- Email: [email protected]
8-O-Methylated flavones from Calycadenia ciliosa (Compositae): Inter- and intrapopulational variation
BiochemicalSystematicsand Ecology,Vol. 14, No. 1, pp. 29-32, 1986.
0305-1978/86 $3.00+0.00 Pergamon Press Ltd.
Printed in Great Britain.
8-O-Methylated Flavones from Calycadenia ciliosa (Compositae)" Inter- and Intrapopulational Variation JEAN K. EMERSON, R. L. CARR*, SUSAN McCORMICKt andBRUCE A. BOHMt$ Biology Department, St. Mary College, Leavenworth, KS 66048, U.S.A.; *Biology Department, Eastern Washington University, Cheney, WA 99004, U.S.A.; tDepartment of Botany, University of British Columbia, Vancouver, British Columbia, V6T lW5, Canada
Key Word
Index--Calycadenia cih'osa;Compositae; Madiinae; flavonoids; interpopulational variation; intrapopulational variation.
Abstract--The non-polar fraction of flavonoids from Calycadenia ciliosa consists of several compounds five of which have been identified as 8-methoxy flavones. The major component, present in all but one of the populations studied, was shown to be 5,3',4'-trihydroxy-3,6,7,8-tetramethoxyflavone. Several different arrays of the pigments were observed in the f'we chromosomal races studied. Three populations of structural heterozygotes were analysed for between-plant variation: two showed invariant patterns while the third exhibited complex pigment patterns.
Results and Discussion The structures of the five compounds in question (1-5) are shown in (Fig. 1). Small amounts of four additional compounds were chromatographically detectable after concentration of pooled material following isolation of the above flavonoids. All absorbed UV characteristic of 3-methoxyflavones. One appears to be a 3',4'-dihydroxy derivative (colour with aminoethyl diphenylborate). Two do not give a colour reaction with the reagent and are likely 4'hydroxyflavones. The fourth was identified as 3. Recent, detailed cytological studies [4] have documented the existence of at least five chromosomal races of C. ciliosa which appear to have arisen through a series of reciprocal translocations. Several populations of each were sampled and subjected to flavonoid profile analysis
Introduction
Calycadenia DC (Compositae, Madiinae) is a small genus of xerophytic annuals found in California and extreme southern Oregon [1]. The 12 or so species are self-incompatible and have chromosome numbers of n=4, 5, 6, 7 and 9 [2]. The genus exhibits complex evolutionary relationships which have been studied by Carr [2, 3]. The species under consideration in the present work, C. ciliosa Greene (n=6), consists of at least five chromosomally distinct races [4]. Moreover, high levels of structural heterozygosity within some populations make racial assignment difficult. An investigation of the flavonoid chemistry of this species has been undertaken to determine the usefulness of these pigments as an additional source of information in the study of interpopulational differences. A complex array of flavones and flavonols has been seen. This paper reports the existence of five 8-methoxyflavones in C. ciliosa. These compounds comprise the major consituents of the non-polar flavonoid fraction of the plant. A preliminary report [5] recorded incorrect structures for two of these compounds. We also report here the existence of interpopulational and intrapopulational differences in the occurrence of certain of these compounds.
Ior~l 2
© FIG. 1. REPRESENTATION OF THE RELATIONSHIPS BETWEEN FIVE POPULATIONS OF CALYCADEN/AC/LIOSABASED UPON FIG. 20 IN REF. [4]. Co-Coming, CI--Ciliosa, DC-Dry Creek, L--Lewiston, P--Pillsbury. 1, 2 and $ are the flavonoids described in the text. Compound 3 was obtained in low concentration from pooled extracts and is excluded from the diagram. Numbers between populations identify the chromosomes involved in translocations [4].
:l:To whom correspondence should be addressed.
(Received 28 September 1984) 29
30
JEAN K. EMERSON, R. L. CARR, SUSAN McCORMICK AND BRUCE A. BOHM
[1(2
~L
t1()
~()11
1
R z =OMc
2
R ~ = OM~', R : - I!
t,~
3
R J =OMc
4
R 1 = }l
R:
5
R ~ = R: = O I I
OII
()Me
R2::(111
()
FIG. 2. STRUCTURES OF THE METHYLATED FLAVONOLS FROM CALYCADENIA CILIOSA.
by thin layer chromatography: six populations were from the 'Dry Creek' race, four populations were sampled for each 'Coming', 'Ciliosa' and 'Lewiston', and three populations were obtained from 'Pillsbury'. Four races were found [4] that exhibit high degrees of structural heterozygosity. These latter individuals were found in south central Shasta Co. and adjacent north central Tahama Co. and occur where the Dry Creek, Coming and Lewiston races overlap. The racial flavonoid chemistry of C. ciliosa exhibits variation which is summarized in Table 1. Five different patterns were observed. All Dry Creek populations had the entire complement of major 8-methoxyflavones (1, 2, 4 and 5). The Coming and Pillsbury races, with four and three populations screened, respectively, were identical in having only 1. Lewiston, with four populations sampled, also exhibited pattern consistency showing only 1 and 4. In the case of the Coming race the situation is more complex in that two different patterns were observed: three populations were identical in possessing 1, 4 and 5, while a fourth population has only 4 and 5. The last population was unique among the populations tested in this study in that it lacked 1. Carr and Carr summarized the cytological relationships between the five races of C. ciliosa by means of a diagram [4, Fig. 20] which has been redrawn here to include the flavonoid data (see Fig. 1). The flavonoid data support the placement of Pillsbury as arising from Ciliosa as deduced from cytological, geographical and morphological data [4]. The relationships of the Coming, Dry Creek, Lewiston and Ciliosa races show multiple losses and gains if the flavonoid data are superimposed on the cytological results. We hope that further insight into these relationships may be
TABLE 1. OCCURRENCE OF 8-METHOXYLATED FLAVONES IN RACES OF CALYCADENIA ClLIOSA Flavonoids
Race
1
2
5
Dry Creek (6)* Coming (3)* (1)* Ciliosa (4)*
~
~
~, i ÷
4
Pillsbury (3)* Lewiston (4)*
~ i
~
Redding (20)t Bowman Road (6)t Anderson (30)t
~ all
variable
4
, none
*Numbers in parentheses indicate number of populations examined for the first six races. tNumber is the number of individual plants examined.
gained when further populations are sampled for flavonoid profiles and when structures can be assigned to the minor constituents. Individuals from three structurally heterozygotic populations were available which allowed us to assess between-plant variation in the occurrence of the 8-methoxyflavones. In the first of these, identified as 'Redding' (R. L. Carr 2134 4), the 20 plants studied were identical in having all four flavonoids. Six plants of the 'Bowman Road' populations (R. L. Carr 2136) were also identical to one another but were very simple in exhibiting only 1. The other extreme of variation was observed with the 'Anderson' population (R. L. Carr 2206). This population consists of several flower colour morphs ranging trom white, through cream, to yellow with some of the plants exhibiting a reddish pigmentation in the proximal portions of the ligulate flowers. This group of plants proved to be the most variable with regard to their non-polar flavonoids. All 30
8-O-METHYLATEDFLAVONESFROM CALYCADENIAClLIOSA
individuals exhibited 1; 4 was absent throughout. Some of the plants of each flower colour type had flavonoids 5 and 6 while others of each type had compound 6 but not 5. There appeared to be no correlation between the occurrence of O-methylated flavones and flower colour. Experimental Plant material. Plants from each of the named chromosomal races of C. ci/iosa Greene [4] were collected from the field. Each population sample comprised 30-40 individuals. Plants from the structurally heterozygous populations were grown in a greenhouse from field-collected seed. Vouchers have been deposited in DAV and in the herbarium of Eastern Washington University. Bulk flavonoid studies were done on 50 g dry wt of R. L. carr 2189 (racial origin unknown). Isolation of flavonoids, The total methanol soluble phenolic fraction of the plant (R. L. Carr 2189) was subjected to Sephadex LH-20 column separation using increasing amounts of MeOH in H20. Fractions containing flavonoids were then resolved by partitioning on cellulose with H20 as the stationary phase and various mixtures of EtOAc and petrol as the mobile phase [6, 7]. LH-20 column fractions consisting of relatively few components could also be resolved by preparative TLC. When it was realized that the plant possessed highly non-polar Omethylated aglycones a modified procedure was adopted. Dried plant material was extracted with CHCI3 by soaking for 48 h. The extract was coned and fractionated on a Polyclar-AT column using increasing quantities of MeOH in CHCI3. Final resolution was achieved by preparative TLC using the Et formate-cyclohexane-BuOAc-HCOOH system referred to above. Structures were determined by employing standard UV and NMR methods [8], mass spectroscopy [13], colour reactions with aminoethyl diphenylborate and chromatography against standards [14]. Compound 1 gave a parent ion with m/z==390 indicating a flavone with three OH and four OMe groups [9]. 1H NMR showed the presence of a 3',4"-0- B-ring (86.4 and 7.1, J=8.5 Hz; b=7.3), the absence of A-ring protons, and 12-O-methyl protons (83.70, 3.77, 3.82, 3.97). Mass fragmentation gave a base peak at m/z 375 [M-15] indicating a methoxy function at C-8 [10, 11]. UV data pointed to a 3',4'-dihydroxy system, substituted 7-oxygenation, unsubstituted 5-OH group, and methoxy group at C-8 (shift of 66 nm of AICI3-HCI peak relative to the MeOH spectrum [12]). The compound is dark under UV (386 nm) which locates a methoxyl at C-3. The other methoxyl can be placed at C-6 (A-ring lacks protons). This substitution pattern is supported by a peak at m/z 137 (from the B-ring) and m/z 211 [A1-15]. These observations lead to the structure 5,3',4'-trihydroxy-3,6,7,8-tetramethoxyflavone for 1. Compound 2 gave a parent ion at m/z 374 (a flavone with two OH groups and four OMe groups). UV shows OH groups at C-5 and 4". MeOH groups were placed at C-3 (compound dark under UV; [M--43] fragment [13], C-7 (no Band-II shift with acetate), and C-8 (80 nm shift of Band I with AICI3-HCI relative to MeOH spectrum; [M--15] ion greater intensity than M peak). The remaining MeO group was placed at C-6 supported by an [A~--15] fragment (m/z 211) as with compound 1. 4'Hydroxylation of the compound is supported by the appearance of a major peak at m/z 121. Compound 2 is thus 5,4'dihydroxy-3,6,7,8-tetramethoxyflavone.
Compound 3 gave a parent ion at m/z404 (flavone with two OH and five OMe functions). The high level of methoxylation agrees with the high mobility of compound 3 in nonpolar solvent systems. UV data place OH groups at C-5 and C-4'. Lack of a borate shift places one OMe at C-3'. This is supported by a B-ring fragment at m/z i51 in the MS. A fragment at m/z 211 was also present which supports remaining OMe at C-6, C-7 and C-8. The structure of 3 is, thus, 5,4'-dihydroxy-3,3',6,7,8pentamethoxyflavone. Compound 4 gave a parent ion at m/z 360 (flavone with three OH and three OMe). UV gave OH groups at C-5, C-3' and C-4'. An MS fragment at m/z 137 agrees with the existence of a di-OH B-ring. Methoxy groups were located at positions-3, 7 and 8 by the same criteria as above. 4 was compared with 5,3',4'-trihydroxy-3,6,7-trimethoxyflavone from Chrysoplenium tetrandrum [14]: it ran faster than the latter on Polyamid DC 6.6 using Et formate-cyclohexance-BuOAc-HCOOH (50:25:23:2). The UV of the C. tetrandrum compound showed no long wavelength absorption with AICI3-HCI and the [M-15] peak in the MS was of a lower intensity than the parent peak. Compound 4 was assigned the structure 5,3',4'-trihydroxy-3,7,8trimethoxyflavone. Compound 5 gave a parent ion at m/z376 which would be expected from a flavone having four OH and three MeO groups. UV placed three OH groups at C-5, C-3' and C-4' and the MeO groups at C-3, C-7 and C-8 (criteria as described above). A strong peak at in/z 137 also argued for dihydroxylalion in the B-ring. The remaining OH group must be located at C-6. This is supported by two lines of evidence. Whereas all compounds in this series with 3',4'-dihydroxylation gave characteristic orange colours with aminoethyl diphenylborate spray reagent, their behaviour after fuming with ammonia vapours were different. The orange colour with 1 and 4 was slightly intensified with ammonia while that with 5 turned to dark brown and then slowly faded back to orange. In our experience with this reagent this is consistent with compounds having 6,3',4'-hydroxylation. Mass fragmentation supports a fully substituted A-ring for this compound: the peak at m/z 197 can be accounted for by a reverse DielsAlder breakdown of the A-ring fragment that results from the facile loss of 15 mass units from 8-methoxy function. The suggested structure for compound 5 is thus 5,6,3',4'-tetrahydroxy-3,7,8-trimethoxyflavone.
Acknowledgements--This study was supported by operating and equipment grants from the Natural Sciences and Engineering Research Council of Canada (to B.A.B.) and by a University of Kansas Biomed Support Grant (to J.K.E.) to whom we express our sincerest appreciation.
References 1. Munz, P. and Keck, D. D. (1959) A CalifomiaFIora. University of California Press, Berkeley. 2. Carr, G. D. (1977) Am. J. Botany64, 694. 3. Carr, G. D. (1976) Evolution 29, 681. 4. Carr, F. and Carr. G. D. (1983) Am. J. Botany70, 744. 5. Emerson, J. K. and Bohm, B. A. (1983) Am. J. Botany 70 Part 2, 90. 6. Wilkins, C. K. and Bohm, B. A. (1976) Can. J. Botany 54, 2133. 7. Gornall, R. J. and Bohm, B. A. (1980) Can. J. Botany 58, 1768. 8. Mabry, T. J., Markham, K. R. and Thomas, M. B. (1970) The
32
JEAN K. EMERSON,R. L. CARR, SUSAN McCORMICKAND BRUCEA. BOHM
Systematic Identification of Flavonoids. Springer, New York. 9. Wollenweber, E. and Dietz, V. H. (1979) Phytochem. Bull. 12, 48. 10. Goudard, M., Favre-Bonvin, J., Lebreton, P. and Chopin, J. (1978) Phytochemistry 17, 145. 11. Goudard, M., Favre-Bonvin, J., Strelisk¥, J., Nogradi, M. and Chopin, J. (1979) Phytochemistry18, 186.
12. Sakakibara, M. and Mabry, 3. J. (1977) Rev. Latinoam. Quim. 8,99. 13. Markham, K. R. (1982) Techniques of Flavonoid Identification. Academic Press, New York. 14. Collins, F. W., Bohm, B. A. and Bose, R. (1977) Phytochemistry16, 1205.
0305-1978/86 $3.00+0.00 Pergamon Press Ltd.
Printed in Great Britain.
8-O-Methylated Flavones from Calycadenia ciliosa (Compositae)" Inter- and Intrapopulational Variation JEAN K. EMERSON, R. L. CARR*, SUSAN McCORMICKt andBRUCE A. BOHMt$ Biology Department, St. Mary College, Leavenworth, KS 66048, U.S.A.; *Biology Department, Eastern Washington University, Cheney, WA 99004, U.S.A.; tDepartment of Botany, University of British Columbia, Vancouver, British Columbia, V6T lW5, Canada
Key Word
Index--Calycadenia cih'osa;Compositae; Madiinae; flavonoids; interpopulational variation; intrapopulational variation.
Abstract--The non-polar fraction of flavonoids from Calycadenia ciliosa consists of several compounds five of which have been identified as 8-methoxy flavones. The major component, present in all but one of the populations studied, was shown to be 5,3',4'-trihydroxy-3,6,7,8-tetramethoxyflavone. Several different arrays of the pigments were observed in the f'we chromosomal races studied. Three populations of structural heterozygotes were analysed for between-plant variation: two showed invariant patterns while the third exhibited complex pigment patterns.
Results and Discussion The structures of the five compounds in question (1-5) are shown in (Fig. 1). Small amounts of four additional compounds were chromatographically detectable after concentration of pooled material following isolation of the above flavonoids. All absorbed UV characteristic of 3-methoxyflavones. One appears to be a 3',4'-dihydroxy derivative (colour with aminoethyl diphenylborate). Two do not give a colour reaction with the reagent and are likely 4'hydroxyflavones. The fourth was identified as 3. Recent, detailed cytological studies [4] have documented the existence of at least five chromosomal races of C. ciliosa which appear to have arisen through a series of reciprocal translocations. Several populations of each were sampled and subjected to flavonoid profile analysis
Introduction
Calycadenia DC (Compositae, Madiinae) is a small genus of xerophytic annuals found in California and extreme southern Oregon [1]. The 12 or so species are self-incompatible and have chromosome numbers of n=4, 5, 6, 7 and 9 [2]. The genus exhibits complex evolutionary relationships which have been studied by Carr [2, 3]. The species under consideration in the present work, C. ciliosa Greene (n=6), consists of at least five chromosomally distinct races [4]. Moreover, high levels of structural heterozygosity within some populations make racial assignment difficult. An investigation of the flavonoid chemistry of this species has been undertaken to determine the usefulness of these pigments as an additional source of information in the study of interpopulational differences. A complex array of flavones and flavonols has been seen. This paper reports the existence of five 8-methoxyflavones in C. ciliosa. These compounds comprise the major consituents of the non-polar flavonoid fraction of the plant. A preliminary report [5] recorded incorrect structures for two of these compounds. We also report here the existence of interpopulational and intrapopulational differences in the occurrence of certain of these compounds.
Ior~l 2
© FIG. 1. REPRESENTATION OF THE RELATIONSHIPS BETWEEN FIVE POPULATIONS OF CALYCADEN/AC/LIOSABASED UPON FIG. 20 IN REF. [4]. Co-Coming, CI--Ciliosa, DC-Dry Creek, L--Lewiston, P--Pillsbury. 1, 2 and $ are the flavonoids described in the text. Compound 3 was obtained in low concentration from pooled extracts and is excluded from the diagram. Numbers between populations identify the chromosomes involved in translocations [4].
:l:To whom correspondence should be addressed.
(Received 28 September 1984) 29
30
JEAN K. EMERSON, R. L. CARR, SUSAN McCORMICK AND BRUCE A. BOHM
[1(2
~L
t1()
~()11
1
R z =OMc
2
R ~ = OM~', R : - I!
t,~
3
R J =OMc
4
R 1 = }l
R:
5
R ~ = R: = O I I
OII
()Me
R2::(111
()
FIG. 2. STRUCTURES OF THE METHYLATED FLAVONOLS FROM CALYCADENIA CILIOSA.
by thin layer chromatography: six populations were from the 'Dry Creek' race, four populations were sampled for each 'Coming', 'Ciliosa' and 'Lewiston', and three populations were obtained from 'Pillsbury'. Four races were found [4] that exhibit high degrees of structural heterozygosity. These latter individuals were found in south central Shasta Co. and adjacent north central Tahama Co. and occur where the Dry Creek, Coming and Lewiston races overlap. The racial flavonoid chemistry of C. ciliosa exhibits variation which is summarized in Table 1. Five different patterns were observed. All Dry Creek populations had the entire complement of major 8-methoxyflavones (1, 2, 4 and 5). The Coming and Pillsbury races, with four and three populations screened, respectively, were identical in having only 1. Lewiston, with four populations sampled, also exhibited pattern consistency showing only 1 and 4. In the case of the Coming race the situation is more complex in that two different patterns were observed: three populations were identical in possessing 1, 4 and 5, while a fourth population has only 4 and 5. The last population was unique among the populations tested in this study in that it lacked 1. Carr and Carr summarized the cytological relationships between the five races of C. ciliosa by means of a diagram [4, Fig. 20] which has been redrawn here to include the flavonoid data (see Fig. 1). The flavonoid data support the placement of Pillsbury as arising from Ciliosa as deduced from cytological, geographical and morphological data [4]. The relationships of the Coming, Dry Creek, Lewiston and Ciliosa races show multiple losses and gains if the flavonoid data are superimposed on the cytological results. We hope that further insight into these relationships may be
TABLE 1. OCCURRENCE OF 8-METHOXYLATED FLAVONES IN RACES OF CALYCADENIA ClLIOSA Flavonoids
Race
1
2
5
Dry Creek (6)* Coming (3)* (1)* Ciliosa (4)*
~
~
~, i ÷
4
Pillsbury (3)* Lewiston (4)*
~ i
~
Redding (20)t Bowman Road (6)t Anderson (30)t
~ all
variable
4
, none
*Numbers in parentheses indicate number of populations examined for the first six races. tNumber is the number of individual plants examined.
gained when further populations are sampled for flavonoid profiles and when structures can be assigned to the minor constituents. Individuals from three structurally heterozygotic populations were available which allowed us to assess between-plant variation in the occurrence of the 8-methoxyflavones. In the first of these, identified as 'Redding' (R. L. Carr 2134 4), the 20 plants studied were identical in having all four flavonoids. Six plants of the 'Bowman Road' populations (R. L. Carr 2136) were also identical to one another but were very simple in exhibiting only 1. The other extreme of variation was observed with the 'Anderson' population (R. L. Carr 2206). This population consists of several flower colour morphs ranging trom white, through cream, to yellow with some of the plants exhibiting a reddish pigmentation in the proximal portions of the ligulate flowers. This group of plants proved to be the most variable with regard to their non-polar flavonoids. All 30
8-O-METHYLATEDFLAVONESFROM CALYCADENIAClLIOSA
individuals exhibited 1; 4 was absent throughout. Some of the plants of each flower colour type had flavonoids 5 and 6 while others of each type had compound 6 but not 5. There appeared to be no correlation between the occurrence of O-methylated flavones and flower colour. Experimental Plant material. Plants from each of the named chromosomal races of C. ci/iosa Greene [4] were collected from the field. Each population sample comprised 30-40 individuals. Plants from the structurally heterozygous populations were grown in a greenhouse from field-collected seed. Vouchers have been deposited in DAV and in the herbarium of Eastern Washington University. Bulk flavonoid studies were done on 50 g dry wt of R. L. carr 2189 (racial origin unknown). Isolation of flavonoids, The total methanol soluble phenolic fraction of the plant (R. L. Carr 2189) was subjected to Sephadex LH-20 column separation using increasing amounts of MeOH in H20. Fractions containing flavonoids were then resolved by partitioning on cellulose with H20 as the stationary phase and various mixtures of EtOAc and petrol as the mobile phase [6, 7]. LH-20 column fractions consisting of relatively few components could also be resolved by preparative TLC. When it was realized that the plant possessed highly non-polar Omethylated aglycones a modified procedure was adopted. Dried plant material was extracted with CHCI3 by soaking for 48 h. The extract was coned and fractionated on a Polyclar-AT column using increasing quantities of MeOH in CHCI3. Final resolution was achieved by preparative TLC using the Et formate-cyclohexane-BuOAc-HCOOH system referred to above. Structures were determined by employing standard UV and NMR methods [8], mass spectroscopy [13], colour reactions with aminoethyl diphenylborate and chromatography against standards [14]. Compound 1 gave a parent ion with m/z==390 indicating a flavone with three OH and four OMe groups [9]. 1H NMR showed the presence of a 3',4"-0- B-ring (86.4 and 7.1, J=8.5 Hz; b=7.3), the absence of A-ring protons, and 12-O-methyl protons (83.70, 3.77, 3.82, 3.97). Mass fragmentation gave a base peak at m/z 375 [M-15] indicating a methoxy function at C-8 [10, 11]. UV data pointed to a 3',4'-dihydroxy system, substituted 7-oxygenation, unsubstituted 5-OH group, and methoxy group at C-8 (shift of 66 nm of AICI3-HCI peak relative to the MeOH spectrum [12]). The compound is dark under UV (386 nm) which locates a methoxyl at C-3. The other methoxyl can be placed at C-6 (A-ring lacks protons). This substitution pattern is supported by a peak at m/z 137 (from the B-ring) and m/z 211 [A1-15]. These observations lead to the structure 5,3',4'-trihydroxy-3,6,7,8-tetramethoxyflavone for 1. Compound 2 gave a parent ion at m/z 374 (a flavone with two OH groups and four OMe groups). UV shows OH groups at C-5 and 4". MeOH groups were placed at C-3 (compound dark under UV; [M--43] fragment [13], C-7 (no Band-II shift with acetate), and C-8 (80 nm shift of Band I with AICI3-HCI relative to MeOH spectrum; [M--15] ion greater intensity than M peak). The remaining MeO group was placed at C-6 supported by an [A~--15] fragment (m/z 211) as with compound 1. 4'Hydroxylation of the compound is supported by the appearance of a major peak at m/z 121. Compound 2 is thus 5,4'dihydroxy-3,6,7,8-tetramethoxyflavone.
Compound 3 gave a parent ion at m/z404 (flavone with two OH and five OMe functions). The high level of methoxylation agrees with the high mobility of compound 3 in nonpolar solvent systems. UV data place OH groups at C-5 and C-4'. Lack of a borate shift places one OMe at C-3'. This is supported by a B-ring fragment at m/z i51 in the MS. A fragment at m/z 211 was also present which supports remaining OMe at C-6, C-7 and C-8. The structure of 3 is, thus, 5,4'-dihydroxy-3,3',6,7,8pentamethoxyflavone. Compound 4 gave a parent ion at m/z 360 (flavone with three OH and three OMe). UV gave OH groups at C-5, C-3' and C-4'. An MS fragment at m/z 137 agrees with the existence of a di-OH B-ring. Methoxy groups were located at positions-3, 7 and 8 by the same criteria as above. 4 was compared with 5,3',4'-trihydroxy-3,6,7-trimethoxyflavone from Chrysoplenium tetrandrum [14]: it ran faster than the latter on Polyamid DC 6.6 using Et formate-cyclohexance-BuOAc-HCOOH (50:25:23:2). The UV of the C. tetrandrum compound showed no long wavelength absorption with AICI3-HCI and the [M-15] peak in the MS was of a lower intensity than the parent peak. Compound 4 was assigned the structure 5,3',4'-trihydroxy-3,7,8trimethoxyflavone. Compound 5 gave a parent ion at m/z376 which would be expected from a flavone having four OH and three MeO groups. UV placed three OH groups at C-5, C-3' and C-4' and the MeO groups at C-3, C-7 and C-8 (criteria as described above). A strong peak at in/z 137 also argued for dihydroxylalion in the B-ring. The remaining OH group must be located at C-6. This is supported by two lines of evidence. Whereas all compounds in this series with 3',4'-dihydroxylation gave characteristic orange colours with aminoethyl diphenylborate spray reagent, their behaviour after fuming with ammonia vapours were different. The orange colour with 1 and 4 was slightly intensified with ammonia while that with 5 turned to dark brown and then slowly faded back to orange. In our experience with this reagent this is consistent with compounds having 6,3',4'-hydroxylation. Mass fragmentation supports a fully substituted A-ring for this compound: the peak at m/z 197 can be accounted for by a reverse DielsAlder breakdown of the A-ring fragment that results from the facile loss of 15 mass units from 8-methoxy function. The suggested structure for compound 5 is thus 5,6,3',4'-tetrahydroxy-3,7,8-trimethoxyflavone.
Acknowledgements--This study was supported by operating and equipment grants from the Natural Sciences and Engineering Research Council of Canada (to B.A.B.) and by a University of Kansas Biomed Support Grant (to J.K.E.) to whom we express our sincerest appreciation.
References 1. Munz, P. and Keck, D. D. (1959) A CalifomiaFIora. University of California Press, Berkeley. 2. Carr, G. D. (1977) Am. J. Botany64, 694. 3. Carr, G. D. (1976) Evolution 29, 681. 4. Carr, F. and Carr. G. D. (1983) Am. J. Botany70, 744. 5. Emerson, J. K. and Bohm, B. A. (1983) Am. J. Botany 70 Part 2, 90. 6. Wilkins, C. K. and Bohm, B. A. (1976) Can. J. Botany 54, 2133. 7. Gornall, R. J. and Bohm, B. A. (1980) Can. J. Botany 58, 1768. 8. Mabry, T. J., Markham, K. R. and Thomas, M. B. (1970) The
32
JEAN K. EMERSON,R. L. CARR, SUSAN McCORMICKAND BRUCEA. BOHM
Systematic Identification of Flavonoids. Springer, New York. 9. Wollenweber, E. and Dietz, V. H. (1979) Phytochem. Bull. 12, 48. 10. Goudard, M., Favre-Bonvin, J., Lebreton, P. and Chopin, J. (1978) Phytochemistry 17, 145. 11. Goudard, M., Favre-Bonvin, J., Strelisk¥, J., Nogradi, M. and Chopin, J. (1979) Phytochemistry18, 186.
12. Sakakibara, M. and Mabry, 3. J. (1977) Rev. Latinoam. Quim. 8,99. 13. Markham, K. R. (1982) Techniques of Flavonoid Identification. Academic Press, New York. 14. Collins, F. W., Bohm, B. A. and Bose, R. (1977) Phytochemistry16, 1205.