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A flavonoid study of the lauraceae Yang, Ji Yong 1998

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A F L A V O N O I D S T U D Y OF T H E L A U R A C E A E by J i Yong Yang B . S c , The University of British Columbia, 1992 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E STUDIES D E P A R T M E N T OF B O T A N Y We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A March 1998 © Ji Yong Yang,1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date fdarcj^ ^ Affr DE-6 (2/88) Abstract A total of 80 compounds, consisting mainly of flavonols, were observed in a leaf chemistry survey of 120 species and 35 genera of Lauraceae. A common flavonoid pattern of quercetin-3-(9-glucoside, quercetin-3-O-xyloside, quercetin-3-O-rhamnoside, kaempferol-3-<9-glucoside and kaempferol-3-O-rhamnoside was widely exhibited in the family. Quercetin and kaempferol di- and triglycosides were the next most common compounds followed by C-glycosylflavones, flavones, flavanones, methylated flavonols and chalcones. Flavonoids, like the morphological characters of the Lauraceae, showed large variation within genera; however, the restricted distribution of certain compounds clarified some relationships within controversial genera. The widespread occurrence of C-glycosylflavones in the expanded tribe Perseeae sensu van der Werff and Richter (1996) supports their new classification of this tribe. The lack of C-glycosylflavones in Sassafras, Umbellularia and Actinodaphne supports Rohwer's (1993b) and van der Werff and Richter's (1996) treatment of these genera in their tribe Laureae. C-Glycosylflavones also supports the transfer of the American species of Cinnamomum back to Phoebe. Lastly, the unique occurrence of chrysin in Hypodaphnis supports the placement of this genus in its own tribe as indicated by Kostermans (1957). In addition, flavonoid data have also revealed the classic eastern Asia-eastern North America disjunct distribution in several species of Persea, Lindera and Sassafras. ii Table of Contents Abstract ii List of Tables v List of Figures vi Acknowledgment vii 1 Introduction 1 1.1 Introduction to the Lauraceae 1 1.1.1 Geographical Distribution and Habitat 1 1.1.2 Fossil Records 5 1.1.3 Economic Importance 6 1.2 Classification of the Lauraceae 7 1.2.1 Classification Problems 8 1.2.2 The Three Most Recent Classification Systems of the Lauraceae 11 1.2.3 Other Sets of Taxonomic Characters 16 1.3 Flavonoids 18 1.3.1 Flavonoids as Taxonomic Characters 18 1.3.2 Previous Flavonoid Studies of the Lauraceae 22 1.4 Objective of the Study 25 2 Materials and Methods 26 2.1 Source of Plant Material 26 2.2 Extraction of Flavonoids 26 2.3 Identification of Flavonols 32 2.3.1 Thin Layer Chromatography (TLC) 32 2.3.2 High Performance Liquid Chromatography ( H P L C ) 34 2.3.3 Further Identification 36 iii 3 Results 37 3.1 Flavonoid Survey of the Lauraceae 37 4 Discussion 83 4.1 Flavonoid Variation Among Genera 80 4.2 Taxonomic Implications of Flavonoids at the Generic Level 89 4.3 Interspecific Relationships 96 4.4 Conclusion 102 Literature Cited 104 iv List of Tables 1.1 Genera with variation in number of stamens and number of anther cells 9 1.2 Kostermans' classification system (1957) 12 1.3 Rohwer's classification system (1993b) 13 1.4 van der Werff and Richter's classification system (1996) 14 1.5 Flavonoids previously identified in the Lauraceae 23 2.1 The plant species used in this study 27 3.1 Index numbers assigned to the flavonoid compounds 60 3.2 Flavonoid distribution in the Lauraceae 63 4.1 Occurrences of flavonoid classes in lauraceous genera 85 4.2 The generic arrangements for the tribe Laureae or Litseeae 91 4.3 The position of Cassytha in the three classification systems 93 4.4 The position of Hypodaphnis in the three classification systems 93 4.5 The position of Beilschmiedia, Endiandra, Dehaasia, Apollonias and Mezilaurus in the three classification systems 95 4.6 The position of Caryodaphnopsis in the three classification systems 97 v List of Figures 1.1 Morphological characteristics of the Lauraceae 2 1.2 Biosynthesis of flavonoids illustrating the different flavonoid classes 20 1.3 Some common variations observed in flavonols 21 2.1 Summary of methods involved in the extraction and identification process of flavonoid analysis 31 2.2 A n example of a 2 D - T L C of Sassafras albidum with known standards 33 2.3 H P L C analysis of Machilus chinensis 35 3.1 The common flavonoids of the Lauraceae shown in 2 D - T L C and H P L C 38 3.2 Chemical structures of some of the common flavonoids observed in the Lauraceae 39 vi Acknowledgment I would like to thank my supervisor, Prof. Bruce Bohm, for his continuing interest, valuable advice, generous financial support and for introducing me to the quaich. I am grateful to the members of my committee, Profs. Fred Ganders and W i l f Schofield, for reading my thesis critically and carefully and providing valuable suggestions for its improvement. I am indebted to the following people who generously provided me with plant materials: Dr. Henk van der Werff from the Missouri Botanical Garden, Dr. Richard Rabeler from the University of Michigan Herbarium and the late Dr. Gerald Straley. I would also like to thank my colleagues and friends. Jon Page for his valuable assistance in the lab, particularly with the H P L C . Alan Reid and Ken Marr for their insightful information about flavonoids. Dean Mulyk for the educational discussions on the various aspects of Sytematic Biology and Nishi Rajakaruna for making the lab a fun place to be. I am truly thankful for the support of my wonderful wife, Carol, who criticized my work and wrote this sentence. Finally, my research was supported by a Natural Sciences and Engineering Research Council of Canada grant to Prof. Bruce Bohm. vii Chapter 1. Introduction 1.1 Introduction to the Lauraceae The Lauraceae is a large, predominantly tropical family of aromatic evergreen trees and shrubs, with the exception of the parasitic twining herb, Cassytha. Currently about 50 genera and 2500-3000 species are recognized, although these numbers are in a constant state of flux. The family can be recognized easily by its distinctive floral morphology. The flowers typically consist of six alternating, trimerous whorls: two whorls of three tepals, four whorls of three stamens and the gynoecium. In the androecium, stamens from the third whorl frequently bear a pair of accompanying glands, and stamens from the fourth whorl are usually reduced to staminodes or are absent. The anthers have either two or four locules and dehisce by flap-like valves (Fig. 1.1). The pistil is unicarpellate with a single pendulous anatropous ovule and the ovary is generally superior. The fruit is a berry or a drupe that is often subtended or completely surrounded by a fleshy cupule (Fig. 1.1). 1.1.1 Geographical Dis t r ibut ion and Habitat The Lauraceae is distributed worldwide, but it is most commonly found in the tropics of America and southeast Asia . North of its tropical area the family is found in North America as far north as southern Oregon (Umbellularia californica) and southern Ontario (Sassafras albidum and Lindera benzoin). Only a single species occurs in Europe, mainly in the Mediterranean region (Laurus nobilis), whereas in northern As i a the family is widely distributed from China to Japan (Litsea and Lindera). South of its tropical area the flowering branch longitudinal section of a drupe flower fertile stamens with anthers of 4 locules longitudinal section of flower stamens of third whorl bearing glands ®(0>&) gland cross section of flower showing two whorls of tepals and four whorls of stamens m whor l 4 staminodes from fourth whorl Fig. 1.1 Morphological characteristics of the Lauraceae. Persea borbonia (after Zomlefer 1994) 3 Chapter 1. Introduction family occurs in southern Chile (Persea lingue) and Argentina. The family is poorly represented in most of Africa (Beilschmiedia, Dahlgrenodendron and Hypodaphnis) but several species occur in Madagascar, including an endemic genus (Ravensara). Numerous species occur in Australia particularly in the rain forests of Queensland and N e w South Wales, while only three species of Beilschmiedia are found in N e w Zealand. The habitat containing the greatest diversity of lauraceous species is the lowland rain forest, but some species also occur at high elevation in tropical montane forests where they appear to dominate the vegetation (Gentry 1988). Lauraceae tend to be rarer in drier environments, although a few species occur in semi-arid habitats (Umbellularia californica). Other species have adapted to various extreme edaphic conditions, for example seasonally flooded forests or almost nutrient depleted sandy areas (Rohwer 1993b). Several genera have a pantropical distribution: Beilschmiedia, Cassytha, Cryptocarya, Litsea, Ocotea and Persea, although in the case of Litsea this is true only because of recent introduction to Africa, Madagascar and N e w Zealand by humans (Rohwer 1993b). Others are restricted to As ia and/or Australia (Actinodaphne, Alseodaphne, Dehaasia, Endiandra, Eusideroxylon, Hexapora, and Neolitsea). Eusideroxylon is restricted to Borneo and parts of Sumatra while Dehaasia and Hexapora are restricted to peninsular Malaysia. Madagascar contains Ravensara and Potameia. The former is endemic to Madagascar while the latter exhibits an interesting disjunct distribution: one species occurs in Nepal, another in southern China and the 4 Chapter 1. Introduction remainder in Madagascar. A even more spectacular disjunction in the family is found in Apollonias. One species occurs in the Canary Islands and the other in India. However, the validity of the Indian species should be questioned since it has been collected only once (Rohwer 1993b). Some of the common western Hemisphere genera include Aiouea, Dicypellium, Licaria, Lindera, Mezilaurus, Nectandra, Ocotea, Pleurothyrium, Rhodostemonodaphne, Sassafras and Umbellularia. Lindera and Sassafras exhibit the eastern North American and eastern Asian disjunction. In addition, a disjunction between As ia and Tropical America is found in Caryodaphnopsis, Cinnamomum and Phoebe. To understand the widespread pantropical distribution of many lauraceous species we need to examine both the dispersal potential and the fossil record of the family. In the tropical rain forests, Lauraceae fruits provide more than 80% of the diet of many fruit eating birds: bell birds (Cotingidae), fruit pigeons (Columbidae), quetzals (Trogonidae), and toucans (Rhamphastidae) (Snow 1981, cited in Rohwer 1993b). These birds provide the dispersal mechanism: they swallow the fruit whole and regurgitate the seed after a short time. In addition, other less specialized birds that feed on lauraceous fruit also contribute to dispersal, as is often the case in temperate regions (Wheelwright et al. 1984, cited in Rohwer 1993b). Monkeys, porcupines and squirrels have also been observed eating the fruits and dispersing the seeds (Kostermans 1957). Kostermans (1974b) also notes that some species of Caryodaphnopsis can disperse by means of water. 5 Chapter 1. Introduction 1.1.2 Fossi l Records The Lauraceae has an extensive fossil history particularly in North America and Europe suggesting that the family was widespread and dominant in these regions in the past. Most fossils are of early Tertiary leaves (Bandulska 1926, Ferguson 1974, Kovach and Dilcher 1984), wood (Wheeler et al. 1977) and flowers (Taylor 1988, Drinnan et al. 1990). Cretaceous fossil records are rare, but a recently discovered lauraceous fossil has been dated back to the mid-Cretaceous from Maryland, U S A (Drinnan et al. 1990). This fossil provides the earliest evidence of trimerous flower parts and endosperm in angiosperms. The flowers and inflorescence are remarkably well preserved and described as Mauldinia mirabilis by Drinnan et al. (1990). The flowers have three small outer and three larger inner tepals and nine two-locular anthers in three whorls with well developed staminode-like appendages. This unique floral structure is also present in some extant Lauraceae, such as Persea cuneata. Drinnan et al. (1990) hypothesize that Mauldinia-like plants were probably widespread around the mid-Cretaceous in North America. However, no reliable palynological record of Lauraceae exists prior to mid-Cretaceous. It appears that pollen from this family is generally poorly preserved; this is attributed to the low concentration of sporopollenin in the mature wall (Hesse and Kubi tzki 1983). There are two different views that attempt to explain the paleodistribution of Lauraceae. Raven and Axelrod (1974) suggest that the Lauraceae had a Neotropical-African-Southeast Asian paleodistribution, with northward migration. The close taxonomic relationship in the fossil records of the subtribe Cinnamomineae between 6 Chapter 1. Introduction North America and the Neotropics may support this hypothesis. However, Taylor (1988) argues that fossils with close affinities to this subtribe have also been found in Europe but at present none have been found in Africa. He suggests that the fossils in North America and Europe may be more closely related to each other than to specimens from anywhere else. Therefore, the subtribe may have had a Boreotropical distribution from North America to Europe and along the Tethys seaway to southeast Asia , with early migration southwards into the Neotropics. Exchange of tropical elements over the North Atlantic has been well established and was possible until the late Eocene (Tiffney 1985). In addition, there is growing evidence of interchange between North and South America (Este and Baez 1985, cited in Taylor 1988) during the late Cretaceous and early Tertiary. More evidence appears to suggest the Boreotropical distribution but only with further discoveries of new fossil records, particularly in the Mediterranean, Asian and African regions, w i l l a more complete paleodistribution of the Lauraceae be established. 1.1.3 Economic Importance The Lauraceae provides a wide selection of valuable economic products. One of the most important products is the avocado (Persea americana) which is cultivated in the tropics and subtropics worldwide for its edible fruits. In addition, numerous barks and leaves are widely used in the culinary world for their aromatic oi l content. Cinnamon is derived from the barks of Cinnamomum verum and C. cassia. Leaves of Laurus nobilis (bay leaves) and Umbellularia californica (California bay) are used for flavouring in Mediterranean cuisine. Aromatic substances are also extracted for perfumery (rosewood 7 Chapter 1. Introduction oil from Aniba rosaeodora and saffrol from Cinnamomum porrectum), and for medicinal properties (Cinnamomum camphora, Aniba coto, and Ocotea rodiaei). Timber is another important economic product from the family. Some species have exceptionally hard and decay-resistant wood that is used for construction, such as stone wood (Mezilaurus itauba and M. navalium) and greenheart (Ocotea rodioei) from Brazi l , and iron wood (Eusideroxylon zwageri) from Indonesia. The wood from some other species is highly esteemed for its fine grain wood and is used for building furniture; these include Imbuia (Ocotea porosa), Queensland walnut (Endiandra palmerstonii), and stink wood (Ocotea bullata). This latter species is now under protection in South Africa because of excessive exploitation in the past (Kostermans 1957, Hutchinson 1963, Gottlieb 1972, Rohwer 1993b). 1.2 Classification of the Lauraceae Despite its economic importance, the number of lauraceous species and their distribution is poorly understood. This is reflected by difficulties in the classification of the family. N o doubt, this lack of knowledge is related to the fact that many species are tall trees with small, inconspicuous flowers that inhabit primary rainforests that are difficult to collect (van der Werff and Richter 1996). This is clearly shown by recent reports of newly discovered species and genera throughout the globe. Hyland's (1989) recent revision of the Australian Lauraceae reported 115 species, of which 46 were new; Rohwer's (1993a) revision of Nectandra involved 114 species, of which 33 were new; Chapter 1. Introduction van der Wer f f s (1993) treatment of Pleurothyrium described 40 species, of which 20 were new; and eleven new species of Aniba are currently known in Ecuador when none were reported in this area by Kubitzki in 1982. Moreover, ten new genera and hundreds of new species, mostly in the neotropics, have been discovered in the last two decades (Burger (1988), Kostermans (1994), Madrinan (1996), van der Merwe et al. (1988), Rohwer etal. (1991), van der Werff (1986, 1987a, 1987b, 1988, 1991, 1993, 1994), van der Werff and Richter (1985) and Wofford (1983)). Some of these genera are Aspidostemon, Dahlgrenodendron, Gamanthera, Paraia, Potoxylon, Povedadaphne, and Williamodendron. A s plant exploration becomes more extensive in the future, hopefully more detailed information on the species number and distribution of Lauraceae w i l l be acquired. 1.2.1. Classification Problems There are numerous suprageneric classifications of Lauraceae, but as stated by van der Werff and Richter (1996) "al l have in common one characteristic: they are not widely accepted". This generic circumscription is based on the limited number of useful morphological characters available to distinguish generic limits. A set of important and commonly used characters includes the number of fertile stamens and number of anther cells on each stamen; however, considerable variation in these characters is exhibited by species within the same genus (Table 1.1). For instance, Caryodaphnopsis is described as having nine 4-celled stamens; however, there are species in the genus that contain nine 2-celled stamens or six 4-celled stamens plus three staminodia. Many other genera also exhibit significant variations in these characters. Therefore, the systematic classification 9 T3 o 13 o CN <u T3 <u •4—* o SH O CN £S 13 13 S - i H cd I-I S - i fi a a +3 (A 1/3 € -2 13 "5 O o CN CN <D .3 a C2 >~. a 10 Chapter 1. Introduction of the Lauraceae at the generic level is far from being settled. In addition, the number of recognized genera is in a state of constant flux; this is reflected in the two most widely accepted generic classifications, those of Kostermans (1957) and Rohwer (1993b). Kostermans applied a broad generic concept and merged numerous widely accepted genera, recognizing a total of 31. Since then, eleven genera have been reinstated, and ten new genera have been found based on either new interpretations of known material previously included in older genera, or on newly discovered species that do not fit the definitions of any known genera. Three of the new genera accepted by Rohwer (1993b), Dahlgrenodendron, Gamanthera and Povedadaphne are not accepted by Kostermans (1990, 1993). Likewise, four recently described genera, Clinostemon, Sinosassafras, Temmodaphne, and Triadodaphne are not accepted by Rohwer (1993b). Various classification systems of the Lauraceae have been published. The earliest classifications by Nees (1836), Meissner (1864), Bentham and Hooker (1880), Mez (1889), Pax (1889), and Hutchinson (1964) are all based on the following morphological characters: inflorescence paniculate versus umbellate; number of anther cells (2 vs. 4); number of stamens; fruit enclosed in perianth versus seated in a cup or free; flowers unisexual or bisexual. However, the resulting classifications varied greatly dependent on the respective author's opinion on the importance of certain characters. For example, in Pax (1889) 2 versus 4-celled anthers is the most important character, while in Mez (1889) and Nees (1836) it is whether the inflorescence is paniculate or racemose. Furthermore, these authors do not explain the reasons for their choices on the importance of these characters (van der Werff and Richter 1996). 11 Chapter 1. Introduction 1.2.2. The Three Most Recent Classification Systems of the Lauraceae Kostermans dedicated much o f his life to collecting and classifying the family, the result of which was a new classification published in 1957. He recognized two subfamilies (Lauroideae and Cassythoideae) and five tribes (Perseeae, Cinnamomeae, Litseeae, Cryptocaryeae and Hypodaphneae). Litseeae is distinguished from the others by its involucrate inflorescence; the other four with exinvolucrate inflorescences are recognized by the development of a cupule or the lack of it. Perseeae is distinguished by a complete absence of a cupule, Cinnamomeae by the presence of a more or less cup-shaped cupule, Cryptocaryeae by having the fruit almost entirely closed by the cupule, and Hypodaphneae by having a truly inferior ovary and the fruit completely enclosed by the hypanthium. Further divisions into subtribes are based solely on the number of anther cells, 2 vs. 4 (Table 1.2). Kostermans' classification (1957) has found general acceptance in the last 30 years owing to the fact that he was highly respected for his knowledge of the family rather than for his choice of characteristics used in the classification (van der Werff and Richter 1996). Some lauraceous systematists have criticized his classification for the difficulties it creates when dealing with generic circumscription in the family (Hyland 1989; Rohwer et al. 1993b; van der Werff and Richter 1996). Recently, features of the wood characters have come to play an important role in the classification of genera. The main work is found in Richter (1981) which subdivided the family into three large groupings of genera based entirely on wood anatomy. Rohwer (1993b) combines the information from wood anatomy with traditional characters to 12 Table 1.2. Kostermans' classification system (1957) Subfamily Lauroideae Tribe Perseeae. Inflorescence exinvolucrate. Cupule absent Subtribe Perseineae. Anthers four-celled Persea* (Alseodaphne, Caryodaphnopsis, Machilus)** Phoebe Subtribe Beilschmiediineae. Anthers two-celled Apollonias Beilschmiedia Dehaasia Hexampora Endiandra Potameia Mezilaurus Tribe Cinnamomeae. Inflorescence exinvolucrate. Cupule present Subtribe Cinnamomineae. Anthers four-celled Actinodaphne Cinnamomum (Neocinnamomum) Ocotea (Nectandra, Pleurothyrium) Sassafras Umbellularia Subtribe Anibineae. Anthers two-celled Aiouea Aniba Endlichera Licaria Phyllostemonodaphne Systemohodaphne Urbqnodendron Tribe Litseeae. Inflorescence involucrate. Cupule present Subtribe Litseineae. Anthers four-celled Litsea Neolitsea Subtribe Lauriineae. Anthers two-celled Laurus Lindera (Parabenzoin) Tribe Cryptocaryeae. Inflorescence exinvolucrate. Fruit almost completely by cupule Subtribe Eusideroxylineae. Anthers four-celled Eusideroxylon Subtribe Cryptocaryineae. Anthers two-celled Cryptocarya Ravensara Tribe Hypodaphnis. Ovary inferior. Fruit fused with floral tube Hypodaphnis Subfamily Cassvthoideae Genus Cassytha New Genera UNCLASSIFIED Genera Dahlgrenodendron, Gamanthera, Povedadaphne, Rhodostemonodaphne, Williamodendron 13 Table 1.3. Rohwer's classification system (1993b) Large group Perseeae. Inflorescences non involucrate, basically thyrsoid; anthers of third whorl basically extrorse. Cryptocarya group Cassytha subgroup: Cassytha* Cryptocarya subgroup: Cryptocarya Aspidostemon subgroup: Aspidostemon Eusideroxylon subgroup: Eusideroxylon Hypodaphnis subgroup: Hypodaphnis Dahlgrenodendron Ravensara Potoxylon Beilschmiedia group Beilschmiedia Hexapora Ocotea group Persea subgroup Alseodaphne Dehaasia Notaphoebe Ocotea subgroup Aiouea Neocinnamomum Rhodostemonodaphne Aniba subgroup Aniba Dicypellium Urbanodendron Mezilaurus subgroup Mezilaurus Anaueria Endiandra Potameia Apollonias Persea (Machilusy Cinnamomum Nectandra Pleurothyrium Licaria Paraia Systemonodaphne Povedadaphne Brassiodendron Caryodaphnopsis Phoebe Endlicheria Ocotea Gamanthera Phyllostemonodaphne Williamodendron Large group Laureae. Inflorescenes involucrate, basically (di) botryoid; anthers of third whorl basically introrse Actinodaphne Laurus Lindera (Parabenzoih) Litsea Neolitsea Sassafras Umbellularia Dodecadenia Iteadaphne Parasassqfras 14 Table 1.4. van der Werff and Richter's classification system (1996) Subfamily Laureae Tribe Laureae. Racemose, often umbellate and bracteolate inflorescences, generally surrounded in bud by bracts (involucre). Absence of marginal (banded) parenchyma and of septate fibers (in some taxa). Presence of phloem fibers. Actinodaphne* Laurus Lindera (Parabenzoin)** Litsea Neolitsea Sassafras Umbellularia Tribe Perseeae. Paniculate-cymose inflorescence. Absence of marginal parenchyma, presence of septate fibers, and presence (in some taxa) of phloem fibers. Aiouea Alseodaphne Cinnamomum Endlichera Mezilaurus Nectandra Persea (Machilus) Pleurothyrium Chlorocaridum Aniba Apollonias Dehaasia Licaria Neocinnamomum Ocotea Phoebe Aspidostemon Tribe Cryptocaryeae. Paniculate ± cymose inflorescence; presence of marginal parenchyma, absence of septate fibers and absence of phloem fibers. Have conspicuous bordered pits, lack scalariform perforation plates, but have sclereids. Beilschmiedia Cryptocarya Hypodaphnis Potoxylon Caryodaphnopsis Endiandra Eusideroxylon Triadodaphne Subfamily Laureae genus New genera genera Cassytha UNCLASSIFIED Dahlgrenodendron, Gamanthera, Povedadaphne, Rhodostemonodaphne, Williamodendron * The genera in bold represent the species examined in this study ** The genera in brackets are considered either subgenera or belonging to the adjacent genera depending on the authors in question 15 Chapter 1. Introduction produce a new, but tentative classification. He recognizes two large groups, Perseeae and Laureae, based primarily on inflorescence type (the former being involucrate and the latter exinvolucrate and to a smaller extent on combinations of wood characters). Perseeae is divided into three smaller groups (the Cryptocarya group, the Beilschmiedia group and the Ocotea group) which in turn are further divided into subgroups based mainly on fruit and floral characters (Table 1.3). The most recent classification is that of van der Werff and Richter (1996) who propose three tribal groups based on a new combination of existing characters (Table 1.4). They review the three most common sets of characters used in establishing generic classifications, inflorescence, androecium and fruit. They determined that inflorescence characters are reliable and useful for classification since individual genera rarely possess more than one inflorescence type. In contrast, androecial characters, particularly the number of stamens and number of anther cells on each stamen, are often found to vary within genera and thus can be considered to be unreliable. They also find that characters related to fruit, such as the degree to which the fruit is covered by the hypanthium, are consistent within most genera and useful in classification. However, they note that the generic classification based on this character differs greatly from classifications based on wood and bark anatomy, and give priority to the latter. Consequently, they dismiss the androecial and fruit characters entirely and concentrate on inflorescence types and on wood anatomy, as two different, but complementary sets of characters useful in establishing suprageneric groups (Madrinan-Restrepo 1996). 16 Chapter 1 . Introduction 1.2.3. Other Sets of Taxonomic Characters Since there are a limited number of useful morphological characters (fruit, floral inflorescence, wood type) available to distinguish generic limits, other sets of taxonomic characters have been examined, including phytochemistry (Gottlieb 1972), palynology (Raj and van der Werff 1988, van der Merwe et al. 1990), karyology (Odaka and Tanaka 1975) and recently cuticular features (Christophel et al. 1996). However, the new information provided by these different fields have not been as useful as some of the morphological characters. Additional taxonomic information, particularly from non-morphological sources, such as flavonoids, may be needed to provide clearer relationships in the Lauraceae. Karyology The main chromosomal study in the Lauraceae is Onaka and Tanaka (1975). They examined a variety of species and found that most exhibited a base chromosome number of x = 12 (2n = 24) with minimal variation in the karyotype. Polyploid numbers were found only in the following genera: Cassytha, Laurus, Sassafras (2n = 48) and Neolitsea (2n = 72). Therefore, because of the lack of variation, chromosome numbers do not improve the classification of the family. Palynology The main pollen studies in the Lauraceae were conducted during the last ten years (Raj and van der Werff 1988; van der Merwe et al. 1990). Raj and van der Werff (1988) provide a comprehensive survey of the pollen morphology of neotropical Lauraceae. 17 Chapter 1. Introduction They report that pollens of many genera in the neotropics are easily identifiable by their size and by the number and organization of the spinules. V a n der Merwe et al. (1990) survey the pollen morphology of south African Lauraceae and review the palynological literature for the family; they based their study mainly on shape and exine sculpture of the grain. Four main, broadly defined pollen types are distinguished. The distribution of these four pollen types corresponds to each of the tribes proposed by Kostermans (1957). Therefore, comparative palynological studies in the Lauraceae have supported certain relationships proposed by Kostermans. However, pollen studies from the rest of the world, particularly from Asia , are much needed to assess completely the pollen diversity of the family. Cut icu lar Study of Lauraceae Leaf cuticular features in the Lauraceae are provided by Christophel et al. (1996). Their aim was to analyze cuticular features of selected Lauraceae species whose generic positions have been predetermined by others, and in some cases questioned. They surveyed a large number o f specimens most o f which were from Australia and used three broad categories of leaf cuticular characters to delimit genera. Preliminary results show the utility of these characters in defining genera such as Endiandra and Beilschmiedia and in confirming biogeographical anomalies, such as in Caryodaphnopsis, where the cuticles confirm placement of disjunct species within one genus. However, these features were not useful for defining many other genera examined. Chapter 1. Introduction 1.3 Flavonoids Flavonoids are a class of low molecular weight phenolic compounds that originate from the phenylpropanoid pathway. Flavonoid synthesis starts with the condensation of one molecule of p-coumaroyl coenzyme A and three molecules of malonyl coenzyme A yielding a chalcone, this reaction is carried out by the enzyme chalcone synthase (CHS). Chalcone is subsequently isomerised by the enzyme chalcone isomerase (CHI) to yield a flavanone. From here the pathway diverges into several side branches, each yielding a different class of flavonoids each catalyzed by a specific enzyme (Fig. 1.2). Flavonoids have been found to exhibit a diverse spectrum of biological functions in plants. This is particularly interesting since only 2% of all the carbon processed by plants is converted into flavonoids (Markham 1982). They protect plants from harmful effects of U V irradiation, deter insect feeding, play an important role in sexual reproduction, attract pollinators and participate in the interaction between plants and other organisms. 1.3.1 Flavonoids as Taxonomic Characters O f all the chemical compounds used in chemosystematics, flavonoids have been arguably the most useful for the following reasons: their structural variability, stability, ease of identification and widespread occurrence within the plant kingdom. The variation in flavonoid structure arises from a number of sources including the level of oxidation, glycosylation, methylation, and sulfate groups. Whether the C-ring is cyclized or not, contributes further variation. To illustrate this structural diversity of flavonoids, some 19 p-Coumaroyl Coenzyme A + 3 Malonyl Coenzyme A CHS Aurone Dihydrochalcone OH O Chalcone t CHI SUGAR OH O C-Glycosylf lavone HO. IFS FNS T OH O Flavanone FHT OH O 2-Hydroxyisoflavanone IFD ,OH HO. OH O Dihydroflavonol (3 -Hydroxyf lavanone) OH O k - ^ Isoflavone OH O Flavonol 'OH OH OH Flavan-3,4-diol F O M T OH O OH O V 0H OH Anthocyanidin Flavonol 3-methyl ether F l a v o n o 1 3-O-glucoside Fig. 1.2 Biosynthesis of flavonoids illustrating the different flavonoid classes 20 Chapter 1. Introduction common variations based on the flavonol structure are illustrated in Fig. 1.3. The chemical stability of flavonoids is one of the main advantages to their use in taxonomic studies. In addition, isolation o f flavonoids is simple and inexpensive and the methods for identification are well known and relatively easy. In most cases flavonoids can be extracted from either fresh or dried plant material and stored in vials for many years for future examinations. Herbarium specimens have been shown to yield excellent results. Flavonoids have also been successfully recovered from angiosperm fossils provided the plants have been fossilized under certain conditions (Giannasi and Niklas 1977). Furthermore, flavonoids occur in virtually all vascular plants including the angiosperms, gymnosperms and pteridophytes. They have also been found to occur in some mosses. This widespread occurrence across the entire spectrum of vascular plants makes flavonoids exceptionally good taxonomic characters. Flavonoids have been used successfully to help solve classification problems at every taxonomic level. A t the family level, flavonoid data strengthen the link between Empetraceae and Ericaceae, and distinguishes them from the other related families, Epacridaceae, Clethraceae and Diapensiaceae. Gossypetin (3,5,7,8',3',4'-hexahydroxyflavone) is present in members of Empetraceae and Ericaceae but not in the other families (Moore et al. 1970). A t the subfamily level, Gesneriaceae is divided into two subfamilies, Gesnerioideae and Cyrtandroideae, primarily based on differences in the developmental fate of the cotyledons. The presence of 3-deoxyanthocyanins in members 21 R' OH O F L A V O N O L S R R ' R " R ' " Aelycones Kaempferol H H H H Quercetin H H O H H Isorhamnetin H H OCH3 H Myricet in H O H O H H Glycosides K-3-0-glucoside G l u H H H Q-3-O-rhamnoside Rha H O H H Q-3 -O-diglucoside G l u - G l u H O H H Q-7-0-galactoside H H O H Ga l Q-3-0-methyl ether OCHs H O H H F ig . 1.3 Some common variations observed in flavonols 22 Chapter 1. Introduction of Cyrtandroideae, but not in the Gesnerioideae supports the division of the Gesneriaceae into these two subfamilies (Harborne 1966). A t the generic level, flavonoids agree with the rbcL sequence data that Itea (native to eastern Asia) is most closely related to Pterostemon (native to Mexico) in the Saxifragaceae. Detailed flavonoid examinations of some 30 genera in the Saxifragaceae revealed that Itea and Pterostemon were the only genera to contain C-glycosylflavones (Bohm et al. unpublished). 1.3.2. Previous Flavonoid Studies of the Lauraceae Information on the flavonoid chemistry of Lauraceae is limited considering the size o f the family. Flavonoids have been reported from only 12 genera out o f the 50 or so currently recognized, and in most cases no attempt was made to use the isolated flavonoids for taxonomic purposes. Previously described flavonoid compounds from the family are summarized in Table 1.5. Flavonols appear to be the most common compounds, particularly derivatives of kaempferol and quercetin. These compounds have been reported from species of Actinodaphne, Beilschmiedia, Cinnamomum, Cryptocarya, Laurus, Litsea, Machilus, Nectandra, Ocotea, Persea and Umbellularia. Other types of flavonoids that occur are isorhamnetin {Beilschmiedia, Cryptocarya and Umbellularia) and myricetin {Ocotea). In addition, unusual flavonols such as quercetin-5-O-methyl ether {Beilschmiedia), quercetin-3-O-methyl ether {Phoebe) and B-ring deoxyflavonols {Aniba and Lindera) have been reported. Derivatives of the flavone luteolin have been found in species of Beilschmiedia. C-Glycosylflavones as well as apigenin and luteolin 23 a s o S1 o w "3 c o « 1/3 a o >> o CO i • co I a cd i -i o I co a I 9 ro "5b i 9 i 13) 6 I • i a _>» i 9 C O *bb O C O I ti cd •a 6 i i o i m i a a co C o o co C O on CO a o > cd x o ca -a bo .g I PQ CD fl O JO "cd u on co d o o U to CO « O o Id o o >H o u cd •a c3 ed -a i o i a a j2 "Bo O bo "bb 6 "? cd C O Tab • O CO I a cd" -a i o I C O a cd CO o cd I co oo ON T3 CD • l-H +-» C CO .12 Si O '> CO >H . CO 'o a o P H la l l 0) N CD T 3 P H CD •s a o H <3 ON I PQ CO CD 'I '1 •S ON NO ON N -a c co u co a o •§ <3 ca s '3 OH C O 00 O N O •a H H S s o S 53 SC K N O O N O N a o u ON > O c3 ,—v N O N O O N ON a a cd cd H H T3 CO CO co CO h-1 (N NO ON 3 cd u H I oo oo ON .3 qo ON ON o ON ON CO % cd PH T3 Cl cd O N O N 1 PH O K S a u s S ti •—. ca a on O •S s bo a CD s s cj J3 24 P4 l-H "bb "bb *3 IR IR O C O "bb cV 13) "bib _>> "bb 1 ja 1 O 1 U 1 o 9 "bb i "bb C O O i on on on cV x, M-3 u, K-3 ^, Flav Flav ^, Flav "3) bfl • "bO m "Hb 9 • e-7 9 9 9 !H Q-3 c o C O C O C O "bb ethe Q-3 cV cV cV cV ethe H ^ ;, Lute, » "bb a glu ;, Lute, '5b >> "bb "bb O cu me 1 9 1 o ;, Lute, 1 O o 1 o e o O C O „ bfl ari, C O ou "a. a l C O 1 C O • 1 1 C O < PH cV ON" 00 T ? O N O N i / s O N O N O N o T - H O N O N O N O N a' O N O N ^ — s ,—N N ^ •~~» »—i ^r Tt- N O o o v — ' ^ — ' r- r- r-- O N PQ Q O N O N O N O N T3 0) ^—' —' s — / ^ — ' ID ikom bosa-cez e • i-H o • rH fford fford fford fford bonn rtinez Neville a Kar Bar Gar Mei Wo Wo Wo Wo Har Mai Neville a fe •S orni § roc a c a c om •S 5 tandra glc ?ea amern s — / u_l a hunberg* tandra glc tea ve//o ?ea amern Hgue (L orbonia umilis (. alustris Si , to 1 a. bellulari Nea Oco Pen P. fl a-i Umt O cd '3 O o o 3 T3 O bO O II ^ Cl II "bb > T3 r2 o ~ - 8 ^ "3) PH II _H oo B 11 bb >. • is o ^ s § " I bO o Cl 0) cd .1 'g ,o 2 <4H Q oo cd o M o II Cl 'B u bO .S cd d '3 o ,cd "S OH jd PH CD bO cd § ^ ». II 3 a oo O .3 H 00 Cl O 'I '> CJ > H „r cd oo 'ob.2-cl bfl "bb o Cl o o I "bb bO ' I 1 H-l 25 Chapter 1. Introduction have been isolated from Persea. Chalcones in Lindera and flavanones, particularly naringenin, in Litsea and Machilus have also been reported (see Table 1.5). 1.4. Objective of the Study The purpose of this study was to survey and characterize the flavonoid profiles of the Lauraceae and to determine i f flavonoids would provide additional information that might assist in arriving at a clearer classification of the family, rcf 26 CHAPTER 2: METHODS AND MATERIALS 2.1 Source of Plant Material Leaves from 120 species representing 35 genera in the Lauraceae were examined for their flavonoid profiles (see Table 2.1). These samples were obtained from the Missouri Botanical Garden (MO) , the University of British Columbia herbarium ( U B C ) and University of Michigan herbarium (MICH) . Most of the neotropical samples were provided by Dr. Henk van der Werff from M O and most of the Asian samples were from U B C and M I C H . 2.2 Extraction of Flavonoids The extraction was based on the procedures of Wilkins and Bohm (1976) with minor modifications. Flavonoids were extracted from dried leaves either from recent collections or from herbarium specimens. The leaves were repeatedly extracted with 80% aqueous methanol at room temperature. The methanol extracts were combined and evaporated to dryness, and the dried residue was extracted with boiling water. The solution was filtered, and the filtrate extracted several times with an equal volume of water-saturated n-butanol. The n-butanol extracts were combined and evaporated to dryness in a rotary evaporator. The residue was taken up in a small volume of methanol and stored for flavonoid analysis by thin layer chromatography or high performance liquid chromatography (see Fig . 2.1 for summary of methods). 27 Table 2.1 The plant species used in this study Species Collector & number Actinodaphne chinensis Nees A. lancifolia (Sieb. & Zucc.) Meissner Shiu Y i n g H u 6340 U B C botanical garden (collected by G . Straley 1995) Aiouea dubia M e z A. obscura van der Werff Madrinan, et al. 728 Herrera5017 Alseodaphne semecarpifolia Nees Cooray & N . Balakrishnan 1148 Aniba firmula (Nees ex Mart.) M e z A. puchury-minor (Mart.) M e z G. Eiten & L . Eiten 5478 M . Crizon & L . Igurgo 8830 Apollonias barbuyana (Cav.) Bornm. Viera & Clavijo botanical garden (collected July 20,1992) Beilschmiedia sp. B. tawa (A. Cunn.) Benth. McPherson 15665 T. C. Brayshaw 59 Caryodaphnopsis burgeri Zamora & Poveda C. theobromifolia (Gentry) van der Werff & Richter G . Herrera 4979 van der Werff 12360 Cassytha filiformis L . C. micrantha Meissner C. pubescens R. Br. V . J . Krajina 620420046 K . I . Beamish 290 K . I . Beamish 785 Cinnamomum burmanii (Nees & Th. Nees) Blume C camphora (L.) J. Presl C. cassia (L.) Blume C costaricanum (Mez & Pittier) Kosterm. C. daphnoides Sieb. & Zucc. C. insularimontanum Hayata C. japonicum Sieb. ex Nees C. neurophyllum (Mez & Pittier) Kosterm. C. osmophldeum Kaneh. & T. H . Hsieh C. zeylanicum Blume C sp L . P. Wan & K . S. Chow 79143 G . Straley 3399 V . J. Krajina 620409005 G. Herrera 3541 G. Murata 12982 T .C . Huang 10160 G . Straley 5126 G. Herrera 5013 U B C herbarium 205495 V . J. Krajina 620317301 G. Herrera 4980 Cryptocarya australis Benth. C. chingii Cheng C. chinensis (Hance) Hemsley U B C herbarium 70382 K . S. Chow 78371 K . S. Chow 78119 28 Table 2.1 (Cont'd) The plant species used in this study Species Collector & number C. concinna Hance C. hypospodia F. Mue l l . C. latifolia Sonder C. mackinnomianum F. Mue l l . C. murrayi F. Mue l l . C. weyleri Stapf Dahlgrenodendron natalense J. Merwe & Wyk Dehaasia cuneata Blume Endiandra sieberi Nees Endlicheria formosa A . C . Sm. Gamanthera herrerae van der Werff Hypodaphnis zenkeri (Engl.) Stapf Laurus azorica (Seub.) Franco L. nobilis L . Licaria applanata van der Werff L. multinervis H . Kurz Lindera aggregata (Sims) Kosterm. L. angustifolia Cheng L benzoin Blume L. chinensis Ching L. chunii Merr. L. citriodora (Sieb. & Zucc.) Hemsley L. communis Hemsley L. erythrocarpa Makino L. glauca (Sieb. & Zucc.) Blume L. megaphylla Hemsley L. robusta (C. Allen) H.P. Tsui L. pbtusiloba Blume L. salitsifoliaJR\\ime) Boerl. K . C . Yang 693 K . I. Beamish 2357 Abbott 6206 K . I. Beamish 2357 K . I. Beamish 2351 Abbott 6205 Abbott 6207 A . D . E . Elmer 20949 Maiden 19991 G. Herrera 4967 G. Herrera 4996 McPherson 15471 Viera & Clavijo botanical garden (collected July 20,1992) Rodes & Adrichen 5738 van der Werff 12190 G. Herrera 4982 S. L . L i u 890163 Kunming Botanical Garden (collected M a y 18, 1991) W . C. M c C a l l a 12165 U B C botanical garden (collected by G . Straley 1995) K . S. Chow 78082 G . Straley 6408 K . S. Chow 78038 B . R. Yinger et al. 3768 Q. Wang 040 M . J . W . 1207 K . S. Chow 78422 K . Yao 11449 U B C botanical garden (collected by G . Straley 1995) 29 Table 2.1 (Cont'd) The plant species used in this study Species Collector & number L. sericea (Sieb. & Zucc.) Blume L. umbellata Thunb. B . R. Yinger et al. 3752 G. Straley 5042 Litsea acutineva Hayata L. akoensis Hayata L. calycaris (A. Cunn.) Benth. L. coreana var. sinensis Yang & P. H . Huang L. cubeba (Lour.) Pers. L. japonica (Thunb.) Jurs. L. sp. K . S. Chow 78490 T. C. Huang 10160 T.C. Bradshaw 56 K . Yao 11427 S. F. Huang 561 K . Iwatsuki et al. 199 Wagner 6646 Machilus chinensis Hemsley M. japonica Sieb. & Zucc. M. thunbergii Sieb. & Zucc. Shiu Y i n g H u 082472 M . Hiroe 14098 B . R. Yinger et al. 3222 Mezilaurus mahuba (Kuhlm.) van der Werff van der Werff 11852 Nectandra antillanira Meissner N. mathewsii Meissner N. purabela Nees H . Stehle & H . Smith 443 N e i l l 9486 A . Lofgren 10517 Neocinnamomum mekongense (Hand.-Mazz.) Kosterm. Kunming Botanical Garden (collected M a y 18, 1991) Neolitsea aurata Koidz . N. hiiranensis L i u & Liao N. sericea (Blume) Koidz . N. buisanensis Yamo. & Kami . K . Yao 11467 Y . B . Cheng 1001 H . Takahashi 1890 T. H . Hsieh 741 Ocotea bullata (Burchell) Meyer 0. floribunda (Sw.) M e z 0. foetens (Aitch.) Benth & Hook 0. quixos (Lam.) Schmidt 0. rivularis Standley & L . O . Will iams 0. rugosa van der Werff 0. schomburgkiana (Nees) M e z 0. sinuata (Mez) Rohwer 0. socliroana van der Werff 0. valeriodes W. Burger 0. sp. 0. sp. Abbott 6208 G. Herrera 5002 Viera & Clavijo botanical garden (collected July 20,1992) N e i l l 9487 G . Herrera 4976 B . Gray & G . Tipas 12429 L . J. Gillespie 1102 G . Herrera 4983 van der Werff 12402 G. Herrera 4986 Aguilar791 van der Werff 12504 30 Table 2.1 (Cont'd) The plant species used in this study Species Collector & number Parabenzoin praecox (Sieb. & Zucc.) Nakai P. trilobum Nakai Persia americana Mi l l e r P. borbonia (L.) Sprengel P. inchangensis (Rehder & E . Wilson) Kosterm. P. indica (L.) Sprengel P. mutisii H B K P. palustris (Raf.) Sarg. P. perseiphylla (C. Allen) van der Werff P. racemosa M e z P. Yunnanensis (Lecomte) Kosterm. P. sp. P. sp. Phoebe hunanensis Hand.-Mazz. P. tavoeyana Hemsley P. mexicana Meisnner P. sheareri (Hesmley) Gamble Pleurothyrium giganthum van der Werff P. insigne van der Werff P. krukovi A . C . Smith P. pauciflorum van der Werff & Hammel P. tomiwahlii van der Werff Povedadaphne quadriporata W . Burger Rhodostemonodaphne kunthiana (Nees) Rohwer R. sp. R. sp. Sassafras albidum (Nutt.) Nees Sassafras tzumu (Hemsley) Hemsley Umbellularia californica (Hook, and Arn.) Nutt. Williamodendron glaucophyllum (van der Werff) Kubitski & H . Richter G . Straley 5077 M . Hiroe 13327 F. C. Hoehne 29827 G. Straley 5203 G. Straley 7665 Viera & Clavijo botanical garden (collected July 20,1992) van der Werff 12332 J. B . N e l s o n 16089 van der Werff 12525 Novaes 10507 G. Straley 7664 van der Werff 12522 G. Herrera 4986 S.L. L i u 890168 K . S. Chow 78326 E . Matuda 1082 T. M . Tsui 755 van der Werff 12363 V D W type tree #11 Krukoff5255 G. Herrera 5006 van der Werff 12365 G. Herrera 5003 van der Werff 12358 van der Werff 12190 Madrinan & Cuadros 647 J. F. logue 2048 G. Straley 8498 G . Straley 6471 G . Herrera 5008 31 u d <D OH HP] AL a? u a H • »-H H-> a o • i-H < <D -t-> < CD S-H o o a CO *c3 'o a o o . CO CO CJ o O I-i CH a o • i-H tS O CJ i cd C o cd c o T3 CO j> | co O $ CO <4—I o GO CN 32 Chapter 2. Methods and Materials 2.3 Identification of Flavonoids 2.3.1. Thin Layer Chromatography (TLC) The flavonoid residue in methanol was spotted and chromatographed two dimensionally by using thin layers of Polyamide 6.6. The solvents used were a mixture of n-butanol-water-acetone-dioxane (40:15:10:5) for the first dimension and 1,2-dichloroethane-methanol-methylethyl ketone-water (50:25:21:4) for the second dimension (Gornall & Bohm 1980). The first solvent system resolves flavonoids according to the nature of their glycosylation (i.e., pentosides, hexosides, diglycosides) with the larger glycosides moving progressively further on the plate. The second system resolves compounds according to the number of free phenolic groups available (see Fig. 2.2 for illustration). After development the plates were dried in a fume hood and examined under U V (360 nm) light. The number and colour o f the flavonoid spots were recorded. Then, under U V the spots were fumed with ammonia and the colour reactions expressed by each flavonoid again recorded. Finally the plates were sprayed with Naturstoffreagent A , diphenylboric acid ethanolamine complex, and examined under U V . This spray reagent produces different colour reactions for flavonoids that depend on their hydroxylation patterns. A mixture of quercetin-3-O-arabinoside, 3-O-glucoside and 3-0-rutinoside was used as a chromatographic standard on each plate. This 2 D - T L C system was found very useful for determining the general flavonoid profile of the species involved; however, it was difficult to assess minor compounds using this method. A second system was used based on high performance liquid chromatography ( H P L C ) to address this problem and is described below. 33 r2 'oO Q TS U « TS a C/5 c U i O o 00 s o i o m i o e c c >> 0 ) « 1 C ca 1-1 •s ycosk 'oside-(9-glucoside >side O-trig] <9-gluc (9-glucoside X O rcetine cone Q-3-m i a Q-3-Q-3-Quei agly o o o o © c 03 DX) © snoanbe cd I co CO O O h-1 H I Q <N cd o ca I X ca CN <N 60 ca id • <—1 0 0 o o "00 9 ca co T3 •3. ^ 3 ca" -5 •55 o 0 1 v 11 O vo co ca" & -" 0 0 11 8 CN 3 ca" "3) •is 5 11 co ca" 1 -o a-a 11 o 0 0 1-1 ca -o cd3 CO a 11 t/3 34 Chapter 2. Methods and Materials 2.3.2. High Performance Liquid Chromatography (HPLC) The methanolic residues were subjected to high performance liquid chromatography ( H P L C ) using a Waters 600 pump controller equipped with Waters 996 photodiode array detector, and Waters 717 plus auto sampler. The system was controlled by Mil lenium 2.1 software and employed a 4 mm x 300 mm (5 mm) C18 column (Merck LiChrosphere) equipped with NovaPak (Waters) pre-column. The solvent system employed (A) tetrahydrofuran-isopropanol (65:35), (B) acetonitrile and (C) water containing 0.5% o-phosphoric acid. Analysis was performed using a gradient elution system modified from Hasler et al, (1992) with the following conditions: 0 min (15:5:80; tetrahydrofuran/isopropanol-acetonitrile-water), 7 min (15:7:78), 7.01 min (12:8:80), 12 min (12:10:78), 16 min (15:13:72), 20 min (5:25:70), 20.01 (5:30:65), 24 min (8:42:50), 24.01 (0:48:52), 30 min (0:78:22). The flow rate was 1 ml/min with detection over the wavelength range 200 to 600 nm and specific detection at 350 nm. Injection volume was 10 pi . Emerging flavonoid peaks were detected on the basis of U V - V i s spectra as determined by photodiode array detection and by comparison of retention time and U V -V i s spectra with those of known standards (see Fig. 2.3 for illustration). Class of flavonoid, i.e., flavone, flavonol, flavanone, etc. was readily established from the combined T L C and H P L C (using diode array detection) analysis. The level of glycosylation was also established although the nature of the di- and triglycosides was not determined. 35 36 Chapter 2. Methods and Materials 2.3.3. Fur ther Identification Unknown flavonoids were isolated using column chromatography on Sephadex L H 2 0 and purified using T L C (thin layer chromatography) according to methods from Gornall and Bohm (1980). For further structural identifications, flavonoid compounds were subjected to ultraviolet absorption analysis using standard techniques (Mabry et al., 1970). These techniques involved spectral shift reagents of sodium acetate, boric acid, aluminum chloride / hydrochloric acid, and sodium methoxide. Another spectroscopic method used, but to a much lesser extent, was mass spectral analysis which involved standard electron impact mass spectrographic techniques (Markham 1982). For identification of sugar molecules, purified glycoside compounds were subjected to total and partial acid hydrolysis using trifluoroacetic ( T F A ) and the sugars identified by using the method described by Ceska and Stiles (1984). 37 Chapter 3: RESULTS 3.1 Flavonoid survey of the Lauraceae A total of 120 species belonging to 35 genera in the Lauraceae were investigated for their flavonoid patterns. Eighty flavonoid compounds have been detected and their respective flavonoid classes identified. In addition, the exact structures of many of these compounds have also been determined. Noticeably, most of the taxa examined exhibit flavonoid patterns consisting of the following compounds from the flavonol class: quercetin-3-O-glucoside, quercetin-3-O-rhamnoside, quercetin-3-O-xyloside, kaempferol-3-0-glucoside and kaempferol-3-O-rhamnoside (see Fig. 3.1). Henceforth I w i l l refer to this particular pattern as 'the common flavonol monoglycosides'. Other compounds observed in this class are isorhamnetin-3-O-glycosides and myricetin-3-O-glycosides. Compounds belonging to four other flavonoid classes are also found to occur: flavones, flavanones, chalcones and dihydroflavonols. Flavones are the most common represented by apigenin, luteolin and chrysin. C-glycosylflavones are also known: orientin, isoorientin, vitexin and isovitexin. Flavanones are the next most common class and are represented by naringenin and pinocembrin. Members of the last two classes of flavonoids, chalcones and dihydroflavonols, are rarely observed in the family. The chemical structures of the common flavonoid compounds found in the family are illustrated in Fig . 3.2. The results of the flavonoid survey are illustrated in Table 3.2, and described over the next 23 pages. Comments on the size and geographical distribution of each genus are also provided. 39 C-glycosylflavones Fig . 3.2 Chemical structures of some of the common flavonoids observed in the Lauraceae 40 Chapter 3: Results 1. Actinodaphne Nees It is estimated that about 100 species Actinodaphne occur from India to Japan to Indonesia. Two species of Actinodaphne, both from China, have been examined for their flavonoid profiles: A. chinensis and A. lancifolia. They share similar flavonoid patterns which consist of the common flavonol monoglycosides. The difference between the two species is that kaempferol-3-O-xyloside is accumulated only mA. chinensis and an unidentified flavanone occurs only in A. latifolia. 2. Aiouea Aublet Aiouea consists of about 20 species found in Central and South America. Two species have been examined for their flavonoid profiles, Aiouea obscura from Costa Rica and A. dubia from Colombia. These two species exhibit very different flavonoid patterns. The pattern in A. obscura is based on only quercetin-3-O-glucoside, quercetin-3-0-rhamnoside and quercetin-3-O-rutinoside; by contrast, that of A. dubia exhibits numerous flavonoids including kaempferol-3-O-glucoside, kaempferol-3-O-rhamnoside, kaempferol-3-O-rutinoside, quercetin-3-O-rutinoside, an isorhamnetin diglycoside and three varieties of kaempferol triglycosides. 3. Alseodaphne Nees About 50 species exist; all are from tropical Asia . The only species of this genus available was A. semecarpifolia from Sri Lanka. Its flavonoid pattern consists of the common flavonol monoglycosides (except for quercetin-3-O-xyloside), 41 Chapter 3: Results quercetin-3-O-rutinoside, quercetin-3-O-diglucoside and kaempferol-3-O-dirhamnoside. 4. Aniba Aublet About 40 species of Aniba are recognized. They occur mainly in tropical South America and only rarely in Central America. Two species of Aniba have been examined for their flavonoid profiles. Both species, A. formula from Brazi l and A. puchury-minor from Ecuador, exhibit similar flavonoid patterns. In contrast to other lauraceous species, no flavonol glycosides were observed. The observed patterns consist solely of C-glycosylflavones, mostly based on isovitexin. Vi texin was observed only in A. puchury-minor. 5. Apollonias Nees Only two species exist, one o f which is endemic to the Canary Islands while the other to India. The only material available for analysis was A. barbuyana from the Canary Islands. This species accumulates the common flavonol monoglycosides, quercetin-3-O diglycoside, kaempferol-3-O-rutinoside and kaempferol-3-O-dirhamnoside. 6. Beilschmiedia Nees Beilschmiedia consists of about 250 species from Chile to Africa to N e w Zealand. Two species were examined for their flavonoid profiles, B. tawa from N e w Zealand and an unidentified Beilschmiedia species from Gabon. Both species exhibited the common flavonol monoglycosides and an acylated-quercetin-3-Oglycoside. The main difference 42 Chapter 3: Results is that quercetin-3-O-diglycoside occurs only in B. taw a while pinocembrin occurs only in the unidentified Beilschmiedia species. 7. Caryodaphnopsis Airy-Shaw Caryodaphnopsis, which consists of about 15 species, has a disjunct pattern of distribution. Seven species occur in As ia from Yunnan to Indochina while eight species occur in Central and South America. Two species, both native to tropical America, have been examined for their flavonoid profiles: C. burgeri from Costa Rica and C. theobromifolia from Ecuador. They showed similar flavonoid patterns, each consisting of quercetin-3-O-glucoside, quercetin-3-O-rhamnoside, quercetin-3-O-rutinoside and two naringenin derivatives. The only difference is that kaempferol-3-O-rhamnoside also occurs in C. theobromifolia. 8. Cassytha L. About 20 species comprise the genus Cassytha, most of which occur in Australia and the remainder in Africa, As i a and North to South America. Two Australian species, C. micrantha and C. pubescens, and one from Hawaii , C. jiliformis, were examined for their flavonoid profiles. The two Australian species share a greater similarity in flavonoid patterns with each other than with C. Jiliformis. The flavonoid profiles of both Cassytha micrantha and C. pubescens exhibit the common flavonol monoglycosides and quercetin-3-O-rutinoside, but the similarity ends there. Cassytha micrantha accumulates quercetin-triglycoside, luteolin-glycoside, isorhamnetin-3-O-glycoside and isorhamnetin-3-O-43 Chapter 3: Results diglycoside while only quercetin-3-O-diglycoside is accumulated by C. pubescens. The flavonoid profile of C. filiformis is unusual for the family in that the common flavonol monoglycosides are not detected. The other compounds observed in C. filiformis are quercetin-3-O-diglycoside, kaempferol-3-O-diglycoside, isorhamnetin-3-O-glycoside and isorhamnetin-3-O-diglycoside. 9. Cinnamomum Schaeff About 350 species exist. These occur mainly in tropical Asia , however some occur in Australia and the Pacific Islands and 60 species occur in Central and South America. Eleven species have been examined for their flavonoid profiles and interesting patterns are observed. The flavonoid profiles of C. burmannii and C. cassia, both from China, and C. osmophloeum from Taiwan showed identical patterns consisting of quercetin-3-0-rhamnoside, kaempferol-3-O-rhamnoside and three kaempferol triglycosides. Cinnamomum insularimontanum from Taiwan and C. zeylanicum from China exhibited similar profiles that consisted of the common flavonol monoglycosides (except for quercetin-3-O-xyloside), quercetin-3-O-rutinoside, kaempferol-3-O-rutinoside, kaempferol-3-O-dirhamnoside, kaempferol-3-O-diglycoside, quercetin-triglycoside and isorhamnetin triglycoside. The differences among these two are the presence of minor traces of quercetin and kaempferol triglycosides only in C. insularimontanum. A n interesting flavonoid pattern is observed in the distribution of C-glycosylflavones. Only the American species of Cinnamomum (C. species (Herrera 4980), C. neurophyllum 44 Chapter 3: Results and C. costaricanum ) exhibit these compounds. A n unidentified Cinnamomum species (Herrera 4980) accumulates the common flavonol monoglycosides (except for quercetin-3-O-xyloside) and share quercetin-3-O-rutinoside and kaempferol-3-O-diglycoside with C. costaricanum. Cinnamomum neurophyllum accumulates kaempferol-3-O-rutinoside, but unlike most other lauraceous species, flavonol monoglycosides are not observed. The flavonoid profiles o f C. camphora and C. daphnoides, both from China, and C. japonicum from Japan consist of the common flavonol monoglycosides. However, quercetin-3-(9-xyloside is present in only C. japonicum while quercetin-3-O-glucoside is absent in C. daphnoides. Cinnamomum camphora also accumulates apigenin glycoside. 10. Cryptocarya R. Br. Cryptocarya consists of about 350 species from Chile to Africa to Australia. Eight species of have been examined for their flavonoid profiles: C. australis, C. hypodaphnis, C. mackinnomianum and C. murrayi are from Australia, C. latifolia and C. weyleri from South Africa, C. concinna from Taiwan and C. chingii from China. Each of these species has a distinct flavonoid pattern, even though all accumulate the common flavonol monoglycosides. Cryptocarya australis, C. hypodaphnis, C. latifolia, and C mackinnomianum accumulate quercetin and kaempferol triglycosides, while C. latifolia exhibits isorhamnetin-3-O-diglycoside and C. mackinnomianum exhibit an isorhamnetin triglycoside. Pinocembrin is observed in C. chingii, C. hypodaphnis, C. latifolia and C. murrayi and two unidentified flavanones are found in C. latifolia. 45 Chapter 3: Results A n unidentified chalcone was observed only in C. mackinnomianum and an unidentified dihydroflavonol in C. hypodaphnis. Cryptocarya weyleri is distinguished by its accumulation of myricetin-3-O-rhamnoside, a compound found in only three other species in the family. 11. Dahlgrenodendron van der Merwe and van Wyk This genus consists of the single species, D. natalense, native to South Africa. The flavonoid profile of this species consist of quercetin-3-O-glucoside, quercetin-3-O-rhamnoside, kaempferol-3-O-rhamnoside, quercetin-3-O-rutinoside, quercetin-3-O-diglycoside, kaempferol-3-O-rutinoside and a naringenin glycoside. 12. Dehaasia Blume This genus consists of about 35 species all of which occur in As i a from Southern China to New Guinea. The only member of this genus available was D. cuneata from Borneo. Its flavonoid profile consists of quercetin-3-O-xyloside, quercetin-3-O-rhamnoside, kaempferol-3-O-rhamnoside, quercetin-3-O-rutinoside and kaempferol-3-O-rutinoside. 13. Endiandra R. Br. About 100 species comprise this genus, most of which occur in Australia with others known in As i a and the Pacific Islands. The single specimen available, E. sieberi from Queensland, Australia exhibits a mixture of quercetin-3-O-glucoside, quercetin-3-0-xyloside, kaempferol-3-(9-glucoside and pinocembrin. 46 Chapter 3: Results 14. Endlicheria Nees About 40 species of Endlicheria are known, all of which are found in Central and South America. The only material available was E. formosa from Costa Rica. Its flavonoid profile consists of quercetin-3-O-glucoside, quercetin-3-O-rhamnoside, kaempferol-3-O-rhamnoside, kaempferol-3-O-rutinoside, a quercetin triglycoside and two kaempferol triglycosides. 15. Gamanthera van der Werff This genus consists of the single species Gamanthera herrerae, native to Costa Rica. The flavonoid profile consists of quercetin-3-O-glucoside, kaempferol-3-C?-glucoside, kaempferol-3-O-rhamnoside, an acylated quercetin-3-(9-glucoside, isorhamnetin-3-O-glucoside, quercetin-3-O-rutinoside, a kaempferol triglycoside and three isovitexin glycosides. 16. Hypodaphnis Stapf. This genus consists of a single species, Hypodaphnis zenkeri, from Central Africa. The flavonoid profile of this species is the most distinctive among the species examined in the family. The profile consists of only kaempferol-3-O-rutinoside and the flavone chrysin, which was not observed in the rest of the family. 17. Laurus L . Two species o f Laurus exist, one in Macaronesia and the other in the Mediterranean region. Both species were examined for their flavonoid profiles: L. nobilis from the 47 Chapter 3: Results Mediterranean area and L. azorica from the Canary Islands. The flavonoid profiles of the two species are almost identical, consisting of the common flavonol monoglycosides, isorhamnetin-3-O-glucoside, quercetin-3-O-rutinoside, kaempferol-3-O-rutinoside, isorhamnetin-3-O-diglycoside, an isovitexin glycoside and a vitexin glycoside. The only difference is that a kaempferol-3-O-diglycoside was observed in L. nobilis but not in L. azorica. 18. Licaria Aublet Licaria consists of about 40 species in Central and South America. Two species have been examined for their flavonoid profiles, L. applanata from Ecuador and L. multinervis from Costa Rica. These two species exhibit very dissimilar flavonoid patterns. The pattern in L. multinervis is based on the common flavonol monoglycosides (except for kaempferol-3-O-glucoside), quercetin-3-(9-rutinoside, kaempferol-3-Odiglycoside, and two isovitexin glycosides. B y contrast, that of L. applanata lacks flavonol monoglycosides and diglycosides but exhibits a quercetin triglycoside and three kaempferol triglycosides. 19. Lindera Thunb. About 100 species comprise the genus Lindera, most of which occur in Asia . The rest occur in Eastern U S A and one in Australia. Fourteen species of Lindera have been examined for their flavonoid profiles and interesting patterns were observed. The flavonoid profiles of L. benzoin from Eastern U S A , L. citriodora and L. umbellata, both 48 Chapter 3: Results from China, show identical patterns consisting of kaempferol-3-O-glucoside, kaempferol-3-O-rhamnoside, kaempferol-3-O-diglucoside, kaempferol-3-O-rutinoside kaempferol-3-O-dirhamnoside, four kaempferol triglycosides and a chalcone. Another set of species that exhibited identical flavonoid profiles is L. glauca and L. sericea, both from Korea, L. megaphylla from Taiwan and L. salitsifolia from China. Their common flavonoid pattern consists of the common flavonol monoglycosides, quercetin-3-0-rutinoside, kaempferol-3-O-rutinoside, and luteolin-7-O-glycoside. This set of compounds minus luteolin-7-O-glycoside is also observed in L. communis, L. angustifolia and L. chinensis. Lindera chinensis also exhibits isorhamnetin-3-O-diglycoside and methylated kaempferol-3-O-glycoside. Lindera robusta from China is the only other species in the genus to accumulate isorhamnetin-3-O-diglycoside. The other compounds observed are quercetin-3-<9-xyloside, kaempferol-3-O-xyloside, acylated quercetin-3-O-glycoside, acylated kaempferol-3-O-glycoside, quercetin-3-O-dirhamnoside and a chalcone. The flavonoid profile of L. erythrocarpa from China consists only of the common flavonol monoglycosides (except for quercetin-3-(9-xyloside). In contrast, the profile of L. chunii from China lacks all of the common flavonol monoglycosides except for kaempferol-3-O-rhamnoside. It also exhibits kaempferol-3-O-rutinoside, a quercetin-triglycoside, and two varieties of kaempferol triglycosides. Finally, L. obtusiloba consists of the common flavonol monoglycosides (except for quercetin-3-O-xyloside), and three unidentified flavanones. 49 Chapter 3: Results 20. Litsea Lam. About 400 species of Litsea are recognized. They occur mostly in As ia , several in Australia and the Pacific Islands, and only rarely in Central and North America. Seven species of Litsea have been examined for their flavonoid profiles. Litsea calycaris from China and L.japonica from Japan exhibit very similar flavonoid patterns. Their patterns consist of the common flavonol monoglycosides (except for quercetin-3-O-xyloside), myricetin-3-O-rhamnoside and quercetin-3-O-rutinoside. The differences lie in the accumulation of an acylated kaempferol-3-O-glycoside, quercetin-3-O-diglucoside and quercetin-3-O-dirhamnoside in L. calycaris but not in L. japonica. The rest of the species exhibit distinctive flavonoid patterns. The pattern in L. cubeba from China consist of quercetin-3-O-rhamnoside, kaempferol-3-O-glucoside, kaempferol-3-O-rhamnoside, isorhamnetin-3-O-glucoside, kaempferol-3-O-diglucoside, kaempferol-3-O-dirhamnoside and two kaempferol triglycosides. Litsea akoensis from Taiwan accumulates the common flavonol monoglycosides, an acylated quercetin-3-O-glycoside, quercetin-3-O rutinoside, kaempferol-3-(9-rutinoside and isorhamnetin-3-O-diglycoside. Litsea acutineva from China accumulates quercetin-3-O-glucoside, kaempferol-3-O-glucoside, kaempferol-3-O-rhamnoside, an acylated kaempferol-3-O-glycoside and kaempferol-3-O-dirhamnoside. The only species to accumulate pinocembrin in this genus is L. coreana. The other compounds observed are the common flavonol monoglycosides (except for kaempferol-3-Oglucoside) and quercetin-3-O-rutinoside. Lastly, the unidentified Litsea species (Wagner 6646) exhibits a flavonoid pattern consisting of quercetin-3-O-50 Chapter 3: Results glucoside, quercetin-3-O-rharnnoside, kaempferol-3-O-rhamnoside and two naringenin derivatives. 21. Machilus Nees (currently placed under Persea) Machilus species occur mainly in temperate Asia . Three species of Machilus have been examined for their flavonoid profiles. Machilus japonica from Japan and M. thunbergii from Korea share identical flavonoid patterns consisting of the common flavonol monoglycosides, a methylated kaempferol-3-O-glycoside, quercetin-3-O-rutinoside and quercetin-3-O-dirhamnoside. The other species, Machilus chinensis from China, exhibit a different flavonoid profile consisting o f quercetin-3-O-glucoside, quercetin-3-0-rhamnoside, kaempferol-3-O-rhamnoside, luteolin-7-O-glycoside, orientin, isoorientin, and vitexin. 22. Mezilaurus kuntze ex Taubert About 20 species of Mezilaurus are known, all of which are found in the tropics of South America. The single specimen available, M. mahuba from Brazi l , exhibited a mixture of the common flavonol monoglycosides (except for quercetin-3-O-xyloside), kaempferol-3-O-rutinoside, kaempferol-3-O-dirhamnoside, vitexin and isovitexin-glycoside. 23. Nectandra Rolander ex Rottb. About 120 species comprise this genus, most of which occur in the tropics and subtropics of Central and South America. Three species of Nectandra, all native to Brazi l , have 51 Chapter 3: Results been examined for their flavonoid profiles. Each species exhibits a distinctive flavonoid pattern; although N. antillanira and N. mathewsii are more similar to each other than to N. purabela. Nectandra antillanira accumulates the common flavonol monoglycosides (except for quercetin-3-O-xyloside), a quercetin-3-O-diglycoside and kaempferol-3-O-dirhamnoside. Nectandra mathewsii accumulates the common flavonol monoglycosides, quercetin-3-O-rutinoside, a quercetin-3-O-diglycoside, and a kaempferol-3-O-diglycoside. B y contrast, the profile of N. purabela contains the common flavonol monoglycosides, a luteolin glycoside, orientin, isoorientin, vitexin and isovitexin. 24. Neocinnamomum H . L i u Neocinnamomum consists of about six species, all o f which occur in As i a from Southern China to Vietnam. The only member of this genus available for analysis was N. mekongense from China Its flavonoid profile consists of quercetin-3-O-glucoside, quercetin-3-O-rhamnoside, kaempferol-3-O-rhamnoside, quercetin-3-O-dirhamnoside and an unidentified naringenin derivative. 25. Neolitsea Merr. It is estimated that about 100 species make up this genus, most of which occur in Indochina with others known in Australia. Four species have been examined for their flavonoid profiles and all share similar flavonoid patterns. Neolitsea hiiranensis from Taiwan and N. sericea from China share identical flavonoid patterns which consist o f quercetin-3-Oglucoside, quercetin-3-O-rhamnoside, kaempferol-3-O-rhamnoside, quercetin-3-O-rutinoside and two derivatives of naringenin. The flavonoid pattern of the 52 Chapter 3: Results unidentified Neolitsea species from Taiwan exhibits the same compounds except for the additional occurrence of kaempferol-3-O-rutinoside and only one naringenin derivative. The pattern of N. aurata from China also exhibits the same compounds, however pinocembrin is observed instead of the naringenin derivatives. 26. Ocotea Aublet About 350 species comprise the genus Ocotea, most of which occur in tropical and subtropical America. The rest occur in Madagascar, a few in Africa and one in the Canary Islands. Twelve species were examined for their flavonoid profiles: O. quixos, O. rivularis, O. floribunda, O. sinuata, O. valeriodes and an unidentified Ocotea species (Aguilar 791), all from Costa Rica, O. rugosa, O. socliroana and an unidentified Ocotea species (van der Werff 12504), all from Ecuador, O. schomburgkiana from Guiana, O. bullata from South Africa and O. foetens from the Canary Islands. Each of these species shows a distinct flavonoid pattern. The pattern in O. bullata consists of the common flavonol monoglycosides, quercetin-3-O-rutinoside, kaempferol-3-O-rutinoside, a quercetin triglycoside and a kaempferol triglycoside. Ocotea foetens is the only other species to accumulate a quercetin triglycoside. It is also the only species to accumulate an isorhamnetin compound, the isorhamnetin-3-O-diglycoside. The rest of its profile consists of quercetin-3-O-glucoside, quercetin-3-O-rhamnoside, kaempferol-3-O-rhamnoside, kaempferol-3-O-rutinoside, apigenin-7-O-glucoside and the following C-glycosylflavones: vitexin, isovitexin, and two isovitexin glycosides. Another species that accumulates these four C-glycosylflavones is O. rugosa. The rest of O. rugosa''s profile 53 Chapter 3: Results consists of quercetin-3-O-glucoside and kaempferol-3-O-glucoside. A n unidentified Ocotea species (Aguilar 791) also accumulates C-glycosylflavones: orientin, isoorientin, vitexin and isovitexin. The rest of its profile consists of quercetin-3-O-glucoside, quercetin-3-O-rhamnoside, kaempferol-3-O-glucoside, kaempferol-3-0-dirhamnoside and an unidentified flavanone. Another unidentified Ocotea species (van der Werff 12504) also accumulates kaempferol-3-O-dirhamnoside plus the common flavonol monoglycosides (except for kaempferol-3-O-glucoside) and quercetin-3-O-rutinoside. The flavonoid profile of Ocotea floribunda consists of quercetin-3-O-rhamnoside, kaempferol-3-O-glucoside, kaempferol-3-O-rhamnoside and quercetin-3-O-rutinoside. Both O. quixos and O. rivularis also accumulate quercetin-3-O-rutinoside and kaempferol-3-O-rutinoside. The former also accumulates quercetin-3-O-rhamnoside, kaempferol-3-O-rhamnoside while the latter accumulates the common flavonol monoglycosides (except for quercetin-3-Oxyloside). The flavonoid profile of O. sinuata is composed of quercetin-3-O-rhamnoside and two derivatives of naringenin. Ocotea socliroana also accumulates a naringenin compound plus quercetin-3-O-glucoside, quercetin-3-O-rhamnoside, kaempferol-3-O-rhamnoside, quercetin-3-O-arabinoside, kaempferol-3-O-arabinoside and an unidentified flavanone. The flavonoid profiles of both O. valeriodes and O. schomburgkiana exhibit a small number of compounds. The former accumulates two compounds, quercetin-3-O-rhamnoside and kaempferol-3-O-rhamnoside while the latter accumulates three: quercetin-3-O-xyloside, quercetin-3-O-rhamnoside, kaempferol-3-O-rhamnoside. Overall, the flavonoid profiles 54 Chapter 3: Results were more similar within the group of tropical American species than with those of South Africa or the Canary Islands. The exceptions are the two species from America that share the C-glycosylflavones with O. foetens from the Canary islands. 27. Parabenzoin Nakai (currently included in Lindera) Parabenzoin species mainly occur in temperate Asia . Two species of Parabenzoin, both from China, were examined for their flavonoid profiles. Both species, P. trilobum and P. praecox, exhibit similar flavonoid patterns consisting of the common flavonol monoglycosides and quercetin-3-O-rutinoside.. The difference is that a quercetin-3-O-triglycoside occurs only in P. trilobum. 28. Persea Mi l l e r About 200 species of Persea are known. They occur in tropical to temperate regions of Asia , North, Central and South America, plus one species in the Canary Islands. Twelve species of Persea have been examined for their flavonoid profiles. The two species from southeastern U S A , P. borbonia and P. palustris, exhibit very similar flavonoid patterns consisting of the common flavonol monoglycosides, quercetin-3-c7-rutinoside, luteolin-7-O-glycoside, two unidentified flavanones and the following C-glycosylflavones: orientin, isoorientin, vitexin and isovitexin. The only difference is that a methylated kaempferol-3-O-glycoside occurs only in P. borbonia. Persea indica, endemic to the Canary Islands, also accumulates the unidentified flavanones mentioned; the rest of the profile consists of the common flavonol monoglycosides (except for quercetin-3-O-xyloside), kaempferol-3-55 Chapter 3: Results O-dirhamnoside and isorhamnetin-3-O-diglycoside. Among the species examined in this genus, it is only species to accumulate isorhamnetin. Another species which accumulates C-glycosylflavones is P. yunnanensis from China. Its profile consists of quercetin-3-0-glucoside, quercetin-3-O-xyloside, kaempferol-3-Oxyloside, quercetin-3-O-rutinoside, an apigenin glycoside, a luteolin-7-O-glycoside, orientin, isoorientin, isovitexin and an isovitexin glycoside. A n unidentified Persea species (Herrera 4987) from Costa Rica also accumulates C-glycosylflavones: isovitexin and vitexin. N o other compounds were observed. Among the South American species examined, P. perseiphylla from Colombia, P. racemosa from Brazi l , P. mutisii and an unidentified Persea species (van der Werff 12522), both from Ecuador, all exhibit similar flavonoid profiles. The observed patterns consist mainly of the common flavonol monoglycosides (except kaempferol-3-O-glucoside is not observed in P. racemosa or in the unidentified Persea species). The differences observed among these species are that kaempferol-3-O-rutinoside occurs only in P. perseiphylla and luteolin-7-O-glycoside occurs only in P. racemosa. Persea americana collected from Brazi l accumulates quercetin-3-O-glucoside, quercetin-3-O-rhamnoside, an acylated quercetin-3-O-glycoside and a quercetin triglycoside. This is the only species in this genus to accumulate an acylated quercetin-3-O-glycoside. Lastly, the flavonoid profile of Persea inchangensis from China consists of the common flavonol monoglycosides (except for kaempferol-3-O-glucoside), quercetin-3-O-rutinoside and a methylated quercetin-3-O-glycoside. 56 Chapter 3: Results 29. Phoebe Nees About 100 species comprise this genus, most of which occur in As i a and a few in Central and South America. In his recent treatment of the Lauraceae, Rohwer (1993b) treats the American species of Phoebe as Cinnamomum. Four species have been examined for their flavonoid profiles. Phoebe hunanensis and P. sheareri, both from China, share identical flavonoid patterns. The observed patterns consist of the common flavonol monoglycosides, kaempferol-3-O-xyloside, apigenin-7-O-glycoside, vitexin glycoside and isovitexin. Phoebe tavoeyana, also from China, accumulates vitexin, vitexin-glycoside, isovitexin and an isovitexin glycoside. In contrast to other lauraceous species, no flavonol glycosides were observed. Phoebe mexicana from Mexico exhibits a unique pattern consisting of the common flavonol monoglycosides, kaempferol-3-O-xyloside, isorhamnetin-3-O-rhamnoside, a quercetin-3-O-diglycoside, an isorhamnetin-3-O-diglycoside and three varieties of isovitexin-glycosides not found in the other three species. 30. Pleurothyrium Nees ex Lindley Pleurothyrium consists of about 45 species found in Central and South America. Five species have been examined for their flavonoid profiles. Each of the five species exhibits a distinct flavonoid pattern. The patterns of P. insigne and P. pauciflorum, both from Costa Rica, contains the common flavonol monoglycosides. The differences between the two species are that kaempferol-3-O-xyloside occurs only in P. insigne while vitexin and isovitexin occur only in P. pauciflorum. Pleurothyrium krukovi from Brazi l 57 Chapter 3: Results also accumulates the common flavonol monoglycosides (except for kaempferol-3-0-glucoside) plus an acylated quercetin-3-O-glycoside, quercetin-3-O-diglucoside, quercetin-3-O-rutinoside, a quercetin-3-O-diglycoside and two quercetin triglycosides. The flavonoid pattern of P. giganthum from Ecuador exhibits kaempferol-3-O rhamnoside, quercetin-3-O-rutinoside, kaempferol-3-O-rutinoside, two kaempferol triglycosides, and two naringenin derivatives. The other species from Ecuador, P. tomiwahlii, exhibits a pattern consisting of quercetin-3-O-glucoside, quercetin-3-0-diglucoside, quercetin-3-O-rutinoside and three quercetin triglycosides. 31. Povedadaphne Burger Povedadaphne quadriporata is a monotypic genus from Costa Rica. The flavonoid profile of this species consists of kaempferol-3-O-glucoside and isorhamnetin-3-0-rhamnoside. 32. Rhodostemonodaphne Rohwer and Kubitzki This genus is estimated to contain 20 species that occur mostly in the tropics of Central and South America. Three species were examined for their flavonoid profiles, although only one species was identified: R. kunthiana from Costa Rica. The other two unidentified species were collected from Ecuador. The flavonoid pattern of R. kunthiana consists of isorhamnetin-3-O-diglycoside, a quercetin triglycoside and two kaempferol triglycosides. The two unidentified species share identical flavonoid patterns consisting of kaempferol-3-O-rhamnoside, quercetin-3-O-rutinoside and kaempferol-3-0-58 Chapter 3: Results dirhamnoside. In contrast to many other lauraceous species, no quercetin-3-0-monoglycosides were observed in these three Rhodostemonodaphne species. 33. Sassafras Nees and Eberm. Three species of Sassafras are known, one in eastern North America and two in China. Two species have been examined for their flavonoid profiles. Both species, S. albidum from eastern U S A and S. tzumu from China, share similar flavonoid patterns consisting of quercetin-3-O-glucoside, kaempferol-3-O-glucoside, kaempferol-3-O-rhamnoside, quercetin-3-O-rutinoside, kaempferol-3-O-rutinoside and kaempferol-3-O-dirhamnoside. The differences between these two species is that quercetin-3-O-rhamnoside and a quercetin-3-O-diglycoside occur only in S. albidum. The similarity in flavonoid patterns between the two species illustrates the well known eastern North America and eastern As i a disjunct distribution pattern. 34. Umbellularia (Nees) Nutt. The single species of Umbellularia, U. californica, occurs in western North America. The flavonoid profile of this species consists of quercetin-3-O-rhamnoside, kaempferol-3-O-rhamnoside, quercetin-3-(9-rutinoside, kaempferol-3-O-rutinoside, an isorhamnetin-3-O-diglycoside, a quercetin triglycoside, a kaempferol triglycoside and an isorharnnetin triglycoside. 35. Williamodendron Kubi tzki and H . Richter Three species exist, all of which occur in Central and South America. Williamodendron 59 Chapter 3: Results glaucophyllum from Costa Rica was the only species sampled. The flavonoid profile of this species consists of quercetin-3-O-glucoside, quercetin-3-O-rhamnoside, kaempferol-3-O-rhamnoside, myricetin-3-O-rhamnoside, quercetin-3-O-rutinoside, kaempferol-3-O-rutinoside, a kaempferol-3-O-diglycoside, a quercetin triglycoside and two kaempferol triglycosides. Table 3 . 1 . Index numbers assigned to the flavonoid compounds F L A V O N O I D C O M P O U N D S F L A V O N O L S Monoglycosides 1 quercetin-3-O-glucoside 2 quercetin-3-O-xyloside 3 quercetin-3-O-arabinoside 4 quercetin-3-Orhamnoside 5 kaempferol-3-O-glucoside 6 kaempferol-3-O-xyloside 7 kaempferol-3-O-arabinoside 8 kaempferol-3-O-rhamnoside 9 quercetin-3-O-methyl ether 10 quercetin-3-O-glycoside A (acylated?) 11 kaempferol-3-O-glycoside A (acylated?) 12 kaempferol-3 -Omethyl ether 13 isorhamnetin-3-O-glucoside 14 isorhamnetin-3-O-rhamnoside 15 myricetin-3-O-glucoside 16 myricetin-3-O-rhamnoside 17 quercetin aglycone 18 kaempferol aglycone Diglycosides 19 quercetin-3-O-diglucoside 20 quercetin-3-O-rutinoside 21 quercetin-3-O-dirhamnoside 22 quercetin-3-O-diglycosides A 23 quercetin-3-O-diglycosides B 24 quercetin-7-O-diglycoside 25 kaempferol-3-O-diglucoside 26 kaempferol-3-O-rutinoside 27 kaempferol-3-(9-dirhamnoside 28 kaempferol-3-O-diglycoside A 29 kaempferol-3-(9-diglycoside B 30 kaempferol-7-O-diglycoside 31 isorhamnetin-3-O-diglycoside A 32 isorhamnetin-3-O-diglycoside B Triglycosides 33 quercetin triglycoside A 34 quercetin triglycoside B 35 quercetin triglycoside C 36 quercetin triglycoside D 37 quercetin triglycoside E 38 quercetin triglycoside F 39 kaempferol triglycoside A 40 kaempferol triglycoside B 41 kaempferol triglycoside C 42 kaempferol triglycoside D 43 kaempferol triglycoside E 44 kaempferol triglycoside F 45 kaempferol triglycoside G 46 kaempferol triglycoside H 47 kaempferol triglycoside I 48 kaempferol triglycoside J 49 isorhamnetin triglycoside A 50 isorhamnetin triglycoside B 51 isorhamnetin triglycoside C F L A V O N E S 52 apigenin-7-O-glycoside 53 apigenin glycoside 54 luteolin-7-O-glycoside 55 luteolin glycoside 56 luteolin glycoside 57 chrysin C-GLYCOSYLFLAVONES 58 isoorientin 59 isoorientin glycoside 60 orientin 61 orientin glycoside 62 isovitexin 63 isovitexin glycoside A 64 isovitexin glycoside B 65 isovitexin glycoside C 66 isovitexin glycoside D 67 vitexin 68 vitexin glycoside FLAVANONES 69 pinocembrin 70 naringenin 71 naringenin based 72 flavanone A 73 flavanone B 74 flavanone C DIHYDROFLAVONOLS 75 flavanone D 76 dihydroflavonol A 77 dihydroflavonol B CHALCONES 78 dihydrochalcone A 79 chalcone A 80 chalcone B 63 o C N 00 o I o U cj cd Cu) a 1 o 00 "2 'o A o I C N o CM + + + + • i + + + + + > • > + • + • + • + • • • + • i + i • + • • i • • • • co + + • i • i • • • • • + + • • • + • T— • • + • + i i • • + • • i • • • • • 1 CD T - • • -i i i • • • i • • • • lO • • • • i i i • • • • • • • • • • T— • • • • • l i l • • • • + • • • • • • • co • • • • i i i • • • i • • • • • • • CM T— • • i i • • • • • t • • • • • • • • • • • • i i i • • • • i • • • • • • o T— • • • • i + + + • • + • • • • • — cn • • • i i i • • • • • i • • • • • • 00 + • • + + + + + • r*. • • • • i i i • • • • i • • • • • • • • • co + • i + l • • i • • • • • • • • m + + + • • + + i • • • • • • + • + + + • + • + • t + + + • • + + + + • + + + + + + • + + co i • • • • • i • i • • • • • • • • • • • • • • CM + + • • + + + + • + + • • • • + • • + + + + • • + + + + + + • + + + • + • + • + • + ORIGIN China China Costa Rica Colombia Sri Lanka Brazil Ecuador Canary Is. New Zealand Gabon Gabon Costa Rica Ecuador Hawaii Australia Australia China China China Costa Rica China Taiwan Costa Rica Japan Taiwan China TAXON 1 Actinodaphne chinensis A. lancifolia Aiouea obscura A. dubia Alseodaphne semecarpifolia Aniba firmula A. puchurv-minor Apollonias barbuvana Beilschmiedia tawa B. so #1 CM . a CO CQ Carvodaphnopsis burqeri C. theobromifolia Cassytha filiformis C. micrantha C. pubescens Cinnamomum burmannii C. camphora C. cassia C. costaricanum C. daphnoides C. insularimontanum E "S -c Q 8 03 c O C. iaponicum C. osmophloeum C. zeylanicum 64 o CM + • • • + • • • + + + • < + + + + • + + < + > • + • at T— • • • • i • < • • • • • CO • • • • • • • i > > • • i i • • • • • i > > • + + • < • • • CO • • i • + i > > • • • • • • > • If) • • • • i i • • • < • • • • • • T— • • • • • i • • i • • • • • • co • • • • • • i • + < + + • • • • • • CM + • • • i l • • • • • • • + • • • • T— + i • • + • i • • • • • • • • • • • O T— • • + • • • • l + • • • • • • • • • CD • • • • i • • • i • i • • • < • • • • • • 00 + + + + + + + + • + + • + + + + + + r~~ • • t • • • • • • i • • • • • • • • • • • • CO • • • • + • • - • + • • i • • < • • • • • • m + + • + i + • + l + • + + • + + • + + + + + + + + + + + + • • + + • + + + • • + + co i + i • + i • • - • • i • • i • i • • • • CM • + + + + • + + i + + • • + + • + + • + • • + • T— + + + + + + + + + + l + + + • + + • + + • + • • + + ORIGIN Costa Rica Australia China Taiwan Australia South Africa Australia Australia South Africa Costa Rica Borneo Australia Costa Rica Costa Rica Gabon Canary Is. Mediterranean Ecuador Costa Rica China USA China China China China Korea TAXON C. sp. (Herrera 4980) Cryptocarya australis C. chingii C. concinna C. hypodaphnis C. latifolia C. mackinnomianum C. murrayi C. weyleri Dahlgrenodendron natalense Dehaasia cuneata Endiandra sieberi Endlicheria formosa Gamanthera herrerae Hypodaphnis zenkeri Laurus azorica L nobilis Licaria applanata L. multinervis Lindera angustifolia L. benzoin L. chinensis L. chunii L. citriodora L communis L. erythrocarpa 65 o CM + + • • + + • i + + + + + • i + + i • + > + + + + + + + • cn T — • • • i > > I i + • • l i i l i < • • • • • • CO > i > > + • i i l i i l i < i < < < • • • 1^ • • • + • i • i i i i • + i • • i i > (O i • • • < I • + • + i i i i • • > > l I > in T- i • • • • • i i i • • • i i i i • • • > • • i • • T t t • • i i i • • i i i i • • • • • < i i > n • < • • • i • i + • i • • l i > • • > l • • CM T- • • • i • • i i • i + + i • > • • i i • T -i • + • + i + • i i • i • i i i • o i + • • i + i • • i i • i • < • • • i i • cn i • i i i • • • i i • i • < • i i • • CO + + i + + + + + + + + + + + + + + + + + + + + + + + + + + i-» i • • • • • • i i • • • • • i i l i • • i i • to i • + • - • I i + • + • i l • • • i in + + • + + + + + + + • + + i + + + + + + • • • i + + • •<t + + • + + + i + + + + + + + + + + + + + + + + + + + + + co • • • • i i • i i • i i • i i • • • i CM + + + i + + I + • + i • + + i • + + + • • • + • • T— + + i + + + + + + + • + + + + + + + + + + + + + + + • + ORIGIN Korea Taiwan China China China Korea China China Taiwan China China China Japan Taiwan Hong Kong Japan Korea Brazil Brazil Brazil Brazil China China Taiwan China Taiwan South Africa Costa Rica Canary Is. TAXON L. glauca L. megaphylla L. robusta L. obtusiloba L. salitsifolia L. sericea L. umbellata Litsea acutineva L akoensis L. calycaris L. coreana L. cubeba L. japonica L. sp. (Wagner 6646) Machilus chinensis M. japonica M. thunbergii Mezilaurus mahuba Nectandra antillanira N. mathewsii N. purabela Neocinnamomum mekongense Neolitsea aurata N. hiiranensis N. sericea N. sp. (Wagner 6514) Ocotea bullata 0. floribunda 0. foetens 66 o CM + + • i • • • • + + + < + + • i + + • + • • • • + + • + o> T — • • i < • • • • i • < • i • • • • < • < • > • + • + CO • • • i • • < • l • • i • + + + > > • • • • • + • < • T — • l • + + • + + • + • • • + • • • • • • • CO • • • i • • • i • • • • • • • • • • • • • • • • u> • < i < • i • • • > • • • • • • • • • T — • • • l < • • l • • • • • > • • • • + • • • • • • CO < i • i • • • • • • • • • • • • • • • • • CM T - • • • l • • • i + • • • • • • • • • • • • • • • • • • • i • • • < • i • • • • • • • • • • • • o • • i < i • + • • • • • • • • • • • • • + • • • • • i • • • • • + • • • • • • • • • • • • • CO + + + • + + • + + + + + + + + + + • + • + + + + + + • l>~ • • i + • • • • • • • • • • • • • • • • • • • • • • • CD • • • i • • • • • • • + • • + • + • + • + • • lO • + + l • • • + + + + • + + + + • • • • + • + + + • • + • + + • + + + + + + + + + + + + + + + + • + + + • + + • co i • • l + • • • • • • i • • • • • • • • • • • • CM • i • + • • • i + + + • + + • + + + + + + • + • + + + • + + • + + i • + • + + + + + + + + + + + + + + • + • + + + • + + + ORIGIN Brazil Costa Rica Ecuador Guiana Costa Rica Ecuador Costa Rica Costa Rica Ecuador China China Brazil USA China Canary Is. Ecuador USA Colombia Brazil China Ecuador Costa Rica China China Mexico China Costa Rica . Ecuador Brazil Costa Rica Ecuador TAXON 0. quixos 0. rivularis 0. rugosa 0. schomburgkiana 0. sinuata 0. socliroana 0. valeriodes 0. sp. (Aguilar 791) 0. sp. (v.d. Werff 12504) Parabenzoin praecox P. trilobum Persea americana P. borbonia P. inchangensis P. indica P. mutisii P. palustris P. perseiphylla P. racemosa P. yunnanensis P. sp. (v.d. Werff 12522) P. sp. (Herrera 4987) Phoebe hunanensis P. tavoeyana P. mexicana P. sheareri Pleurothyrium insigne P. giganthum P. krukovi P. pauciflorum P. tomiwahlii o CM • • + + + + + + c n • • • • • CO • • r» T— • • CO < • • • + m • • • + ro V • • • • • CM T— • • • T -• + • • o • • • • c n • • • • CO • • + + + + + + h - • • • • • CD • • • m + • + + • • • • + + + ro • • • • • • CM • • • • + + • + ORIGIN Costa Rica Costa Rica Ecuador Ecuador USA Taiwan USA Costa Rica TAXON Povedadaphne quadriporata Rhodostemonodaphne kunthiana R. sp. (v.d. Werff 12190) R. sp. (Madrifian & Cuadros 647) Sassafras albidum S. tzumu Umbellularia califomica Williamodendron glaucophyllum 68 o • • 1 • • • + + + + cn CO • 1 • • • • • + + • + CO co • • r~. co • i • • + • • • • • • co co • i • • • • • • • + in CO • • • i • • • • • + + • + T t CO • i • • • • • • • • • • • • CO CO • i • • • • CM co • + • • + • • • • • • • • CO • • • i • • • + • • • o CO • • • i • • • • • • • • cn CM • • i • • • • • • • • • • • co CM • • + • • • • + • + • + CM i + + • • + + • • • + CO CM • + • + + + + • « • + + + + • + w CM • • i • • • • • « • • • • • • • T t CM • • • i • • • • • • • • • • • CO CM • i i • • • • • • • • • • • • • • CM CM • i • + + • • + • • • • • • T-CM • i i • • i 1 + • • • • • • • ORIGIN C !c O CD c !c O 03 O be CD -*—' CO O o CD J D E o o O 03 ^ c 03 _l *c CO N CD i CQ o T3 (D 3 O LU JO £r 03 c 03 o T3 c CD CD CD N ro z c o _Q CD CD c o X ) 0! (D ro o be CD -*-» CO o O 1 o "D CD 3 O LU TO TO X ro ro i _ -.—' CO 3 < ro ro u . CO 3 < 03 c x: CJ CD t= - C CJ ro c O TO O be ro to o O ro c !c O 0! 03 H ro o be ro to o CJ c 03 C L 01 —5 c 03 CD \— 03 !c CJ TAXON y> 55 c CD O CD C t o .c o .CD o c: ro C J CO -Q O CO CD 3 O •5 .CD •5 3 .ro I e-ro o ro E ro CO ro c •c Q . « O CD (o <. CD 3 I CD £ L _ O .c E 3 -c o 3 C L TO c 1 •e CD -Q CO .ro 5 o o Q. C L i JS ro "3 £ o CO 'ro CQ C L co CQ CN C L CO CQ c CD s> 3 •Q .<o 55 & c C L •8 CD o .co I E 8 -Q O CD £ .<o 1 CO 1 CO co CJ co £ d CO c ro o CO ro -Q 3 a Ci ' £ ro g 3 -Q £ 3 s o E ro c .c 0 2 o •c 1 ro o CJ .CD 55 CO CD u CJ E 3 C co .o c & CO O o d CO CD :o 5 c •c C L • g d E 3 C iS c o .E c CD 3 CO . £ d E 3 .O 5 o C L .TO — i d E "5, •c 3 ro c d E 3 CD O & E CO o d E 3 .O £ CD c£ N d 69 o l • • • • + • • • I • 1 + + + + • cn co l + + 1 < • • + • • • 1 • • • + • + co co l • • • + • • 1 • + • • • r» co l + + • • • • 1 • • • • cd co l • • • • - < • • 1 + • • • • 10 co i • • • • • + • • 1 • • • • • • co l • • • • • • • • 1 • • co co l • • • • • 1 • • cm co l • • • • • 1 • • • T— co l • • • + • • • + + • + • • o co l • • • • 1 • • • cn cm l • • • • • + • • • • 1 • • • • co cm + • • • • • + + • • 1*. cm I • • • • • • • • 1 • • • + • • + • co cm l • • • + • + + + + + m cm i • • • • • 1 + + cm 1 • • - i • • • 1 • • • 1 • co cm l • • • • i • • 1 • • • 1 t cm cm l • • + i i • • • 1 • 1 1 • cm • • i • . • • • • 1 • • • 1 1 ORIGIN CO o TO -*—. 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C •£ o '£ 3 •§ 2 .o c: o .to £ 3 E E o o -J CO & CO o s 1 CD — i 70 o T t l • • + • • • + • • • • + • cn ro l • • • • + • • • - • • • CO CO l • • • • • • • • • • + • co 1 • • • • • • • CO co 1 • • • • • • • • • • • • m CO l • • • • • • • • • • • • • • • • • + T t CO l • • • • • + • • • • • • • • CO CO l • • • • • • + • • • • • • • • CM co l • • • • • • • • • • • • • • T-co l • + • • • + • • • • • • • + o co l • • • • • • • • • • • • • • cn CM l • • • • • • • • • • • • • • 00 CM l • • • • • • • • • • + • • • • • • f -CM i • • • • + • + • • • • • • • CO CM + + • • + • + + • • + m CM • • • • + • • + • • • • • • • • • T t CM • • • • 1 1 • • • • • • • co CM • • • • • • 1 • 1 • • • • • CM CM i • i • • 1 1 • • • • • • • T— CM t • + • • 1 • + • 1 - + + • • + • • • ORIGIN ro cu i o ro ro h -ro c x: O ro c x: O ro c x: O ro 2 2 ro c x: O ro c jc O c ro ro H ro c x: O ro c x: O ro cz x: O c CO Q. ro —3 CZ ro ' r o \— cn c o ^ cn CZ o c ro CL ro ro 2 o N 2 CQ N ro CQ 2 CQ N 2 CQ ro c x: O ro CZ x: O c ro <: ro H CD c x: O CZ ro ro \— ro o *i_ -CZ "Z3 O CO ro o be ro -*—> CO o O ro c ro O TAXON ro o co ca ro ^ Q. ro o> CD E .CO CO -Q 2 ro •Q '55 3 -Q O .ro I ro CO -J ro CD .o c CD CO —j iS -2 ro -Q E 3 ro .c 3 O ro ro ro <2 Ci .CO 55 c ro ro Mi CC ro ro o —i CD c ro o o ro -Q CD •Q 3 o co ,o c o Q. .CD - J CD TT CD CD i CD CZ cn ro Q. U) Mi 55 c CD .C -5 o CO 3 s CD .O 5 o Q. .CD —i : | CD -Q C 3 si CD -Q 3 • C CD E CO 5 3 -2 N I .2 c ro C CD 1 C •S o i ro ro E 2: •2 CD -Q 2 3 CD CO C ro C35 c CD S E 3 E o S co c .c o o 1 -2 2 3 CD CD CD O 3 CO 55 c CD C 1 2: ro ro .o c ro CO 2: in CO i— ro CZ cn ro a. CO 2: -S -2 3 -Q CO -2 o o o c 3 :Q C o Ci CO c .CD CD d 71 o l • • • • • • • • • • • • • • 1 cn co l • • • • • • • 1 00 co 1 • • • • + • • • • • • • + i - . co l • • • • • • • • • • • • • • • • + CO CO l • • • • • • • • • 1 m CO l • • • • • • • • + CO l • • • • • • • • • • • • • • • • • • + • co CO l • • • • • • • + • CM co 1 • • • • • • • • • + • T -CO l • • • • • • • • • • • • • • o co l • • • • • • • + • • • cn CM l • • • • • • • • • • • 00 CM l • • • • • • • • • • • • r» CM 1 • • • • • • • + • • • • • • CD CM + • • • + IO CM 1 • • • • • • • • • • • •«* CM l • i • • • • • • • • • • • • • • • co CM l • • • • i • • • • • • CM CM l • i i • • • • • • • • • + T— CM l i • • i • • • • • • + • • 1 ORIGIN N ro i _ CO CO o be CD -4—' CO o O \ o "O CO ZJ o LU CD c CD 'zj O CD 0 be CD -*—' CO 0 0 1 0 •0 CO ZJ 0 LU CD 0 al CO -*—' CO 0 0 CO 0 al CD ^—' CO 0 0 0 T3 CO ZJ O U J CO cz 'sz 0 CO c. 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CD C .to J5 c 0 C -c Q. •8 o c o e to o "o o •c cc O <y> CN i at 5 •b CL to CC CD CO O -a CO 3 o o3 c co ;c T > CD d to CC E 3 CD to CD CO CO CD CO 3 s 3 •H CO CD E TO O CO c CD . 3 "cB -Q i E 3 & o 3 CO CD C 1 o E .CD i 83 Chapter 4. Discussion 4.1 Flavonoid Variation Among Genera Too numerous for manageable comparison at the generic level, the 80 flavonoid compounds observed have instead been grouped by flavonoid classes: chalcone, dihydroflavonol, flavanone, flavone, and flavonol. In turn the last two of these classes have been further subdivided: flavone into C-glycosylated flavones and non C-glycosylated flavones, flavonol into kaempferol, quercetin, isorhamnetin and myricetin plus the different levels of glycosylation (monoglycosides, diglycosides and triglycosides) and methylation. A total of thirteen flavonoid groups are identified, and Table 4.1 summarizes their presence or absence in the 35 genera of the Lauraceae. The distribution of the flavonoid groups are compared to the classification systems of Kostermans (1957), Rohwer (1993b), and van der Werff and Richter (1996). A s in the case of morphological characters, only certain flavonoids are found to be useful taxonomically. Occurrences of the remaining flavonoids are too widespread throughout the family to illuminate patterns of relationships among genera. I w i l l begin by discussing the distribution of flavonoid groups that are found to be taxonomically useful, and then those groups that are not. Distribution of C-glycosylflavones: The C-glycosylflavones identified in this study are the common pairs, vitexin / isovitexin and orientin / isoorientin. The more common of these is the vitexin / isovitexin pair which were observed in twelve genera: Apollonias, Cinnamomum, Gamanthera, Laurus, Licaria, Machilus, Mezilaurus, Nectandra, Ocotea, Persea, Phoebe, and 84 Chapter 4. Discussion Pleurothyrium. The other pair, isoorientin / orientin, were observed in four genera: Cinnamomum, Mezilaurus, Nectandra and Persea (see Table 4.1). C-Glycosylflavones are perhaps the most useful taxonomic character of all the flavonoid groups examined. A l l of the genera listed above, except for Laurus, are placed in the tribe Perseeae by van der Werff and Richter and in the Ocotea group by Rohwer (although in three different subgroups). In contrast, these genera, except for Laurus, are split into two separate tribes, tribe Cinnamomeae and Perseeae, by Kostermans (1957) (see Table 1.1-1.3). Therefore, the distribution of C-glycosylflavones in the Lauraceae supports Rohwer's and van der Wer f f s classifications in preference to Kostermans'. The unexpected grouping of Laurus with the above genera by C-glycosylflavones w i l l be further discussed in section 4.2. Distribution of flavones: The flavones of Lauraceae are based upon apigenin, chrysin and luteolin. The most common is luteolin which occurs as 7-O-glucoside and as two unidentified glycosides. This set of luteolin compounds was observed in six genera: Cassytha, Cryptocarya, Lindera, Mezilaurus, Neocinnamomum, and Persea. These six genera are placed in at least three different tribes or groups in the three classification systems (see Table 1.1-1.3). Therefore, the distribution of luteolin is too scattered in the family to be useful taxonomically (see Table 4.1). However, apigenin, which occurs as the 7-<9-glucoside and as an unidentified glycoside, is taxonomically more useful. This set of compounds was observed in four genera: Cinnamomum, Ocotea, Persea and Phoebe. A l l four genera 85 MET + + DES—1 TRI + + + + + + + + + t'COSI] DIG + + + + + + + + + + + + + + + + + + f—GL1 MON + + + + + + + + + + + + + + + + + + + 3NOLS 1 MYR + + + 3NOLS 1 ISO + + + + + + + FLAVi QUE + + + + + + + + + + + + + + + + + + + + i KAE + + + + + + + + + + + + + + + + + + + + ONESl CGL A+ A+ A+ A+ A'0+ [FLAV ORD +L _ i < + +L +L FVN d+ +N N'd+ N+ d+ +C +Vp N'd+ DHF + + + + + CHL + + GENERA Actinodaphne Aiouea Alseodaphne Aniba Apollonias Beilschmiedia Caryodaphnopsis Cassytha Cinnamomum Cryptocarya Dahlgrenodendron Dehaasia Endiandra Endlicheria Gamanthera Hypodaphnis Laurus Licaria Lindera Litsea Machilus 86 1 1 a O u >< -o MET + TRI + + + + + + DIG + + + + + + + + + + + + + MON + + + + + + + + • + + + + + + MYR + 1 1 1 1 1 1 1 CZ5 -J ISO + + + + QUE + + . + + + + + + + + + + + + [FLAVONES1 f -FLAVONi KAE + + + + + + + + + + + + + [FLAVONES1 f -FLAVONi CGL +0,V A'0+ A+ A'0+ A+ A+ [FLAVONES1 f -FLAVONi ORD +L +A _ i < + V+ [FLAVONES1 f -FLAVONi FVN zz + N+ N+ +Vp N+ [FLAVONES1 f -FLAVONi DHF + + [FLAVONES1 f -FLAVONi CHL [FLAVONES1 f -FLAVONi GENERA Mezilaurus Nectandra Neocinnamomum Neolitsea Ocotea Parabenzoin Persea Phoebe Pleurothyrium Povedadaphne Rhodostemonodaphne Sassafras Umbellularia Williamodendron 87 Chapter 4. Discussion are placed in the Ocotea group by Rohwer (1993) (although in two different subgroups) and in the tribe Perseeae by van der Werff and Richter (1996). In contrast, the four genera are split into two different tribes, tribe Cinnamomeae and Perseeae, by Kostermans (1957) (see Table 1.1-1.3). Therefore, the distribution of apigenin in the Lauraceae supports Rohwer's and van der Werff s classifications rather than Kostermans'. The remaining flavone, chrysin, was observed only in Hypodaphnis. This is the only genus in the Lauraceae to have a completely inferior ovary, which led Kostermans to place the genus in its own tribe. However, Rohwer (1993b) and van der Werff and Richter (1996) believe that an inferior ovary is not a decisive enough character, and that a separate tribal position for Hypodaphnis is not warranted. The sole occurrence of chrysin in Hypodaphnis supports Kostermans classification for this genus. Distribution of flavanones: The major flavanones that were observed in the Lauraceae are naringenin, pinocembrin and two unidentified polar flavanones (see Table 4.1). Naringenin is the most common flavanone in the family and it occurs as naringenin aglycones and naringenin-glycosides. These compounds were observed in eight genera: Caryodaphnopsis, Cryptocarya, Dahlgrenodendron, Litsea, Neocinnamomum, Neolitsea, Ocotea and Pleurothyrium. The next most common flavanone is pinocembrin which was observed in five genera: Beilschmiedia, Cryptocarya, Endiandra, Litsea, and Neolitsea. The two unidentified polar flavanones were found in two genera, Lindera and Persea. 88 Chapter 4. Discussion Overall, the occurrence of naringenin and pinocembrin is too scattered throughout the family to be taxonomically useful and the two polar flavanones were observed in two unrelated genera. Distribution of flavonol derivatives: Quercetin and kaempferol occur almost ubiquitously in the Lauraceae as 3-0-monoglycosides, diglycosides and triglycosides. Povedadaphne and Aniba are the only genera that do not exhibit kaempferol, and the latter does not exhibit quercetin. The other flavonol derivatives observed were isorhamnetin and myricetin. Isorhamnetin occurs as a 3-0-monoglycosides, diglycosides and triglycosides, all of which were found in eleven genera: Beilschmiedia, Cassytha, Cryptocarya, Gamanthera, Laurus, Lindera, Litsea, Phoebe, Povedadaphne, Rhodostemonodaphne and Umbellularia. In contrast, myricetin, which only occurs as monoglycosides, was found in three genera: Cassytha, Cryptocarya, and Litsea (see Table 4.1). Overall, these four flavonol derivatives are too scattered throughout the different tribes of the family to be taxonomically useful. Distribution of methylated flavonols and glycosides: Like aglycones, flavonol monoglycosides and diglycosides also occur throughout the Lauraceae (see Table 4.1). However, monoglycosides were not observed in Hypodaphnis and in Aniba which lacks flavonols altogether. In addition, diglycosides were not observed in Actinodaphne, Endiandra, Povedadaphne and Aniba. Triglycosides were common and observed in sixteen genera: Aiouea, Caryodaphnopsis, Cinnamomum, 89 Chapter 4. Discussion Cryptocarya, Endlicheria, Gamanthera, Licaria, Lindera, Litsea, Parabenzoin, Persea, Pleurothyrium, Rhodostemonodaphne, Umbellularia and Williamodendron. The methylated flavonols that were observed in the family are of kaempferol and quercetin-3-methyl ether. They were observed in three genera: Cryptocarya, Lindera and Persea (see Table 4.1). Overall, the occurrence of these methylated flavonols and the different levels of flavonol glycosides are too scattered throughout the different tribes of the family to be taxonomically useful. Distribution of dihydroflavonols and Chalcones: Three unidentified dihydroflavonols were observed in seven genera of the Lauraceae: Actinodaphne, Cinnamomum, Cryptocarya, Lindera, Litsea, Ocotea and Phoebe. Unidentified chalcones were observed in three genera: Cryptocarya, Litsea and Lindera (see Table 4.1). Both of these compounds are too scattered throughout the different tribes of the family to provide any useful information for addressing problems of relationships in the Lauraceae. 4.2 Taxonomic Implications of Flavonoids at the Generic Level In this section, the specific contributions that flavonoid data can make toward a unified classification system for the Lauraceae are highlighted. Specifically, the flavonoid evidence supporting—or contradicting—each of the three main classification systems is considered for the following genera: Sassafras, Umbellularia, Actinodaphne, 90 Chapter 4. Discussion Cassytha, Hypodaphnis, Beilschmiedia, Endiandra and Caryodaphnopsis. The discussion treats the most pronounced discrepancies between these three classification systems. First consider the implications of the distribution of C-glycosylflavones for the tribes to which the genera Sassafras, Umbellularia and Actinodaphne should be assigned. Along with several other genera (Lindera, Litsea, etc. (see Table 1.1-1.3)) Sassafras, Umbellularia and Actinodaphne are assigned by Rohwer (1993b) and van der Werff and Richter (1996) to the Laureae group or the tribe Laureae respectively. In contrast, Kostermans (1957) assigns these last three genera to the Cinnamomeae (see Table 4.2), and the remainder to the Litseeae. With respect to Actinodaphne, Madrinan-Restrepo (1996) states that Kostermans (1957) mistakenly considers the deciduous involucral bracts to be lacking in the genus and thus concludes that Kostermans' placement of Actinodaphne in the Cinnamomeae is incorrect. Flavonoid analysis reinforces the conclusion of Madrinan-Restrepo (1996). Specifically, C-glycosylflavones are absent in all the genera, except for Laurus, that Kostermans' (1957) ascribes to the Laureae. Yet C-glycosylflavones are present in most genera belonging to the Cinnamomeae (again following Kostermans classification). The exceptions are the three controversial genera Sassafras, Umbellularia and Actinodaphne, which all lack C-glycosylflavones. The similarity of the flavonoid profiles of these three genera with the genera placed by Kostermans in the Laureae - the absence of C-glycosylflavones - therefore supports Rohwer's and van der Werff and Richter's placement of these three genera in the same ON ON S-cu -a S3 C5 CU u e CU « CU CU a « cu i . s « -J u .o a CU £ CU ox) e a k. u « u cu C CU bxi CU - C H C N CU 3 H ON ON CU o PH cu s cu -Q <3 53 H < i < ^ V 03 CU S-s O ca ca -O C3 O ^ < o * s l > in ON i—1 CU Vl cu S3 cu « Vi s 13 U N H CU CU • n VI q S3 cn H >-) ^3 s cu c >* C3 •5 CJ J3 ~ 3 00 O J3 ^ OQ o cu « CU s o S CS a a cu Xi ha H c -s: (a, -a o K •»-»» (53 (-1 CD c CD 00 cd CD 1-1 ^ _: K C Q CD O CJ . . CD CD CO 92 Chapter 4. Discussion tribe as the genera that they ascribe to the Laureae. This is not to say that flavonoid analysis entirely supports Rohwer's and van der Werff and Richter's definition of the Laureae. Laurus, a genus from the Laureae, contains C-glycosylflavones which are the characteristic compounds for the Cinnamomeae. Moreover, recent studies using D N A data (mat K gene) also show that Laurus is closely related to members of the Persea or Ocotea group (Rohwer, personal comm.). Therefore, further examinations of the morphological characters of Laurus should be performed to determine its correct placement within the Lauraceae. Next consider one of the most controversial differences among the three classifications: the placement of Cassytha. Kostermans (1957) and van der Werff and Richter (1996) place Cassytha in its own subfamily on the basis of its herbaceous, parasitic nature. In contrast, Rohwer (1993b) places Cassytha in the Cryptocarya group (along with Cryptocarya, Hypodaphnis, etc. (see Table 1.2)) based on similarities in flower, fruit, and pollen morphology. A t first glance, the occurrence of both isorhamnetin and myricetin in Cassytha and Cryptocarya appears to support Rohwer's classification of Cassytha in the Cryptocarya group (see Table 4.3). However, these two compounds also occur in Litsea, a genus that is unrelated to either Cassytha or members from the Cryptocarya group. In addition, isorhamnetin and myricetin are found separately in other genera; the former is found in six other genera and the latter in three. In summary, the occurrence of isorhamnetin, myricetin or any other flavonoids are too scattered in the ON ON C ON ON U o Pi ON e S-cn U t/2 o <8 cd ta 8 e?^ 5 £ 8 £ o a +-> C3 §- b © O o OH 0> cn S C/3 CD cd S - i C D C ~ <U CD t-i ,o O CN -8 •s H CD CD cn e CD • c o • i-H Id o ' t o cd CD CD CD O o a o cn O Cl, CD H CD n H M5 ON ON a es > ON ON S-cu o ON «5 a a i-O o u a = o u OX) C J co £ "S a- o o CD cd I-H CD C CD 00 o a ^ 0 B ^ •8 b H <L, cn O -*-» CD 3 a C~> CD a « ^ H o « „ I-I « & CN CD X3 cd H CD CD t o 94 Chapter 4. Discussion family to assist in clarifying the classification of Cassytha. This is not the case with the genus Hypodaphnis, which, as noted above, Rohwer (1993b) places in the Cryptocarya group. V a n der Werff and Richter (1996) make the identical placement while Kostermans instead places Hypodaphnis in its own tribe because of its unique inferior ovary (Table 4.4). The unique occurrence of chrysin in Hypodaphnis provides strong support for the placement of this genus in its own tribe as suggested by Kostermans (1957). Another controversial difference among the three classifications is the placement of Beilschmiedia and Endiandra. Kostermans (1957) and Rohwer (1993b) place both genera in the subtribe Beilschmiediineae and in the Beilschmiedia group, respectively. However, Kostermans (1957) also includes in his subtribe three other genera, Apollonias, Dehaasia, and Mezilaurus, which are placed in the Ocotea subgroup by Rohwer (1993b). Van der Werff and Richter (1996) however, place Beilschmiedia in the tribe Cryptocaryeae (along with Endiandra, Cryptocarya, Hypodaphnis and Caryodaphnopsis (see Table 4.5)). Among the genera listed above, Endiandra and Beilschmiedia share the most similar flavonoid pattern, with pinocembrin, and kaempferol and quercetin glycosides in common. This result provides support for Rohwer's Beilschmiedia group consisting of just Beilschmiedia and Endiandra. The other three genera included in Kostermans' subtribe Beilschmiediineae exhibit different flavonoid patterns: Apollonias and Dehaasia lack pinocembrin, and Mezilaurus accumulates C-glycosylflavones, which ON s-c u PS3 P H c u -d O > c u CS CU u o a u CU .Q • P H I . H o i 8 2 O CD FA, co CJ g &3 53 C FA, co pP 3 o CO oq t»q <C (D U CO CD ci C -M CL) <LJ M l CU « CU CU CO P H c u C O PH o C2 s 0) PI D C ^ P H o CJ „ b H co CO Xi ON 0\ <U Xi o tf a 3 o P H O 2 -3 cu • P H S -c u co "S pa cu S eq c»q 2 -3 g CJ DX) a. 3 O a o ^ o ca s s P 2 C O PH o s S-H ca o pc C O b H co u CO CO cd CO CO ON SO C P H c u - * • » CO O o CO C U 773 CJ C O $ m C U r t ; < u - § P H H •Si" <3 ca C eq fa. 13 « ? P « o ^ 3 ^ 1 96 Chapter 4. Discussion are the characteristic compounds of the Ocotea group (following Rohwer's classification). The other three genera in van der Werff and Richter's classification also exhibit very different flavonoid patterns. Hypodaphnis, as mentioned previously, exhibits a unique flavonoid pattern. Cryptocarya accumulates pinocembrin but, like Caryodaphnopsis also contains numerous flavonoid types that are not present in either Beilschmiedia or Endiandra. Lastly, the classification of Caryodaphnopsis has also been controversial. Both Rohwer (1993b) and Kostermans (1957) place the genus within the Perseeae or the Persea subgroup, respectively (along with Persea, Phoebe, etc. (see Table 1.1-1.2)). In contrast, van der Werff and Richter (1996) place Caryodaphnopsis in the tribe Cryptocaryeae (along with Beilschmiedia, Cryptocarya, etc. (see Table 4.6)). The occurrence of naringenin in Caryodaphnopsis and Cryptocarya and the absence of this compound from members in the Perseeae (following Kostermans classification) support van der Werff and Richter's treatment of Caryodaphnopsis. In addition, many members in the Perseeae accumulate C-glycosylflavones. However the absence of these compounds in the Caryodaphnopsis profile and members of Cryptocaryeae (following van der Wer f f s classification) further support van der Werff and Richter's classifications. 4.3 Interspecific Relationships Except for the kaempferol and quercetin-3-O-monoglycosides, flavonoid patterns vary greatly among the 120 species surveyed. However, there are some species that share oC ON ON 4> JS (J 4) >H 4> a S3 4> « « o U H K o -t—» 0 < Co C? I-C CO CD o o CD bO CD o o co" 1 - 1 I-I .CO cS -c • o ^ w CD 0 CQ H I 8 < . co ON ON s-4> © 9 P O S K o u o 5 5 2 + 3 CD CO 00 ca »C> ca O a. CD I-i O C3 CN CD 5 b H i 2 CD ON vi C 93 4) O 73 4> .g vi .52 8 » I 4) PH O 4) 4) C ** >H 5f fc £ "S 4> -S O SH H C3 o +-» CD ca" •ft ca O ft. 2 0 0 o E <3 -ca ca 0 • • ro o l -co CD <, (0 98 Chapter 4. Discussion almost identical flavonoid patterns, and the relationships among these species are discussed below. In particular, the classical eastern Asia-eastern North America disjunct distribution is well illustrated by flavonoid data in several species of Persea, Lindera, and Sassafras. In addition, other inter-species relationships that provide taxonomic information useful in settling classification problems in Laurus, Ocotea, Cinnamomum and Phoebe are also discussed. A s noted, a particularly interesting relationship observed in the occurrence of flavonoids is the disjunct distribution of closely related species from eastern As i a and eastern North America. This disjunction pattern was first pointed out by Asa Gray in 1859 (Boufford and Spongberg 1983) and is an example of vicariance biogeography in which a continuous distribution has been fragmented. The disappearance of the land bridge (Beringia) between As i a and North America in late Miocene and the development of a summer-dry climate in western North America at the end of the Miocene created such a fragmentation (Graham 1972). Regarding the occurrence of this disjunctive distribution in particular sets of species, D N A data provide evidence for the occurrence of this distributional pattern in Cornus (Xiang et al. 1994), and against it in Magnolia (Qiu et al. 1995). Flavonoid data similarly support the occurrence of this distributional pattern in Agastache (Vogelman 1984), in Elliottia (Bohm et al. 1978) and in the following genera from this study: Persea, Lindera and Sassafras. Three species from Persea, P. borbonia, P. palustris (both from southeastern U S A ) and P. yunnanensis (from eastern 99 Chapter 4. Discussion China) share almost identical flavonoid patterns. A l l three exhibit quercetin-3-O-glucoside, quercetin-3-O-xyloside, quercetin-3-O-rhamnoside, kaempferol-3-c9-glucoside and kaempferol-3-O-rhamnoside and four types of C-glycosylflavones: isoorientin, orientin, isovitexin and vitexin. These three species differ only in that two unidentified polar flavanones occur only in P. borbonia and P. palustris. The similarity between the two North American species has been identified previously (using morphological and flavonoid analysis, by Pax (1889) and Wofford (1974) respectively) while this is the first time the close relationships among all three have been identified. Next consider Lindera. Lindera benzoin is a small, common tree in the eastern U S A ; L. citriodora is from eastern China, L. umbellata is from Japan. These three species exhibit almost identical flavonoid patterns consisting of kaempferol-3-(9-glucoside, kaempferol-3-O-rhamnoside, kaempferol-3-O-diglucoside, kaempferol-3-O-rutinoside, kaempferol-3-O-dirhamnoside, four kaempferol triglycosides and a chalcone derivative. Note that—in contrast to almost all other lauraceous species—these three species do not contain quercetin-3-t9-monoglycosides. The identical flavonoid patterns observed in these three species suggest their close relationships within this genus. Another lauraceous genus to exhibit this disjunct distribution is Sassafras. Three species of Sassafras are known, one in eastern North America and two in China. For this study, S. albidum from eastern U S A and S. tzumu from China have been examined for their flavonoid profiles. Both species share similar flavonoid patterns consisting of quercetin-3-O-glucoside, kaempferol-3-O-glucoside, kaempferol-3-(9-rhamnoside, quercetin-3-O-rutinoside, kaempferol-3-O-rutinoside and kaempferol-3-0-100 Chapter 4. Discussion dirhamnoside. The differences between these two species is that quercetin-3-0-rhamnoside and a quercetin-3-O-diglycoside occur only in S. albidum. Lastly, the genus Caryodaphnopsis consists of seven species in the neotropics and the other eight in southeast Asia . Unfortunately, only the neotropical species were available (C. burgeri from Costa Rica and C. theobromifolia from Ecuador) for this flavonoid study. These two species exhibit almost identical flavonoid profiles consisting of quercetin-3-O-glucoside, quercetin-3-(9-rhamnoside, quercetin-3-O-rutinoside and two naringenin derivatives. The only difference is that kaempferol-3-O-rhamnoside also occurs in C. theobromifolia. For future comparison studies, flavonoid surveys of Asiatic Caryodaphnopsis would be of interest. Aside from illustrating the eastern Asia-eastern North America disjunct distribution, flavonoid profiles have been also useful taxonomically at the species level. For example, consider the economically important genus Laurus (bay leaf), which consists of two species: L. nobilis from the Mediterranean region and L. azorica from the Canary islands. These two species share almost identical flavonoid profiles, thus supporting Rohwer's (1993b) view that L. nobilis and L. azorica would be better treated as two subspecies. The flavonoid profiles consist of the common flavonol monoglycosides, isorhamnetin-3-O-glucoside, quercetin-3-O-rutinoside, kaempferol-3-O-rutinoside, an isorhamnetin-3-O-diglycoside, a vitexin glycoside and an isovitexin glycoside. The only difference is that a kaempferol-3-O-diglycoside also occurs in L. nobilis. 101 Chapter 4. Discussion Another species from the Canary Islands, Ocotea foetens, shares identical C-glycosylflavones (vitexin, isovitexin, and two isovitexin glycosides) with O. rugosa from Ecuador. Among the twelve Ocotea species examined, only these two species are observed to accumulate this set of C-glycosylflavones. Another Ocotea species, an unidentified species from Costa Rica, also accumulates C-glycosylflavones but only those based on vitexin and isovitexin. Thus flavonoid analysis suggests a close relationship among these three species whether or not this is confirmed by morphological or D N A analysis would be of interest. In addition, establishing whether or not other Ocotea species also exhibit C-glycosylflavones would also be useful. Next, flavonoids have illuminated relationships within certain species of Cinnamomum. Three Cinnamomum species share identical flavonoid profiles: C. burmannii, C. cassia and C. osmophloeum. The profiles consist of quercetin-3-0-rhamnoside, kaempferol-3-O-rhamnoside and four kaempferol triglycosides. These three Cinnamomum species are native to Asia , although C. cassia was collected from Hawaii where it is grown in botanical and private gardens. Another set of three, Cinnamomum species (C. neurophyllum, C. costaricanum, and an unidentified Cinnamomum sp. (Herrera 4980)) all of which are from Costa Rica, show interesting chemical similarity within the genus. These neotropical species are the only species within Cinnamomum to contain C-glycosylflavones. The seven other Cinnamomum species tested are all from eastern As i a and lack C-glycosylflavones. 102 Chapter 4. Discussion Unt i l recently, the American species of Cinnamomum were placed in the genus Phoebe (Rohwer 1993b). Phoebe is mainly an Asiatic genus and all four species examined in this study, P. hunanensis, P. tavoeyana, P. mexicana, and P. sheareri, accumulate C-glycosylflavones. A s the only Cinnamomum species to contain C -glycosylflavones are the American species, the flavonoid data support the transfer of the American species of Cinnamomum back to Phoebe. AA Conclusion The Lauraceae has the reputation of being one of the most difficult families of angiosperms to classify: the delimitation of genera is problematic and the species themselves are quite difficult to identify. These problems mainly arise from the ambiguity of the key morphological characters used in classification. Other types of taxonomic characters are therefore badly needed; this is the impetus for this study of the flavonoids in this family. A survey of leaves from 120 species and 35 genera of the Lauraceae has been performed using new and old tools of flavonoid analysis and eighty flavonoid compounds have been at least partially identified. The predominant compounds that were found are quercetin-3-O-glucoside, quercetin-3-O-rhamnoside, quercetin-3-O-xyloside, kaempferol-3-<9-glucoside and kaempferol-3-O-rhamnoside. Other types of flavonoids, such as flavones, C-glycosylflavones, flavanones, chalcones, dihydroflavonols and methylated flavonols, occur more sporadically in the family. Since flavonoid patterns vary considerably across species within the same genera, flavonoids 103 Chapter 4. Discussion have not been found to provide universally useful set of characters. Nonetheless, flavonoids do illuminate some interesting distribution patterns of related species and help to elucidate relationships within certain groups of controversial genera. For example, some significant results provided by flavonoids are as follows. The lack of C -glycosylflavones in Sassafras, Umbellularia and Actinodaphne support Rohwer's (1993b) and van der Werff and Richter's (1996) treatment of these genera in their tribe Laureae. The sole occurrence of chrysin in Hypodaphnis and in no other genera of the Lauraceae supports the placement of this genus in its own tribe as indicated by Kostermans (1957). Flavonoid data also reflect the eastern Asia-eastern North America disjunct distribution from several species of Persea, Lindera and Sassafras. Lastly and perhaps the most significantly, flavonoid analysis supports the transfer of the American species of Cinnamomum back to Phoebe. 104 Literature Cited Bandulska, H . 1926. On the cuticles of some fossil and recent Lauraceae. 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