profiles are intermediate between the other twotypes (Seigler and Wollenweber 1983).Flavonoid races were also observed in a study ofLasthenia californica (Bohm et al. 1989; Desrochers & Bohm1993). Different flavonoid profiles were observed inseveral populations in California. The geographical1 04^=72(d)2n = 72(c)ic)0Figure 1.^ 1 1A flavonoid study of three diploid Asplenium species andtheir alloploid progenyAlthough (e)(A.x kentuckiense) has an altered chromosomenumber compared to its ostensible tetraploid (d)(A.rhizophyllum x A. montanum) and diploid (c)(A. platyneuron)sources, its ancestry is verified by a perfectly additivechromatographic pattern, including the "original"contributions from (a)(A.x rhizophyllum) and (b)(A.montanum).patterning of these pigment types within the populationswere invariant over a period of 10 years.Individuals - Flavonoid differences among individualswithin a species may be greater than differences amongspecies or even among higher level taxa. Such a situationwas seen in the Hawaiian members of the genus Bidens (Ganders et al. 1990). The rich and seemingly randominfraspecific expression of flavonoids within this genussuggests genetic plasticity, possibly in response to theadaptive pressures of relatively recent colonization.Flavonoids DefinedFlavonoids are phenolic compounds based on the 1,3-diphenylpropane skeleton (Figure 2). A number of structuralclasses exist based on whether the three-carbon bridge iscyclized (as in [2] where flavanones have R = H anddihydroflavonols have R = OH) or not (as in [1], achalcone). Further variation is based upon the level ofoxidation of ring-C, the degree of oxygenation of rings-Aand B, the level of glycosylation, the presence of 0-methylgroups and certain other functionalities. Occurrences offlavonoid classes in plants range from very common, as inthe case of flavones (in [2] R = H^213), flavonols (in [2]R = OH^2/3) and anthocyanidins [3], to comparatively rare,as in the case of flavonoid sulfates. Table 1 presents a12Figure 2.Flavonoid Structure GuideOHOH 0[1]HOOH[2]HOOHOH[ 3 ]general overview of where many of the flavonoid types occurwithin the plant and lists possible functions.Table 1. Classes of Flavonoids and Possible FunctionsClass^Major Organ^Suggested FunctionFlavones^Leaves^U.V. ScreenFlavonols^Leaves U.V. Screen, HormoneAnthocyanins^Flowers^Pollinator AttractantChalcones^Flowers Pollinator AttractantAurones Flowers^Pollinator AttractantIsoflavonoids^Leaves, Roots^PhytoalexinDihydroflavonols Leaves, Wood^PhytoalexinFlavanones^Leaves^Feeding DeterrentThe first flavonoids, chalcones, are formed by thecondensation of three molecules of malonyl CoA with one ofp-coumaroyl CoA catalyzed by chalcone synthase (CS). Theflavonoid A-ring arises by cyclization of the six-carbonpolyketide portion of the precursor; the B-ring and three-carbon bridge arise from the p-coumaroyl unit. Chalconesare cyclized to flavanones through the action of chalconeisomerase (CI). An alternate cyclization of chalcones mayoccur to yield aurones which are characterized by havingfive-membered C-rings. The bridge double bond of chalconesmay be reduced to give dihydrochalcones.14Flavanones serve as branch point intermediates. Lossof hydrogens from carbons-2 and 3 yields flavones (flavonesynthase, FS). Flavanone 3-hydroxylase (F3H) catalyzes thehydroxylation of flavanones at C-3 to yield 3-hydroxy-flavanones (dihydroflavonols). Loss of hydrogens from C-2and C-3 of dihydroflavonols yields flavonols in a reactionanalogous to the formation of flavones. Different enzymesare involved, however. A third fate of flavanones involvesmigration of the phenyl function from C-2 to C-3 to giveisoflavones (isoflavone synthase).Dihydroflavonols also serve as substrates for severaldifferent processes. Dehydrogenation to flavonols hasalready been mentioned. Dihydroflavonols may also undergoreduction of the carbonyl function to yield flavan-3,4-diols(dihydroflavonol 4-reductase) which can in turn be convertedthrough a complex series of reactions to anthocyanidins.Flavan-3,4-diols are also involved in the biosynthesis ofproanthocyanidins, so-called condensed tannins.A-Ring hydroxyl groups at positions-5 and 7 areprovided by the precursor acetyl CoA units while the 4'hydroxyl group comes from the p-coumaroyl CoA unit.Flavonoid 3'-hydroxylase catalyzes the placement of an OHgroup at position-3' of the B-ring to yield the 3',4'-dioxygenated, or quercetin type, substitution pattern.Further hydroxylation at position-5' by a related enzyme15yields the 3',4',5'-trioxygenated, or myricetin-type,substitution pattern. 0-Methylation, to yield compoundssuch as isorhamnetin, is catalyzed by a series of flavonoid0-methyl transferases that appear to be highly selectivewith regard to the position methylated. S-Adenosyl-methionine serves as the cofactor in this reaction.0-Glycosylation is often the final step in flavonoidbiosynthesis. Diglycosides, such as rutinosides (rhamnosyl-glucosides), are formed sequentially. The first reaction,in the formation of rutinosides, involves transfer ofglucose unit from uridine diphosphoglucose (UDPG) to the 3-OH of quercetin catalyzed by flavonol 3-0-glucosyl-transferase. The second sugar, rhamnose in this case, isplaced on the glucose by a rhamnosyltransferase specific forthe 6-hydroxyl group of glucose. Elongation of thediglycoside to a triglycoside would involve transfer of thenext sugar in the same fashion.16Figure 3.Collection Sites of UmbellulariaIIIXIII, XIVII. MATERIALS AND METHODSSource of Plant MaterialUmbellularia californica was collected from 21 sites inCalifornia, one in Oregon and one in West Vancouver, BritishColumbia. In the case of the Californian and Oregon samplesplant material was collected from individual plants andplaced in a plant press. The B.C. specimen was extractedwithout drying. Voucher specimens have been deposited inUBC. Two wood samples were collected, one from a shrub atthe Mud Flat site (BAB-1960) and one from a large tree fromSanta Clara County (Schofield s.n., Dec. 1992). Table 2shows details of the collection sites.collection sites.Figure 3 shows theTable 2.^Collection SitesPop'n. Area County 1No.^Form2 Wt.I B.C. W. Vancouver - 2004^T^20II OR Rogue River Josephine WBS s.n.^T^20III CA Smith River Del Norte WBS s.n.^T^20IV CA Berry Summit Humboldt WBS s.n.^T^20V CA Mud Flat Tehama BAB s.n.^S^20VI CA Mud Flat Tehama 1956^S^13VII CA Mud Flat Tehama 1957^S^19VIII CA Mud Flat Tehama 1958^S^13IX CA Mud Flat Tehama 1960^S^201819^X^CA^Mendocino^Mendocino WBS s.n.^T^20XI^CA^Hop Land^Mendocino WBS s.n.^T 20XII^CA^Napa Napa^WBS s.n.^T^20XIII^CA^Butts Cyn Rd^Lake^1963^S^7XIV^CA^Butts Cyn Rd^Lake^1964^S 18XV^CA^Butts Cyn Rd^Napa^1965^S^8XVI^CA^Pope Val Rd^Napa^1966^S^15XVII^CA^Calistoga^Napa^WBS s.n.^T^20XVIII^CA^Jasper Ridge^San Mateo 1973^T 19XIX^CA^Los Altos^San Mateo WBS s.n.^T^19XX^CA^Switzer's Park L.A.^1974^T^20XXI^CA^Switzer's Park L.A.^1975^T^19XXII^CA^Claremont^L.A.^2005^T^20XXIII^CA^Feather River Butte^2006^?^31) Numbers are B.A. Bohm collections; WBS are W.B.Schofield collections.2) T is tree form, S is shrub form.Extraction ProceduresWhen available, 20 grams of dry leaves were removedfrom the branches and immersed in dichloromethane for twominutes with intermittent stirring. For a few samplessmaller amounts were available (see Table 2). This washingwas repeated once. The combined dichloromethane extractswere filtered and evaporated to dryness under reducedpressure in a rotary evaporator. Residues were taken up inca. three mis of dichloromethane and stored in small vials.After removal of the dichloromethane, the extractedleaves were air dried and crushed in liquid nitrogen toachieve maximum disruption of cells. The leaf powder ofeach sample was extracted at room temperature six times withca. 400 ml of methanol for each extraction. This number ofextractions were needed to remove all colour (chlorophyll)from the ground leaves. The pooled methanol extract fromeach sample was evaporated to dryness as above. The residuewas shaken vigorously with two 250 ml volumes of boilingdistilled water. The resulting aqueous extract was filteredthrough paper to remove waxy solids. Occasionally somechlorophyll or chlorophyll degradation products passed intothe water extract. These caused no problems in subsequentstages of the isolation procedure. Flavonoids were removedfrom the aqueous extract by several extractions with water-saturated n-butanol. After evaporation of the combinedbutanol extracts of each sample, the residues were taken upin a few mis of methanol and stored in small vials.Flowers (ca. five grams) of U. californica (pooledsample) were extracted with methanol and subjected to two-dimensional TLC as for the leaf extracts.20Ground debarked wood samples were extracted by soakingin methanol at room temperature for several days. Soakingwith new methanol was repeated until the solution was nolonger coloured. Combined methanolic extracts wereevaporated to dryness under reduced pressure. The woodextract (shrub form) was chromatographed on LH-20 usingmethods to be described for vacuolar flavonoids. The woodextract from the tree was compared to the shrub using TLC.Thin-layer Chromatographic MethodsThe leaf surface extracts were chromatographed onedimensionally using a mixture of ethyl formate/ cyclohexane/n-butyl acetate/ formic acid (50:25:23:2). The TLC platesused consisted of ca. 0.25 mm thick Polyamid 6.6 spread onglass. All plates were home-made.Two solvent systems were used for the vacuolarflavonoids. The first, referred to as the aqueous system,consisted of water/n-butanol/acetone/dioxane (70:15:10:5).The second system, referred to as the organic system,consisted of dichloroethane/methanol/butanone/water(50:25:21:4). Two-dimensional chromatography was done byrunning plates in the first direction using the aqueoussystem and then in the second direction using the organicsystem. The plates were air dried between the first andsecond runs. To confirm the extent of glycosylation of the21isolated flavonoids commercial microcrystalline celluloseplates were used with 6% aqueous acetic acid. Sugaranalyses were done using commercial silica gel K 60 platesdeveloped in chloroform/methanol/water (16:9:2).The butanol-soluble extract from each plant sample wasspotted onto a thin layer plate and chromatographed in twodirections. After thorough drying in the air the phenolicprofile was viewed under ultraviolet light (366nm) underthree conditions: (1) no spray, (2) fumed with ammonia, and(3) after spraying with 0.1% Naturstoffreagent (ethanolaminediphenylborinate) in 1:1 methanol/water (NR). The behaviourof each compound/spot was recorded and a copy of thechromatogram was made using tracing paper.All of the plant samples were analysed in this way.Due to the similarity of all of the two-dimensional profilesthe butanol extracts were combined (except for smallquantities saved for reference purposes), evaporated todryness and suspended in about 50 mis of 30% methanol inwater and filtered through a pad of Celite filter-aid inpreparation for chromatographic column loading.22Isolation of Vacuolar FlavonoidsA glass column (70 x 3.5 cm) was packed with a slurryof Sephadex LH-20 in 30% methanol in water. The extract wasloaded onto the column with a Pasteur pipette. Thirtypercent methanol was run through the column at anapproximate rate of five mis per minute. Two fractions (ca.200 mis) were taken before changing to 40% methanol. Threeadditional fractions, totalling ca. 200 mis, were taken;then fifty percent methanol was run for seven fractions(total volume ca 500 mis). Next sixty percent methanol wasrun for four fractions (ca. 300 mis total), seventy percentfor five fractions (ca. 450 mis total), eighty percent forsix fractions (ca. 450 mis total), and one hundred percentfor two fractions (ca. 300 mis total). The final fractionconsisted of 200 mis of acetone. All thirty fractions wereevaporated to dryness under reduced pressure and theresidues were taken up in a few mis of methanol and storedin capped vials.These fractions were spotted on thin-layer polyamideplates and chromatographed in one direction in the aqueoussolvent system. A duplicate plate was run in the organicsystem. As before, the behaviour of the spots was recordedunder UV, etc.23Partition column chromatography was used to separatethe compounds in LH-20 column fraction No. 9 (whichcontained the two major flavonoids). A 20 x 2.5cm glasscolumn was packed with a slurry of microcrystallinecellulose-water (2:1) in a water-saturated mixture of ethylacetate and petroleum ether (60:40). Final packing of thecolumn was done under slight air pressure (air line). Afterloading the fraction-9 material the column was developedwith mixtures of of ethyl acetate in petroleum ether (60:40,70:30, 80:20, 100:0). Approximately 100 ml fractions weretaken. After evaporation to dryness and redissolving in afew mis of methanol, the 22 fractions were chromatographedone dimensionally in the organic system. Spots werevisualized and recorded in the usual manner.LH-20 Column fractions, other than No. 9, were resolvedinto component flavonoids using TLC. Final purifications ofindividual compounds was also done by preparativechromatography on polyamide plates using the solventsdescribed above. In all cases bands of purified compoundswere scraped off the thin-layer plates and eluted usingmethanol.Sugar AnalysisThe level of glycosylation of each flavonoid wasdetermined, in part, by its TLC behaviour. Flavonoid24monoglycosides, diglycosides and triglycosides havecharacteristic mobility in the aqueous system with themonoglycosides being the slowest moving, triglycosides beingthe fastest moving and diglycosides being intermediate. SeeFigure 4.Saccharide identity was determined by hydrolysis offlavonoid glycosides with trifluoroacetic acid, TLC of thecleaved sugars on silica gel using the chloroform/methanol/water system followed by spraying with a reagent thatconsists of thymol (0.9 g) in 19 mis 95% ethanol and 1.0 mlH2SO4 . Heating the sprayed chromatograms for a few minutesat 105 ° develop the characteristic colours of the sugars.Appropriate standards are used for comparison. See Figure 5for a graphic representation of a sugar analysis.Spectroscopic MethodsCompounds were subjected to ultraviolet absorptionanalysis using standard techniques (Mabry et al. 1970).These techniques involve spectral shift reagents: sodiumacetate, boric acid, aluminum chloride/HC1, and sodiummethoxide. Mass spectral analysis involved standardelectron impact mass spectrographic techniques (70ev) asdescribed by Markham (1972).25Figure 4.U.V. fluorescence of one dimensional chromatogramof main flavonoids of Umbellularia californicaversus glycoside standards:^M = Quercetin-3-0—Gle,D = Isorhamnetin rutinoside, T = Isorhamnetin-3-0—Gal—Rha—Rha.(after reagent spray)0 = greenO = blue= yellow4^9y 9g^13^24y 24g 24g'^26y 26g 30^M D T24g' = fraction 24, upper green^ 9y = fraction 9, yellow cs,Rhamnose(dark pink)Xylose(violet)Glucose(pink)Galactose(puce)• •Aglyconeresidue(brown)Si^ S2Arabinose ^(dark violet:dark pinkpink•Figure 5.One dimensional chromatogram of cleavedsugars from isorhamnetin triglycoside(of fraction 9)27III. RESULTSTwo-dimensional TLC analysis of all 23 collections ofU. californica revealed that they were virtuallysuperimposable both qualitatively and quantitatively.Figure 6 is a graphical representation of the profile. Twokinds of spots were observed, blue fluorescent ones that arepresumably lower molecular weight phenols, and UV absorbingones (dark). The UV absorbing spots fluoresced green in thepresence of ammonia fumes which indicated that they wereeither 5-OH flavones or 3-0-substituted flavonols with a 4'-OH (Markham 1982, p. 19). Upon spraying the chromatogramwith NR some of the dark spots gave a green colour and somegave a yellow colour. Green indicates the presence of a 4'-hydroxyl group, while a yellow colour indicates 3',4'-dihydroxylation.28= Dark before sprayGreen after spray= Dark before sprayYellow after sprayO14Org8 9_  2121311•756AquFigure 6.Two dimensional chromatogramof Umbellularia californica leaf extract.29Figure 6. (continued)(Key to spot numbers)1. Quercetin tetraglycoside2. Isorhamnetin tetraglycoside3. Quercetin triglycosides plus diglycosides4. Isorhamnetin triglycosides plus diglycosides5. Quercetin diglycosides plus monoglycosides6. Quercetin monoglycoside7. Isorhamnetin diglycoside8. Quercetin monoglycoside9. Isorhamnetin monoglycoside10. Quercetin aglycone11. Isorhamnetin or Kaempferol aglycone12. Quercetin diglycoside or monoglycoside13. Quercetin diglycoside30Column chromatography of the combined butanol-solublephenolic fractions from 21 specimens (The West Vancouver,Claremont and Feather River collections were not used) gavethe following general elution results: fractions 3 and 4,presumed tetra-glycosides; fractions 5-10, triglycosides;fractions 11-22, diglycosides; fractions 23-28monoglycosides; and fractions 29 and 30, aglycones. Bluefluorescent compounds appeared in many fractions. SeeFigure 7 for a diagrammatic representation of these data.Work on selected fractions resulted in isolation andidentification of nine compounds. Kaempferol and quercetinderivatives were identified by means of their characteristiccolour reactions with NR, their ultraviolet spectralcharacteristics and by direct comparison with knowncompounds. The structure of isorhamnetin, suggested byultraviolet data, was confirmed by mass spectroscopy(molecular ion = 316, molecular ion minus methyl = 301, A-ring fragment = 152/153, B-ring fragment = 151).The monoglycoside fractions yielded a flavonol andeither glucose or rhamnose on hydrolysis. The compoundsidentified were kaempferol and quercetin 3-0-glucosides andkaempferol and quercetin 3-0-rhamnosides. The diglycosidefraction yielded two major compounds which gave eitherkaempferol or isorhamnetin on acid hydrolysis plus equalamounts of glucose and rhamnose. These compounds had Rf31Figure 7.U.V. fluorescence of two one dimensional chromatograms of^Umbellularia californica^leaf vacuole column fractions 1-15(after reagent spray) S = Quercetin 3-0—Glc1^ 5^ 10U = blue= yellow0 = green15= Blue= Yellow= GreenFigure 7. (continued)U.V. fluorescence of two, one dimensionalchromatograms of Umbellularia californicaleaf vacuole column fractions 16-30.(after reagent spray) S = Quercetin 3-0—Glc16^ 20^ 25^ 30behaviour equal to that of rutinosides (rhamnosylglucosides)but confirmatory studies on the isolated diglycosides werenot done. A very small amount of what has been assumed tobe the equivalent quercetin 3-diglycoside was also observed.The triglycoside fraction gave two compounds that gave, onacid hydrolysis, either quercetin or isorhamnetin andglucose and rhamnose. Inspection of sugar chromatogramsfrom these compounds suggested that glucose was present inhigher amounts than rhamnose which would mean that thesecompounds were flavonol 3-0-(glucose, glucose, rhamnose).The order of arrangement of the sugars was not determined.Partial hydrolyses of these compounds, done using lowconcentrations of acid at room temperature for short periodsof time, gave ambiguous results. Very small spots were seenin the 2D chromatograms that had higher Rf values in theaqueous solvent than the triglycosides. Attempts to obtainthese presumed tetraglycosides failed, however. Figure 8shows the structures of the flavonoids identified.Attempts to relate the compounds identified withindividual spots on the 2D chromatogram were only partiallysuccessful. Figure 6 shows an attempt to correlate thevarious flavonol glycosylation levels identifed with spotson a representative chromatogram. The triglycoside spotsare the most reliably assigned. It is also clear thattetraglycosides would be expected to run higher than thetriglycosides in the aqueous solvent. The diglycosides34identified co-chromatographed with known flavonoldiglycosides in the aqueous system so the assignment of thediglycoside region on the 2D TLC is reasonable. Themonoglycoside region on the 2D chromatograms was morecomplex than the identified compounds would suggest.Insufficient plant material was available to solve thisproblem. Small amounts of the aglycones, presumablykaempferol, quercetin and isorhamnetin, were observed alongthe organic system axis on the 2D chromatograms and in LH-20column fraction 30. Whether these compounds are naturallyoccurring or are artifacts of the isolation procedures wasnot investigated.35Figure 8. Major Flavonoids of Umbellularia californica36H 0Kaempferol, R . = HQuercetin, R = OHIsorhamnetin, R = OMeIsorhamnetin 3-0-triglycoside (Glc, Glc, Rha)Quercetin 3-0-triglycoside (Glc, Glc, Rha)Isorhamnetin 3-O-diglycoside (Glc, Rha)Kaempferol 3-0-diglycoside (Glc, Rha)Quercetin 3-0-diglycoside (Glc, Rha)Quercetin 3-0-monoglucosideKaempferol 3-0-monoglucosideQuercetin 3-0-monorhamnosideKaempferol 3-0-monorhamnosideFlower FlavonoidsFlowers from several collections were pooled andextracted with methanol. Chromatography of the concentratedextract gave a 2D profile that was similar to, but somewhatsimpler than, the profile seen with the leaf samples. Also,there were fewer blue fluorescent spots in the flowerprofiles. As in the case of the leaf chromatograms,spraying with NR produced intense green and yellow spots(kaempferol and quercetin- type flavonols, respectively) inthe triglycoside position. These were accompanied by smallputative tetraglycosides. The monoglycoside position wasoccupied by intense green and yellow spots and it seemslikely that the diglycosidic position was also comprised ofthe same flavonol pair. Traces of aglycones were alsoobserved.Leaf Surface CompoundsThe dichloromethane extracts from the leaf surface ofUmbellularia were chromatographed one dimensionally in theethyl formate-cyclohexane solvent system. No flavonoidspots were seen in any of the samples. There wasconsiderable streaking of blue fluorescent material in mostfractions. No differences were noted between tree and shrubforms.37After several weeks a copious white crystallineprecipitate had accumulated in each sample of the cuticularextract. This material was collected and subjected toinfrared analysis. The IR spectrum exhibited bands thatcould be assigned to a carbonyl group, a cyclopropyl groupand an ether linkage all of which are consistent with thetwo major compounds reported from the oil fraction ofUmbellularia, namely umbellulone, which has both acyclopropyl ring and a ketone group and eucalyptol whichcontains an ether linkage.Wood ComponentsThe shrub wood sample from Mud Flats, California (1960)yielded 39 fractions by column chromatography. The onedimensional chromatogram of these fractions, run using thedichloroethane-based organic system, showed severalprominant compounds (see Figure 9). The compound infraction-16 was dark before spraying and green with ammoniavapour, indicating a 3-0-substituted flavonol. The 3-position is not glycosylated, however, since migration inthe aqueous system for all wood compounds was nil. Thefraction-16 compound gave a yellow colour with NR suggestinga quercetin-type B-ring.38Fraction 22 contained two major compounds both of whichwere yellow-green fluorescent before spraying; neithershowed any change with ammonia fumes. Spraying with NR gaveyellow and green colours suggesting quercetin and kaempferoltype aglycones, respectively.The main compound in fraction 25 was blue beforespraying and showed no change with ammonia, which couldsignify a 5-substituted isoflavone. This compound gave areddish reaction with NR; this behaviour does notimmediately suggest a structure.Two-dimensional chromatograms of crude butanolicextracts of wood samples from tree and shrub forms were runusing the dichlorethane solvent system in both directions.This served to show the profile similarity between the twosamples. Tree and shrub samples appeared the same,therefore, column chromatography of the tree specimen wasnot done.39Figure 9.U.V. fluorescence of one dimensional chromatogramof Umbellularia californica shrub wood fractions 1-39(after reagent spray) S = Quercetin-3-0—GlcredCD = greenyellow0 = blueS^35^30^ 255^10^15^20 OIV. DISCUSSIONThe first two questions asked above concerned thenature of the flavonoids of Umbelullaria californica andwhether any differences exist between the compounds presentin the two growth forms of the species. Twenty-threecollections of U. californica were made, 13 of the tree formand 9 of the shrub form (we did not see the Feather Riversample). The 2D chromatograms of all of these werevirtually superimposable. The only differences seen wereminor quantitative ones. Samples of wood were obtained fromtwo U. californica plants, one a tree and one a shrub.Again, no differences were seen in the phenolic profiles ofthe two growth forms.The tree form of U. californica is a member of the wetcoastal forest vegetation of southwestern Oregon south tocentral California, whereas the shrub form is found in drierhabitats such as occur in the inner coast mountains ofCalifornia. The species also occurs in the westernfoothills of the Sierra Nevada but only one sample wasavailable from this area. Several of the drier sitesoccupied by U. californica are characterized by serpentine-derived soils. Kruckeberg (1984) includes the shrubby formof U. californica in his list of taxa that have local orregional indicator status for ultramafic substrates inCalifornia.41It is clear from the flavonoid analyses that neitheredaphic features nor availability of water have any apparenteffect on the flavonoid profile of the species. It ispossible that other sources of information may indicate thatsufficient differences exist between the two growth forms toargue for taxonomic recognition. Flavonoids do not. It isclear from the uniformity of pigment profiles in thesegrowth forms that the flavonoid biosynthetic pathways in U.californica had been established before divergence of thetwo forms.The evolutionary origin of Umbellularia is obscure(Raven 1977). It is possible that it may have had itsorigin from some element of the South American lauraceousflora. Lauraceae is well represented in the present floraof South America. It is also possible that Umbellularia mayhave had a Eurasian origin. Laurus nobilus, the true bay,is a native of the Mediterranean region.Cronquist (1981) stated that fossil wood from lateupper Cretaceous (Mastrichtian Epoch, 72 million yr. b.p.),from California, falls within the range of variation of theLauraceae and that Eocene wood from Yellowstone may belauraceous. Raven and Axelrod (1978) commented that theEocene and early Oligocene floras (35-50 million yr b.p.)that existed from Washington southward comprised severalsubtropical families, Lauraceae included. Persea 42(podalinia) and Umbellularia (californica) were componentsof the late Miocene age Remington Hill flora (Condit 1944)and the Pliocene age Oakdale and Turlock Lake floras(Axelrod 1944). Late Miocene (13 million yr. b.p.) fossilsshow that Persea, Nectandra, and Ocotea, as well asUmbellularia, were part of the oak-laurel forest inCalifornia (Axelrod 1977). Umbellularia has also beenidentified as a component of the late Pliocene ageMulholland and Oakdale floras dating to about 5 million yrb.p. (Axelrod 1944). Persea, Ocotea and Nectandra are largegenera, with the latter two being well represented in thepresent South American flora (Mabberley 1987).Flavonoids of some members of South American generahave been reported, although sampling is poor and in atleast one case only aglycones were identified. From thefruits of Nectandra glabrescens Barbosa-Filho and coworkers(1989) obtained kaempferol and quercetin. Wofford (1974)reported kaempferol and quercetin 3-0-glucosides fromseveral species of Persea, while Merici and coworkers (1982)identified quercetin 3-0-glucoside and 3-0-diglucoside, aswell as the flavones apigenin and luteolin, from P.americana leaves. With the exception of the flavonesreported from P. americana the flavonol derivativesidentified from U. californica are similar to thosedescribed from these South American genera. It can bepointed out that quercetin 3-0-rhamnosylglucoside (rutin)43was also shown to exist in leaves of the European speciesLaurus nobilus (Makarov 1971).An intriguing observation comes from the work of Kochand Ktinig (1981) on Aniba rosaeodora which is a SouthAmerican species although it appears not to be known fromthe California fossil record. These workers identifiedkaempferol and quercetin 3-0-rhamnosylglucosides and aquercetin 3-0-tetraglycoside. The tetraglycoside gave equalamounts of glucose and rhamnose on acid hydrolysis. Theflavonoid profile of Umbellularia californica includesrutinosides, flavonol 3-0-triglycosides with only glucoseand rhamnose, and trace amounts of compounds that have thechromatographic properties of tetraglycosides. The amountsof these putative tetraglycosides were too small to allowpurification, however. The comparative rarity of flavonoltetraglycosides makes this similarity between Umbellularia and Aniba noteworthy.Flavonoids of Umbellularia and Other Lauraceae ComparedIn the discussion below the flavonoids of the twogrowth forms of U. californica will be described. Followingthat will be a comparison of the flavonoids of Umbellularia with other genera in the family. Next, the flavonoidchemistry of the Lauraceae will be compared with thechemistry of other families of the Laurales. Finally, the44flavonoid chemistry of the Laurales will be discussed interms of the known chemistry of the Magnoliidae.Since the two growth forms of U. californica exhibitidentical patterns of flavonol glycosides, the major ones ofwhich are shown in Fig 8, no arguments for recognizinginfraspecific taxa can be made.In order to set the stage for comparisons at highertaxonomic levels, it is first necessary to present theappropriate taxonomic framework. Kostermans' (1957)taxonomic treatment of the family, which involves twosubfamilies, five tribes and eight subtribes, will be used.The constitution of the Laurales and its position in theMagnoliidae will be given according to Cronquist (1981).These relationships are presented in Table 3.45Table 3. Taxonomic Placement of Lauraceae Showing Generafrom which Flavonoids have been IdentifiedClass MAGNOLIOPSIDASubclass MAGNOLIIDAEOrder LAURALESAmborellaceae^IdiospermaceaeCalycanthaceae LauraceaeGomortegaceae^MonimiaceaeHernandiaceae TrimeniaceaeLauraceaeSubfamily Cassythoideae^Only Cassytha (NFD 1 )Subfamily LauroideaeTribe PerseeaeSubtribe Perseineae^PerseaMachilus (incl. in Perseaby Kostermans)Phoebe Beilschmiediinae ApolloniasBeilschmiedia 5 other genera (NFD)Tribe CinnamomeaeSubtribe Cinnamomineae^Actinodaphne CinnamomumNectandra (incl. in Ocoteaby Kostermans)Ocotea46Subtribe AnibineaeUmbellularia 2 or 3 other genera (NFD)Aniba6 other genera (NFD)47Tribe LitseeaeSubtribe Litseineae^Litsea 1 other genus (NFD)Subtribe Laurineae^Laurus Lindera Tribe CryptocaryeaeSubtribe Eusideroxylineae Eusideroxylon (NFD)Subtribe Cryptocaryineae Cryptocarya 1 other genus (NFD)Tribe Hypodaphneae^Hypodaphnis (NFD)1) NFD = No flavonoid data availableTable 3 presents the taxonomic frameworks into whichthe Lauraceae and its constituent genera fit (afterKostermans and Cronquist). Table 4 presents a detailedaccount of flavonoid reports from members of the familytaken from the literature and includes the current resultsfrom Umbellularia. Finally, Table 5 summarizes theflavonoid information in terms of generic profiles andpresents the chemical information in terms of types offlavonoids or unique structural features present in eachgenus.Table 4.CassythoideaeLauroideaeTribe I. PerseeaePerseineaeDistribution of Flavonoids in the Lauraceaeusing the Taxonomic System of KostermansCassvtha^No flavonoid dataPerseaP. americana (L)^Apig,Lute & 7-G1c,K-3-Glc,Q-3-DiGlcP. lingue ^K & QP. borbonia K-3-Glys,Q-3-Glys,Flavone-C-GlysP. humilis ^K-3-Glys,Q-3-Glys,Flavone-C-GlysP. palustris^K-3-Glys,Q-3-Glys,Flavone-C-GlysP. sp. (W)^Flavan-3-ols,DiarylpropanesE. sp. (L) Flavone-C-GlysPhoebe cinnamomifolia Q-3-Me etherMachilis (incl. in Persea by Kostermans)M. thunbergii (L) K-3-Rhm,Q-3-Ara,Q-3-RutM. thunbergii (W)^NaringeninK & Q,DiHK & DiHQFlavan-3-olsMerici et al. (1992)Wofford (1974)Wofford (1974)Wofford (1974)Wofford (1974)Suarez et al. (1988)Harborne (1966)Martinez 0. et al. (1990)Park et al. (1990)Karikome et al. (1991)Beilschmiediineae Beilschmiedia B. miersii (L)Tribe II. CinnamomeaeCinnamomineae^ActinodaphneA. angustifolia (L)A. madraspatana (W)(1979)Lute-O-GlyK-3-Rhm,Q-3-Gln,Q-3-Rhm,Q-5-MeIsorhamnetinQuer-3-Rha-/578-FlavoneHarborne & Mendez (1969)Kubitzki & Reznik (1966)Bhakuni et al. (1971)Adinarayama & GunsekarAnibineaeCinnamomumC. cassia (B)^Flavan-3-olsFlavan-3-ols,ProcyanidinsC. sieboldii (L)^K-3,7-GlysNectandra (incl. in Ocotea by Kostermans)N. glabrscens (F)^K & QUmbellularia (L)^K, Q, IR GlysAniba A. riparia (W)^-/357-Flavone2'/4'6'-Chalcone,5/7-Flavanone,-/57-Flavanone,3/57-Flavanone35/7-Flavone-/357-FlavoneMiyamura et al. (1983)Yazaki & Okuda (1990)Nakano et al. (1983)Barbosa-Filho et al.(1989)Present workFranca et al. (1976)Fernandes et al. (1978)A. rosaedora (W)^57/-Flavanone^cited by Fernandes et al.(1978)Tribe III. LitseeaeLitseineae^Litsea L. glutinosa (L)Laurus L. nobilus (L)C10-ChalconeK-3-Glc-Rhm,Q-3-Rut,Q-3-(G1c2,Rhm2)K-3-G1c,K-7-Glc,Q & Q-3-Rhm,Nar & 7-Rhm,Pel-5-GlcQ,Q -3 -Rutde Alleluia et al. (1978)Koch & Kiinig (1981)Mohan et al. (1977)Makarov (1971)Lindera L. ervthrocarpa (T)^2'/3'4'5'6'-Chalcone,-/5678-Flavanone,Modified A-ring^chalcones^Liu et al. (1973a,b)L. lucida (L)^-/5678-Flavone,-/5678/3'4'-MDO-Flavone57/68/3'4'-MDO-^Flavone^Lee & Tan (1965)L. lucida (R)^Modified A-ringchalcones Lee & Tan (1965)L. pipericarpa (R)^Modified A-ringchalcones^Kiang et al. (1962)L. umbellata (L)L. umbellata (B)Tribe IV CryptocaryeaeEusideroxylineaeCryptocaryineae Crvptocarya C. bourdilloni (R)2'4'6'//3'-Menthyl-chalcone2'6'/4'/3 1 -Menthyl-chalcone2'4'6'/-DiHchalcone,2'6'/4'-DiHchalcone2'6'/4'-Chalcone,2'6 1 /4'/3'-Menthyl-chalcone,57/-Flavanone,5/7-FlavanoneNo flavonoid dataModified A-ringchalconeIchino (1989)Tanaka et al. (1984)Shimomura et al. (1988)Govindachari et al.(1973)Tribe V Hypodaphneae^ No flavonoid dataExplanation of abbreviations:Apig = apigenin; Lute = luteolin.; K = kaempferol; Q = quercetin;DiHK = dihydrokaempferol; DiHQ = dihydroquercetin; Pel = pelargonidin;Me = methyl; MDO = methylenedioxy; Gly = glycoside; Glc = glucoside;Gin = glucuronide; Ara = arabinoside; Rhm = rhamnoside; Rut = rutinoside;L = leaves; B = bark; R = root; F = fruit; W = wood; T = all parts;Hydroxy positions/Methoxy positions/Other substituent(s)Table 5.^Flavonoidl Occurrences in Lauraceous GeneraFLAVONE FLV'OL^GLYGenus Sp. 2 CHL FVN DHF ORD CGL K Q I A M D T P MET MDO BRD XOX OTHERPersea^7/150^+ + + +^+ + CatMachilus 1/- + + + + + CatPhoebe^1/70^+ Apollonias^1/2 + +Beilschmiedia 1/200^+^+ + + + +^+Actinodaphne 2/70 + +^+ +^+ +Cinnamomum^3/250 + + + CatOcotea^1/200^+^+ +Nectandra^1/100 + +Umbellularia^1/1 + + +^+ + + ? +Aniba^2/41 + + + + + + +^+ +Litsea 2/400^+^+ +^+ PelLaurus^1/2 + +Lindera 4/80 + +^+^+ + + + + +Cryptocarya^1/200 + + + +1) CHL = chalcone; FVN = flavanone; DHF = dihydroflavonol; ORD = ordinary flavone(e.g. apigenin); CGL = C-glycosylflavone; FLV'OL = flavonol (K = kaempferol,Q = quercetin, I = isorhamnetin, A = azaleatin); GLY = glycoside ( M = mono-,D = di-, T = tri-, P = tetra-); MET = 0-methyl; MDO = methylenedioxy; BRD = B-ringdeoxy flavonoid; XOX = extra A-ring oxygenation2) Number of species for which flavonoid data are available/Number of species known for the genus3) Quercetin 3-methyl etherThe first thing that is clear from Table 5 is thatmembers of the family are primarily flavonol accumulators.The presence of flavonol derivatives as the major flavonoidtype present in Umbellularia is consistent with this. Fourdifferences from the base pattern of kaempferol andquercetin have been observed. First, Umbellularia andBeilschmiedia mearsii (Harborne and Mendez 1969) are theonly taxa that have been shown to produce isorhamnetin(quercetin 3'-0-methyl ether). Beilschmiedia is furtherdistinguished within the family by its capacity to methylatethe 5-OH of quercetin to give azaleatin. The thirddifference is seen in Phoebe cinnamomifolia which has beenshown to produce quercetin 3-methyl ether (Martinez 0. etal. 1990). The fourth feature is the presence of 0-methylated derivatives of the unusual B-ring deoxyflavonolgalangin in Aniba riparia (Franca et al. 1976; Fernandes etal. 1978).0-Methylated derivatives of other flavonoid types havealso been reported from members of the family. 0-Methylatedflavanones were identified from Aniba riparia (Fernandes etal. 1978), Lindera erythrocarpa (Liu et al. 1973a, 1973b)and L. umbellata (Shimomura et al. 1988) while 0-methylatedflavones have been found in Actinodaphne madraspatana (Adinarayama and Gunsekar 1979) and Lindera lucida (Lee andTan 1965). 0-Methylated chalcones have also been reportedfrom Aniba riparia (Fernandes et al. 1978) and from species53of Lindera (Ichino 1989; Shimomura et al. 1988). An 0-Methylated dihydrochalcone was identified from Lindera umbellata by Tanaka and coworkers (1984).The most commonly observed flavonol glycosides arethose that are linked through the 3-OH group. Most flavonolglycosides in the Lauraceae, including those fromUmbellularia, are of this type. The only exceptional caseis the report of kaempferol-3,7-diglycosides in leaves ofCinnamomum sieboldii (Nakano et al. 1983). The most commonglycosides of flavones and flavanones, on the other hand,are linked through the 7-OH. This is the case with regardto luteolin 7-0-glucoside from leaves of Persea americana (Merici et al. 1992) and naringenin 7-0-glucoside fromleaves of Litsea glutinosa (Mohan et al. 1977). Mohan andcoworkers (1977) also identified the common anthocyaninpelargonidin 5-0-glucoside from L. glutinosa.The present findings of kaempferol and quercetinderivatives in Umbellularia are in agreement with thegeneral observations that simple flavonols are the majorflavonoid group in the family which, in turn, is inagreement with observations that other members of theLaurales are characterized by such a simple profile. Theprofiles are not identical, however, as some other flavonoidtypes have been found in some of the other families.Sterner and Young (1980) found kaempferol and quercetin54glycosides in members of the Calycanthaceae, whilekaempferol, quercetin and isorhamnetin have been reportedfrom the Monimiaceae (Gornall et al. 1979; Bombardelli etal. 1976). Derivatives of kaempferol and quercetin(including isorhamnetin) along with C-glycoflavones havebeen reported from members of the Hernandiaceae (Gornall etal. 1979). Amborella trichopoda, the sole member of theAmborellaceae, accumulates glycosides of kaempferol alongwith procyanidin (Young 1982). Young and Sterner (1981)obtained quercetin glycosides as well as flavone derivativesand C-glycosyl-flavones from Idiospermum australiense, whichis the sole member of the Idiospermaceae.Despite the variation seen in this group of families afundamental feature is the absence of myricetin (3',4',5'-trihydroxy B-ring) derivatives. The lack of myricetinderivatives in members of the Magnoliiflorae (sensu Gornallet al. 1979) was discussed as a significant factor indistinguishing this group from Hamamelidiflorae andDilleniiflorae (Gornall et al. 1979).General ConclusionsWhat general evolutionary conclusions can be drawn fromthis work? One can speculate that several millions of yearsago the adaptation of U. californica flavonoids to theenvironment reached its pinnacle and that conditions since55then have not changed with regard to the evolutionarypressures involved in shaping the flavonoid profile. Thegenetic basis for the growth forms of U. californica wouldappear to be more plastic compared to the supposedly fixedgenes of flavonoids biosynthesis.Since we don't have flavonoids from fossil U.californica plants, it is impossible to say with certaintyjust how far back the simple flavonol profile, defined bythis work, existed. Studies of several genera of fossilplants, Ouercus, Celtis, Ulmus, Zelkova (Giannasi andCrawford 1986) showed that these 15-20 million year oldfossils exhibited essentially the same flavonoid profiles asextant species. Owing to the apparent stability offlavonoid patterns over time it is not unreasonable toassume that the present flavonoid profile of Umbellularia isrepresentative of the plant over its history.Future studies.It would seem reasonable that a morphometric study ofU. californica be undertaken in order to see if consistentdifferences between the two growth forms exist. Somedifferences in leaf shape observed in the field suggest sucha study.56Variation in the size of some of the minor spots onchromatograms suggest that a quantitative study would beuseful. This would be best done with plants from the rangeof the species maintained in a common garden environment.This would also offer an opportunity to study variation ofvolatile leaf components. Natural variation in volatilecomponents has not been done.57V. LITERATUREBarbosa-Filho, J. M., Yoshida, M., and Gottlieb, 0. R.(1989) Lignoids from Nectandra amazonum and N. glabrescens,Phytochemistry, 28(7): 1991Bate-Smith, E. C. (1962) The phenolic constituents of plantsand their taxonomic significance. I. Dicotyledons, BotanicalJournal of the Linnean Society 58: 95-1873.Bhakuni, D. S., Gupta, N. C., Satish, S., Sharma, S. C.,Shukla, Y. N., and Tandon, J. S. (1971) Chemicalconstituents of Actinodaphne augustifolia, Croton sparsifolus, Duabanga sonneratiodes, Glycosmis mauritania,Hedyotis auricularia, Lyonia ovalifolia, Micromelumpubescens, Pyrus pashia, and Rhododendron niveum,Phytochemistry, 10: 2247-2249Challice, J. S. (1973) Phenolic compounds of the subfamilyPomoideae: a chemotaxonomic survey, Phytochemistry 12: 1095-1101.Crins, W. J., Bohm, B. A. and Carr, G. D. (1988) Flavonoidsas indicators of hybridization in mixed populations of lava-colonizing Hawaiian tarweeds (Asteraceae: Heliantheae:Madiinae), Systematic Botany 13: 567-571.58Cronquist, A. (1981) An Integrated System of Classificationof Flowering Plants, Columbia University Press, New York, p.77.de Alleluia, I. B., Braz Fo., R., Gottlieb, 0. R.,Megalhaes, E. G., and Marques, R., (1978) (-)-Rubraninefrom Aniba rosaeodora, Phytochemistry 17: 517-521Drake, M. E. and Stuhr, E. T. (1935) Some pharmacologicaland bactericidal properties of umbelliferone, J. Am. Pharm.Assn. 24: 196-201.Fernandes, J. B., Gottlieb, 0. R. and Xavier, L. M. (1978)Chemosystematic implications of flavonoids in Aniba riparia.Biochemical Systematics and Ecology, 6: 55-58Ganders, F. R., Bohm, B. A. and McCormick, S. P. (1990)Flavonoid variation in Hawaiian Bidens, Systematic Botany15: 231-239.Giannasi, D. E. and Crawford, D. J. (1986) BiochemicalSystematics. II. A Reprise, Evolutionary Biology 20: 25-248.Giannasi, D. E. and Niklas, K. J. (1977) Flavonoid and otherchemical constituents of fossil Miocene Celtis and Ulmus (Succor Creek Flora), Science 197: 765-767.59Giannasi, D. E. and Niklas, K. J. (1981) Comparativepaleobiochemistry of some fossil and extant Fagaceae, Amer.J. Bot. 68: 762-770.Gornall, R. J., Bohm, B. A. and Dahlgren, R. (1979) Thedistribution of flavonoids in the angiosperms, BotaniskaNotiser 132: 1-30.Harborne, J. B. (1966) Comparative biochemistry offlavonoids-II. 3-Deoxyanthocyanins and their systematicdistribution in ferns and gesnerads, Phytochemistry 5: 589-600.Harborne, J. B. (1966) in Comparative Phytochemistry (Swain,T., ed.) p. 271, Academic Press, London.Harborne, J. B., and Mendez, J. (1969) Flavonoids ofBeilschmiedia miersii, Phytochemistry, 8: 763-764Harborne, J. B., Boardley, M. and Linder, H. P. (1985)Variations in flavonoid patterns within the genusChondropetalum (Restionaceae), Phytochemistry 24: 273-278.Harborne, J. B., Williams, C. A. and Smith, D. M. (1973)Species-specific kaempferol derivatives in ferns of theAppalachian Aspleniium complex, Biochemical Systematics andEcology 1: 51-54.60Karikome, H., Mimaki, Y. and Sashida, Y. (1991) Abutanolide and phenolics from Machilus thunbergii,Phytochemistry, 30(1): 315-319King, F. E. (1962) J. Chem. Soc. 1192. This citation wastaken from R. Hegnauer, Vol. IV. Chemotaxonomie derPflanzen, p. 367. A search in the journal showed that thecitation was incorrect. Further searches for papers by Kingor for papers on Ocotea usambarensis were unsuccessful.Kostermans, A. J. G. H. (1957) Lauraceae, Reinwardtia 4,193-256.Lawrence, B. M., Bromstein, A. C. and Langenheim, J. H.(1974) Terpenoids in Umbellularia californica,Phytochemistry 13: 2009.Martinez O., E., De Diaz, A. M. and Joseph-Nathan, P. (1990)3-Methyl ether of quercetin from the leaves of Phoebecinnamomifolia (HBK) Nees, Rev. Colomb. Quim. 19: 123-125.Merici, F., Merici, A. H., Yilmaz, F., Yunculer, G. andYunculer, 0. (1992) Flavonoids of avocado (Persea americana), Acta. Pharm. Turc. 34: 61-63.61Miyamura, M., Nohara, T., Tomimatsu, T. and Nishioka, I.,(1983) Seven aromatic compounds from bark of Cinnamomumcassia, Phytochemistry, 22(1): 215-218Moore, D. M., Harborne, J. B. and Williams, C. A. (1970)Chemotaxonomy, variation and geographical distribution ofthe Empetraceae, Bot. J. Linn. Soc. 63: 277-293.Munz, P. A. (1959) A California Flora, University ofCalifornia Press, Berkeley, p. 77.Nakabayashi, T. (1952) Partition chromatography of tanninsand pigments. XV. Isolation of quercetrin and astilbin fromyoung leaves of Litsea glauca, J. Agr. Chem. Soc. Japan 26:469-472.Nakano, K., Takatani, M., Tomimatsu, T., Nohara, T., (1983)Four kaempferol glycosides from leaves of Cinnamomumsieboldii, Phytochemistry, 22(12): 2831-2833Ornduff, R. (1974) Introduction to California Plant Life.Raven, P. H. (1977) The California Flora in Terrestrial Vegetation of California (M.G. Barbour and J. Major eds),Wiley Interscience, New York, p 110-139.62Raven, P. H. and Axelrod, D. I. (1978) Origin andRelationships of the California Flora, University ofCalifornia Publications in Botany, 72, 1-134.Robeson, D. J. and Harborne, J. B. (1977) Pisatin as a majorphytoalexin in Lathyrus, Z. Naturforsch. 32c: 289.Smith, D. M. and Levin, D. R. (1963) A chromatographic studyof reticulate evolution in the Appalachian Aspleniumcomplex, Amer. J. Bot. 50: 952-958.Suarez, M., Diaz D., P. P., Fonseca, G., and Castano, S.(1988) 1,3-Diarylpropane and flavonols from Persea sp.,Rev. Latinoamer. Quim. 19(2): 83-84Wofford, B. E. (1974) The systematic significance offlavonoids in Persea of the southeastern United States,Biochemical Systematics and Ecology, 2: 89-91Seigler, D. S. and Wollenweber, E. (1983) Chemical variationin Notholaena standleyi, Amer. J. Bot. 70: 790-798.Sterner, R. W. and Young, D. A. (1980) Flavonoid chemistryand the phylogenetic relationships of the Idiospermaceae,Systematic Botany 5: 432-437.63Yazaki, K. and Okuda, T. (1990) Condensed tannin productionin callus and suspension cultures of Cinnamomum cassia,Phytochemistry, 29(5): 1559-1562Young, D. A. (1982) Leaf flavonoids of Amborella trichopoda,Biochem. Syst. Ecol. 10: 21-22.Young, D. A. and Sterner, R. W. (1981) Leaf flavonoids ofprimitive dicotyledonous angiosperms: Degeneria vitiensis and Idiospermum australiense, Biochem. Syst. Ecol. 9: 185-187.64

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