( ~ Pergamon BiochemicalSystematicsand Ecology,Vol. 22, No. 1, pp. 101-107, 1994 Copyright © 1994Elsevier Science Ltd Printed in Great Britain. All rights reserved 0305-1978/94 $6.00 + 0.00 Flavonoids of Diplusodon(Lythraceae)* CECILIA T. T. BLATT,t ANTONIO SALATINO,t$ MARIA L. F. SALATINO,t MARIA A. DEL PERO MARTiNEZ§ and TACIANA B. CAVALCANTIII l"lnstituto de Bioci~ncias, Universidade de S&o Paulo, C. Postal 11461, CEP 05422-970, S~o Paulo, SP, Brazil; §Centro de Estudios Farmacol6gicos y Bot~nicos, Serrano 665, (1414) Buenos Aires, Argentina; IICENARGEN/EMBRAPA, C.Postal 10.2372, CEP 70770, Brasilia, DF, Brazil Key Word Index--Diplusodon; Lythraceae; flavonoids; chemotaxonomy. Abstrect--Flavonoids of 27 species of Diplusodon were identified. O-Mono- and diglycosides of apigenin, luteolin, kaempferol, quercetin and myricetin were found. Different glycosidic patterns were observed and they seem to be consistent as taxonomic characters at the species level. Aglycone structures may also be taxonomically relevant, since two main groups of species are sharply discerned by a mutual exclusiveness of flavone and flavonol glycosides. Flavonoid chemistry does not reflect the systems of classifications of the genus and supports the assumption that the foliar pinnately veined pattern is plesiomorphic in Diplusodon, Introduction Diplusodon is a genus of shrubby and sub-shrubby plants endemic to Brazil. They are distributed mainly in "campos rupestres', an ecosystem that occurs at altitudes between 1000 and 2000 m, with rocky and sandy soils, chiefly in the states of Minas Gerais, Bahia and Goi&s, but some species may also appear in the states of Espirito Santo, Mato Grosso and S~o Paulo. The genus comprises 74 species and, in the Lythraceae, is second in size after Cuphea. Two sectional divisions have so far been proposed for Diplusodon, both based on leaf venation patterns. Koehne (1903) recognized four sections, namely Palmatinerves, Palmat/penninerves, Penninerves and Subuninerves. Lourteig (1989) divides the genus into sections Diplusodon, Palmatinerves and Penninerves. Evidences from disciplines other than morphology (e.g. chemistry) are needed to test and refine the available systems of classification of Diplusodon. Apart from a report of alkaloids (Raffauf and Altschul, 1968), seed lipids (Graham and Kleiman, 1987) and a chemotaxonomic survey based on the distribution of foliar alkanes (Blatt etal., 1991), very little is known about the chemistry of Diplusodon. Our knowledge about the flavonoids of the Lythraceae is also very limited. Flavone C-glycosides occur in Cuphea ignea (BateSmith, 1962) and Lythrum salicaria, and flavone and flavonol glycosides were reported for Ammania coccinea (Graham et al., 1980). The present paper reports the distribution of leaf flavonoids of 51 samples, representing 27 species of Diplusodon. Sections Diplusodon, Palmatinerves and Penninerves (sensu Lourteig, 1989) are represented in this study by 6, 6 and 15 species, respectively. Materials and Methods Samples were collected in areas of "campos rupestres" of the states of Minas Gerais and Goias. All samples were air and oven (60°C) dried before chemical procedures. Voucher specimens are deposited in the Herbarium of the Institute of Biosciences, University of S~o Paulo (SPF). Herbarium specimens of D. virgatus collected 40 years ago in the state of S~o Paulo were also used for chemical analysis. *Part of Ph.D. Dissertation of C. T. T. Blatt. :~Author to whom correspondence should be addressed. (Received 30 December 1992) 101 102 C T T. BLATT ETAL. Samples (1-84 g) were powdered and extracted three times with refluxed 80% aqueous methanol for 60 min, The pooled extracts were concentrated under reduced pressure, The flavonoids were isolated by unidimensional PC using BAW and 15% AcOH. Identification of the compounds followed standard procedures (Mabry et al., 1970; Markham, 1982). Results and Discussion A list of flavonoids obtained from specimens of Diplusodon is presented in Table 1. The distribution of the compounds in the specimens investigated appears in Table 2. The relatively large number of compounds (41) is due mainly to the great variability of glycosidic patterns rather than to aglycone diversification. Nearly all the sugars normally found in flavonoid glycosides are listed in Table 1, including glucuronic acid. In addition, both monoglycosides and diglycosides occur in Diplusodon. In contrast, only the most common flavone and flavonol aglycones were found in the samples studied. The mutual exclusiveness of flavone and flavonol glycosides found among the species of Diplusodon permits a clear-cut chemical characterization of two major groups in the genus, one including seven species with flavones and other with 20 species containing flavonols (Table 2). The taxonomic significance of the reciprocal exclusiveness of flavones and flavonols has already been stressed. For example, Schilling and Heiser (1981) suggested the division of Luffa into a group of flavone- and a group of flavonol-bearing species. The prevalence of flavonols in species of Diplusodon is in agreement with observations of Gomall etal. (1979) that this group of compounds is characteristic of Myrtales. However, C-glycoflavones, found in other lythraceous members (see Introduction), were not detected in Diplusodon. TABLE 1. LIST OF FLAVONOID GLYCOSIDES OF SAMPLES OF DIPLUSODON 7-Dicjlycoside of api£1enin (1) Glucosylrhamnoside 7-Glycosides of luteolin Mono£11ycoside (2) Glucoside Di£lycosides (3) Glucosylglucoside (4) Glucosylrhamnoside 3-Glycosides of kaempferol Mono~lycosides (5) Arabinoside (6) Galactoside (7) Glucoside (8) Glucuronide (9) Rhamnoside Diglycosides (10) (Glucose-xylose)* (11) Glucosylglucuronide (12) Glucosylrhamnoside (13) Rhamnosylrhamnoside 3-Glycosides of quercetin M°n°91yc°sides (14) Arabinoside (15) Galactoside (16) Glucoside (17) Glucuronide (18) Rhamnoside 3-Glycosides of quercetin (Conbnued) Di~lycosides (19) (21)) (21) (22) (23) (24) (25) (26) (Arabinose-glucose) * (Galactose-glucose)* (Galactose-rhamnosel* (Glucose-xylose)* (Glucosylglucoside) (Glucosylg[ucuronide) (Glucosylrhamnoside) (Rhamnosylrhamnoside) 3-Glycosides of myricetin Mono£11ycosides (27) Arabinoside (2a) Galactoside (29) Glucoside (30) Glucuronide (31) Rhamnoside (32) Xyloside Diglycoside s (33) (Arabinose-glucose) ~ (34) (Arabinose-xylose)* (35) (Galactose-glucose)* (36) (Galactose-rharn nose) ~ (37) (Glucose-xylose)* (38) (39) (40) (41) *Relative position of sugars not determined Glucosylglucoside Glucosylglucuronide Glucosylrhamnoside Rhamnosylrhamnoside FLAVONOID OF DIPLUSODON (LYTHRACEAE) 103 TABLE 2. LIST OF SAMPLES OF DIPLUSODON POHL AND RESPECTIVE FLAVONOIDS. See Table 1 for list and codes of compounds. Number of voucher specimens* are given after the corresponding binomials Samples A (d) Lu (m) Lu (d) Section Diplusodon 1. O. astictus Lourt. Cavalcanti 401 2. O. ciliiflorus Koehne CFSC 9630 3. O. cilliflorus Koehn CFSC 10159 4. O. hexander DC var. angustifolius (DC) Koehne CFCR 9567 5. D, hexanderDC var. hexanderCFCR 8552 6. D. hexanderDC var. hexander CFCR 9449 7. D. aft. myrsinites Mart. ex DC CFCR 9122 8. D. uninervius Koehne CFCR 3098 9. D. uninervius Koehne CFCR 5413 10. D. uninervius Koehne CFCR 9266 11. D. uninervius Koehne CFCR 10185 12. D. uninerviusKoehne CFCR 10313 13. D. uninervius Koehne CFCR 10484A 14. D, virgatus Pohl Hoehne 1036 15. D, virgatus Pohl Hoehne 2247 16. D, virgatus Pohl Joly 203 K (d) Q (m) Q (d) 14.18 My (m) My (d) 27.31 24.25 40 17 16 25 29 25 16 10,12 11 11 25 40 40 29 40 20,25 35 24 39 24 39 24 39 24 39 24 39 17 Section Palrnatinerves 17. D. glaucescensDC CFCR 10477 18. D. orbicularis Koehne var. brachyander Koehne CFSC 10163 19. D. rotundifolius Mart. ex DC CFCR 9529 20, D. rotundifolius Mart. ex. DC CFCR 10600 21. O. sordidus Koehne Cavalcanti 30 22. O, speciosus (H. B. K.) DC Cavalcanti 403 23. D. vilosissimus Pohl M. 9arreto 3373 Section Penninerves 24. D.argenteus Cavalcanti 400 25. D, helianthemifolius DC var. helianthemifolius Cavalcanti 230 26. D. helianthemifolius DC vat. pemphoides (DC) Koehne CFCR 8528 K (m) 11 24 11 24 11 24 25 36 15, 17, 16 25 2 12 16,18 12, 16,18 19,22,23, 24,25 19,22,23, 24,25 21,25 33,37,39, 40 3 ~ 37,39, 40 36,40 18 27, 29, 31, 32 18 31 4 24 39 104 C. T. T. BLATT ETAL. TABLE 2--CONTINUED Samples 40. D. helianthemifolius DC vat. pemphoides (DC) Koehne CFCR 9650 D. helianthemifolius DC var. pemphoides (DC) Koehne CFCR 10175 D. hirsutus (Chain. et Schl.) DC CFCR 5766 D. incanus Gard. CFCR 9596 D. incanus Gard. CFCR 10811 D. lanceolatus Poht vat. alutaceus Koehne CFSC 9636 D. lanceolatus Pohl var. alutaceus Koehne Cavalcanti 390 D. /anceo/atus Pohl var. alutaceus Koehne Cavalcanti 414 D. leucocalycinus LourL Cavalcanti 384 D. cf. macrodon Menezes 1208 D. oblongus Pohl Pirani 2085 D. oblongus Pohl Pirani 2129 D. parvifolius Mart. ex DC Harley 26083 D. parvifolius Mart. 41. ex DC Cavalcanti 303 D. pulchellus Koehne 42. M. Barros 1018 D. ramosissimus Pohl 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. A (d) Lu (m) Lu (d) K (m) K (d) Q (m) Q (d) My (m) My (d) 24 30 39 24 12 25 12 24, 25 39 40 24 3, 4 1 2 4 2 4 14 27, 31 1 6, 7, 9 15, 18, 16 28, 29, 31 9 14 7, 9 16, 18 18 2 31 29, 31 4 25 var. decip[ens Koehne M. Barros 1023 D. rosmarinifo/ius St. Hil. CFCR 9881 44. D. rosmarinifohus St. Hil. CFCR 10144 45. D. rosmarinifolius St. Hil. M. Barros 1014 46. D. smithfiLourt. CFCR 3465 47. D. smithiiLourt CFCR 9931 48. D. smithiiLourt. CFCR 10763 49. D. smithfi Lourt. 43. 50. 51. CFSC 9683 D. stn~?osusPohl Cavalcanti 473 D. thymifolius Mart. 2 4 2 4 2 4 10, 12 19, 22, 23, 25 26 13 16, 17, 18 13 2 33, 37, 38, 40, 41 34, 41 30 26 34, 41 4 24, 25 ex DC CFCR 3078 * CFCR = Collection Flora of Campos Rupestres CFSC Collection Flora of Serra do Cip6 K = Kaempferol; Q = quercetin; My = myricetin; A = apigenin; = Lu = luteolin; m = monoglycosides; d = diglycosides. FLAVONOID OF DIPLUSODON(LYTHRACEAE) 105 Myricetin is not common in non-woody taxa (Harborne, 1977). Nonetheless, it can be regarded as an important aglycone in the flavonoid profile of Diplusodon, especially if the number of glycosides is taken into account. Indeed, 15 glycosides of myricetin were found, in comparison to 13 of quercetin, nine of kaempferol, three of luteolin and one of apigenin. Myricetin has already been detected in some families of Myrtales, e.g. Combretaceae, Melastomataceae and Onagraceae (Dahlgren and Thorne, 1984), but the present paper represents the first report of myricetin for the Lythraceae, and also the first finding of flavones in this plant family. The apparent incapacity of Diplusodon to produce methylated flavonoid aglycones is another important chemical feature of the genus, as is also the presence of rare glycosidic combinations involving glucuronic acid. The distribution of flavonoids does not reflect the sectional divisions so far proposed for Diplusodon. Similar results were also noted by Blatt et aL (1991) in a study of foliar alkanes of Diplusodon. Section Penninerves may be regarded as distinct from Diplusodon and Palmatinerves by the occurrence of flavones. It must be pointed out, however, that only six out of 15 species of Penninerves surveyed yielded flavones. It is also important to mention that D. vilosissimus, a species of section Palmatinerves, yielded also a flavone glycoside (Table 2). This species was formerly included in section Palmatipenninerves by Koehne (1903). Recently, Lourteig (1989) sank section Palmatipenninerves as synonymous with Palmatinerves. As far as D. vilosissimus is concerned, the flavonoid chemistry is consistent with neither Koehne's nor Lourteig's systems of classification. The possession of a flavone (Table 2) makes this species anomalous in section Palrnatinerves (Lourteig's system) because all the species of the latter yielded only flavonol glycosides. At the same time, there is no chemical support for the recognition of D. vilosissimus (a member of Koehne's section Palmatipenninerves) as distinct from a group of species of section Penninerves, since they are also flavone-bearing taxa. This result may suggest an intermediate position for this taxon. Foliar alkanes did not show a discriminating capacity powerful enough to permit the erection of clear infrageneric groupings in Diplusodon (Blatt et aL, 1991). In addition, the present work suggests a usefulness of the aglycones as a taxonomic aid at the species level and below. Although myricetin glycosides are very common among the flavonol-bearing species of Diplusodon, they seem to be consistently not detectable in D. virgatus and were also not found in D. hirsutus, D. orbicularis, D. ramosissimus and D. thymifolius (Table 2). On the other hand, the presence of kaempferol is not a general feature of the flavonol-bearing species, and it characterizes 10 taxa: D. hirsutus, D. incanus, D. aft. myrsinites, D. oblongus, D. parvifolius, D. rotundifolius, D. smithii, D. speciosus, D. uninervius and D. virgatus (Table 2). With regard to the glycosylation patterns, diglycosides predominate over monoglycosides, both in number of compounds (Table 1) and in the frequencies they occur (Table 2). It is worth observing that most species seem to specialize in the synthesis of either monoglycosides or diglycosides, a valid consideration at least for the flavonol-bearing group. For example, D. astictus, D. leucocalycinus, D. hirsutus and D. parvifolius appear consistently as monoglycoside-yielding species, whereas diglycosides were consistently obtained from D. glaucescens, D. aft. myrsinites, D. sordidus, D. thymifolius and D. virgatus (Table 2). D. hexander var. hexander and D. rotundifolius are examples of taxa that contain both groups of glycosides. glycosides. Glucuronic acid was not found in flavone glycosides of Diplusodon (Tables I and 2), although such glycosidic combinations are known in other taxa (e.g. Schulz et aL, 1985, EI-Habashy et aL, 1989). The taxonomic value of the glycosylation patterns of the flavonols of Diplusodon is 106 C T. T. BLATT ETAL reinforced by the fact that a considerable proportion of species are specialized in linking certain sugars at position 3 of each flavonol aglycone they accumulate. For example, among the monoglycoside-bearing group, D. oblongus and D. parvifolius are remarkable for containing glycosides of galactose, glucose and rhamnose for all the three flavonol aglycones and for kaempferol and quercetin, respectively. Diplusodon astictus contains arabinosides and rhamnosides of quercetin and myricetin and D. helianthemifolius var. helianthemifolius contains solely rhamnosides of these two aglycones (Table 2). One specimen of D. uninervius (CFCR 10484A) yielded glucuronides of kaempferol and quercetin (Table 2). Among the diglycosidebearing group, glucosylglucuronide is the exclusive or the commonest pattern of D. helianthemifolius, D. uninervius and D. virgatus, while rutinoside holds this position for D. hirsutus and D. incanus; two specimens of D. smithfi (CFCR 9931 and CFSC 9683) show a clear predilection for the dirhamnoside pattern (Table 2). The flavone-bearing species appear chemically as a very homogenous group. Indeed, most of the species are indistinguishable from the flavonoid viewpoint, containing luteolin 7-glucoside and rutinoside (Table 2). The flavonol-bearing species seem to form a much more diverse group. However, it is possible to recognize three pairs of species with close chemical affinities. Despite some intraspecific variation, it is possible to note a common possession of kaempferol, quercetin and myricetin rhamnosides by D. oblongus and D. parvifolius. Similarly, D. hirsutus and D. incanus share the possession of kaempferol and quercetin rutinosides (Table 2). Two other species (D. rotundifolius and D. smithil) present the most complex flavonoid profiles among the species investigated; taking into consideration the flavonoids obtained from all the specimens investigated, there can be seen ten compounds in common for this pair of species (Table 2). Flavonoid chemistry does not support the recognition of intraspecific categories inside D. hexander; specimens of varieties angustifolius and hexander here investigated are chemically identical (Table 2). On the other hand, there seems to be chemical grounds to split D. helianthemifolius into varieties helianthemifolius and pemphoides. While the former contains only monoglycosides based on rhamnose, the latter has chiefly glucosylglucuronides. However, definite conclusions about the contribution of flavonoid chemistry at the intraspecific level of these species is not yet feasible due to the paucity of the samplings. A matter of serious concern for grouping species of Diplusodon based on flavonoid characteristics is related to the intraspecific variation. In this regard, one can notice in Table 2 that some taxa have a high degree of consistency of their flavonoid profiles, as is the case of D. helianthernifolius var. pemphoides, D. rosmarinifolius, D. rotundifolius and D. virgatus. In contrast, in other taxa (D. ciliiflorus, D. hexander var. hexander, D. parvifolius, D. smithii and D. uninervius) several degrees of intraspecific variation in flavonoid profiles can be noted. The latter group of taxa offers possibilities of biosystematic investigations for which the flavonoid chemistry might represent a rewarding contribution. Several degrees of intraspecific variation or a high degree of consistency of alkane profiles in Diplusodon have also been noted by Blatt et al. (1991), depending on the species considered. Both groups of compounds (alkanes and flavonoids) agree in pointing out D. ciliiflorus and D. smithii as chemically heterogenous. Morphologically, the latter is also a "problematical" species. However, although D. rosmarinifolius and D. virgatus did not show appreciable intraspecific variation of their flavonoid composition, they are among the species with a high phenotypic plasticity as to their alkane patterns. It is generally agreed that evolution at low hierarchic levels proceeds by simplification of chemical profiles. In terms of flavonoid chemistry, this hypothesis was first postulated by Mabry (1973), becoming later an object of criticism by Harborne and Turner (1984). Averett and Raven (1984) and Averett et al. (1979, 1990, 1991) suggested FLAVONOID OF DIPLUSODON (LYTHRACEAE) 107 that in Onagraceae (another family of the Myrtales) the flavonoidic plesiomorphic state corresponds to a c o m p l e x profile that c o m b i n e s flavones and flavonols; chemical evolution led to a progressive loss of the former. It seems that a similar trend ocurred in D i p l u s o d o n , w i t h the difference that losses of both classes of flavonoids p r o b a b l y h a p p e n e d in this genus, in distinct groups of species. However, contrary to the situation of Onagraceae, the plesiomorphic chemical state is no longer apparent in D i p l u s o d o n (at least, it was not detected in the present work). The ancestor of the extant species of D i p l u s o d o n might have been a plant with flavones and flavonols, as well as leaves with a pinnately-veined pattern, since both classes of flavonoids are c o m m o n l y seen o n l y in section Penninerves. 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