Methylated C-glycosylflavones as taxonomic markers in orchids of the subtribe ornithocephalinae

Pergamon

0031-9422(94)00479-x

Phytochemastry, Vol 37, No. 4, pp. 1045-1053, 1994 Copyright 0 1994 Elsevler science Ltd Printed in Great Britao. All rights nserved 0031-9422/94 s7.OO+o.a)

METHYLATED C-GLYCOSYLFLAVONES AS TAXONOMIC MARKERS IN ORCHIDS OF THE SUBTRIBE ORNITHOCEPHALINAE CHRISTINEA. WILLIAMS,A. L. TOSCANODE BRITO, JEFFREY B. HARBORNE,JOHN EAGLES*and

PETER G. WATERMAN? Department of Botany, School of Plant Sciences,University of Reading, Whiteknights, Reading RG6 2AS,U.K.; *AFRC Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, U.K.; TPhytochemistry Research Laboratories, Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow Gl lXW, Scotland, U.K. (Received 19 May 1994) Key Word Index-Zygostates;

Ornithocephalus; Ckytroglossa; Phymatidium; Rauhiella; Ornithocephalinae; Orchidaceae; flavone C-glycosides; apigenin 7,4’-dimethyl ether 6-C-glucoside-2”-0-rhamnoside.

Abstract-In a leaf flavonoid survey of 15 Brazilian orchid species from the subtribe Ornithocephalinae, flavone Cglycosides were found to be the major constituents in all except two Ornithocephalus taxa. In Zygostates cornuta a rare glycoside, apigenin 7,4’-dimethyl ether 6-C-glucoside-2”-0-rhamnoside, was identified. This also occurred in 2. alleniana, Z. lunata, Z. pellucida and Z. pustulata. Two further similar apigenin 7,4’-dimethyl ether C-glycoside-Orhamnosides, possibly with different C-linked sugars, were detected in Z. multiflora and Z. grandijora. In Z. cornuta, apigenin 4’-methyl ether 6-C-glucoside-2”-0-rhamnoside was found to co-occur with the apigenin 7,4’-dimethyl ether derivative. Ornithocephalus myrticola was distinguished by the presence of an apigenin I-C-methylpentoside and three apigenin 7-methyl ether I-C-glycoside derivatives. In 0. Bicornis and 0. kruegeri, another apigenin 7,4’-dimethyl ether C-glycoside was found, while from Phymatidumfalcifolium, apigenin 7,4’-dimethyl ether and apigenin 7-methyl ether C-glycosides and their 0-arabinosides were isolated. Chytroglossa marileoniae also contained an apigenin 7,4’dimethyl ether C-glycoside, which appeared to be the same as that in P.falci$olium. Rauhiella siluana, on the other hand, could be distinguished from all the other taxa studied by the absence of methylated C-glycosylflavones and presence of isovitexin. 6-Hydroxyflavone 0-glycosides, which are characteristic leaf constituents of some Oncidium species in the related subtribe Oncidinae were not detected in the present sample. However, the high frequency of methylated flavone C-glycosides suggests a chemically advanced evolutionary position for the Ornithocephalinae, which is in agreemeyt with evidence from their complex floral and vegetative morphology.

INTUODUCTION

latter

usually

interpreted

as a putative

close

relative

cz 31.

The Ornithocephalinae is a small subtribe of the family Orchidaceae comprising 15 genera and about 80 species. Five genera, Dunstervillea Garay, Hintonella Ames, Hof meisterella Rchb.f., Oakes-Amesia C. Schweinf. and P.H. Allen and Platyrhiza Barb. Rodr., are monotypic and only three, Phymatidium Lindl. (1 l), Zygostates Lindl. (18) and Ornithocephalus Hook. (ca 20), have more than 10 species (Toscano de Brito, in prep.). Because of the diminutive size of the plants and the rarity of some of the species, very little is known about their biology. They are epiphytic herbs mostly found in the tropical rainforests of Central and South America with high proportion of genera (40%) and species (ca 50%) restricted to the Atlantic coastal forests of Brazil. The Ornithocephalinae is traditionally recognized as one of the most advanced in the Orchidaceae [e.g. 1, 23. Indeed, the complexity and diversity of floral and vegetative morphology of some of the species of the Ornithocephalinae seem to have reached a level which is apparently only paralleled by some members of the Oncidiinae, the

The genera of the Ornithocephalinae are distinguished on the basis of their floral morphology, but gross leaf morphology, leaf anatomy and habit also play an important role in distinguishing groups of genera. The only available overview of this subtribe is that of one of us (Toscano de Brito, in prep.) who has identified some apparent misfits in certain genera that might be raised to generic level by other taxonomists with a narrower generic concept. However, in view of the still inadequate material available for examination, he has adopted a more conservative approach and has attempted to point out these problematic groups rather than simply segregating them as new genera. Thus, the genus Phymatidium has been divided into four sections, two of them being monotypic. Each section possesses a set of rather distinctive features and one could argue that they deserve generic recognition. Another polymorphic genus is Zygestates which has been divided into two subgenera, Zygostates and Dungsiella, and several sections, some of the latter comprising species with unique and highly

1045

1046

C.

A. WILLIAMS

complex floral morphology. As with Phymatidium, the segregation of some of the distinct groups of Zygostates as autonomous genera could be advocated. However, such a step would enforce the application of the same approach to other genera in the subtribe culminating in the creation of a number of monotypic genera, which would certainly not clarify our understanding of this subtribe. Although a taxonomic revision of the genus Ornithocephalus was beyond the scope of Toscano’s study, the floral and vegetative morphology of this genus was thoroughly investigated. It has similar floral traits to those of Zygostates, differing from the latter only in a few features. However, the habit, vegetative morphology and leaf anatomy of Ornithocephalus are rather distinct and readily distinguish this genus from Zygostates. Rauhiella Pabst & Braga (two species) and Chytroglossa Rchb.f. (three species) are two further genera of this subtribe which share a similar habit and leaf anatomy, but have distinct floral morphology. There has been only one major survey of leaf flavonoids of the Orchidaceae [4] in which 142 species from 72 genera were examined. Flavone C-glycosides were mostly found in the subtribe Vanillinae and advanced members of the subfamily Epidendroideae whereas flavonol glycosides were more frequent in the tribe Neottieae and the subfamilies Orchidoideae and Spiranthoideae. The Ornithocephalinae was not included in this survey and no other Aavonoid study has been carried out on this subtribe. However, in the related Oncidiinae, all five species surveyed contained unusual 6-hydroxyflavones as their only leaf flavonoids. These included scutellarein 6methyl ether and pectolinarigenin 7-rutinosides from Oncidium excavatum Lindl. and 0. sphacelatum Lindl. and pectohnarigenin 7-glucoside from 0. excavatum. The only other record of 6hydroxyflavones in the Orchidaceae is of pectolinarigenin and its 7-glucoside from Eria javanica Blume (tribe Podochileae, subtribe Eriinae) [4]. In the present study seven Zygostates and five Ornithocephalus species, Chytroglossa marileoniae, Phymatidium falcfolium and Rauhiella silvana were analysed for their leaf flavonoids as part of a systematic study of the subtribe Ornithocephahnae. Unfortunately, samples of representative species of other genera of this subtribe were unavailable for study.

RESULTS

The results of the leaf flavonoid analysis of 15 orchid species from the subtribe Ornithocephalinae are presented in Tables 1 and 2. Flavone C-glycosides were the main leaf flavonoid constituents in all except two taxa, Ornithocephalus gladiatus and 0. lankesteri, where no flavonoids were detected but two unidentified dark to dark (in UV + NH,) phenohc compounds were isolated, which from their UV spectra could be phenanthrene derivatives. There was insufficient plant material for further characterization. However, a large variety of phenanthrenes and bibenzyls have been reported from

et

al.

some other subtribes of the Orchidaceae i.e. Coeiogyninae, Dendrobiinae, Bulbophyllinae and Bletiinae [S] but not so far from the Ornithocephahnae. Sixteen flavone C-glycosides (I-16) were isolated from the remaining 13 orchid taxa. These were all apigenin based structures, most of which were 0-methylated and 0-glycosylated. A rare glycoside, apigenin 7,4’-dimethyl ether 6-C-glucoside-2”-0-rhamnoside (1) and the corresponding apigenin 4’-methyl ether 6-C-glucoside-2”-Orhamnoside (2) were characterized from Z_vgostates cornuta. These compounds could not be separated by TLC, HPLC or UV spectral analysis and so an inseparable mixture was analysed by ‘HNMR. The presence of a mixture was also apparent from the FAB-MS, which gave a major molecular ion at m/z 606 (C&H14014 required 606) and a minor molecular ion at m/z 592 (C28H32014 required 592). The structure of 1 as an apigenin 7,4’-dimethyl ether 6C-glycoside followed from a comparison of R, and UV data with vitexin, isovitexin and other related C-glycosides and was confirmed by treatment with pyridinium chloride which gave an apigenin 6-C-glycoside. Dimethylation at the 7- and 4’-positions was apparent from R, mobilities and UV spectral shifts. Demethylation gave a product with similar R/s to isovitexin on TLC in 15% HOAc and water. The presence of a rhamnose moiety linked to the 6-carbon sugar was apparent from its release by mild acid hydrolysis. The nature of the 6-carbon sugar however in 1 and 2 could only be established following NMR analysis. This latter analysis also confirmed all other structural assignments in 1 and 2. NMR spectra were run in pyridine-d, on a mixture of 1 and 2. The presence of the para-substituted B-ring was clear from the presence of an A,B, aromatic system and the 4’-methoxyl identified by a NOESY spectrum which revealed interaction with H-3’ and H-5’. The H-3 signal was recognized by the ‘5 interaction to C-2 in the HMBC spectrum [6], which correlated with the 3J coupling of H2’ and H-6’ to the same carbon. The signal unsubstituted A-ring position could be assigned as C-8 in the light of the highly deshielded methine carbon [7] and the ‘5 coupling to C-9 in the HMBC spectrum. The H-l” proton of the C-glycoside hexose was observed as a doublet (.I = 9.6 Hz, typical of glucose) with ‘5 and 3J couplings to C-6 and C-5/C-7, respectively. The COSY-45 spectrum revealed coupling from H-l” to triplet for H-2” which, in the HMBC spectrum showed a long-range coupling (3J) to a methine carbon at ca 103 ppm (66.33, broad singlet). This single is typical of H1”’ of a-rhamnose. Using COSY-LR and TOCSY spectra it was possible to trace and assign the ‘H resonances from H-l around the entire rhamnose system. After assignment of the rhamnose signals it was possible to return to the COSY-45 spectrum and assign the remainder of the C-hexose. This showed H-l” to H-4” all to be axial and confirmed the structure as /I-glucose. On this basis the compound was assigned structure 1. A significant amount of the 7-dimethyl compound (2) was also present leading to duplication of many of the signals (see Table 3).

Chemot~onomy

of the orchids from the subtribe ~mith~phalinae

?-

H

dci

3 8 tT!

a I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

1

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

f-b

I

I

1

I

I

I

I

I

I ++

I

I

I

I

I +i-

I

I

I

I

I f-k

I

I i-

I

I ++

I

I

I

1

I

I

I

I

I

I

1

I

I

I

I

I

I

I

i

I

s

I

I

1

I

I

I

f

1

I

i

I

I

I

f

1048

C. A. WILLIAMS et al. Table 2. Flavone

Compound number

C-glycostdes

Flavone

1 2 3 4 5 6 7 8 9 10

1% 14 15 16

identified

of the subtribe

acyl group*

X”-0-XylGlc$

Table 3. Chemical

2 3 4 5 6 7 8 9 10 1’ 2’16 3’15’ 4’ 1” 2” 3” 4” 5” 6’ 1”’ 2” 3”’ 4” 11, 5 ,I, 6 7-OMe 4’-OMe

taxa

7,4’-dimethyl ether 6-C-glucoside-2”-O-rhamnoside 4’-methyl ether 6-C-glucoside-2”-O-rhamnoside 7,4’-dimethyl ether C-glycoside-X”-O-rhamnoside* 7,4’-dtmethyl ether C-glycoside-X”-O-rhamnostde 7-methyl ether C-glycoside + unidentified acyl group* I-C-methylpentoside 7-methyl ether C-glycoside-X”-O-glucoside* 7-methyl ether C-glycoside-X”-O-rhamnoside* IT-methyl ether C-glycoside-X”-O-rhamnoside + unidentified 7,4’-dimethyl ether 6-C-glycoside* 7,4’-dimethyl ether 8-C-glucoside-X”-O-arabinoside*t 7,4’-dimethyl ether 8-C-glucosidet 7-methyl ether C-glycoside-X”-0-arabinoside* 7-methyl ether C-glycoside*

*Identity of C-sugar not determined. tIdentity of C-sugar not confirmed by NMR. fOrder of O-sugars not determined. §Two closely related compounds which could not be separated available.

C/H

in some

C-glycoside

Apigenin Apigenin Apigenin Apigenin Apigenin Apigenm Apigenin Apigenin Apigenm Apigenin Apigenin Apigenin Apigenin Apigenin Isovitexin Isovitexin

II§ 12

identified or partially Ornithocephalinae

‘H

6.9717.08

6.9417.05

7 97 d (8.9) 7.12 d (8.9) 56415.76 d (8.9) 5.23/5.54 t (8.9) 4.40 m 4.16 t (8.9) 4.40 M 4.40/4.55 14.62 m 6.331647 br s 4.8014.82 br s 4.3 l/4/40 m 4.09 m 32313.42 m 1.2711.29 t (6.8) 3.88 s 3.77 s

from the small amount

shift values for a mixture ‘Y 164.9 105.3 183.4/183.8 164.6 111.8/112/l 163.51166.4 92.1196.2 158.2/l 58.7 106.2 124.5 129.0/129.1 115.4 163.5 74.3 76.6178.3 83 5 73.5 82.0/82.3 64.0 102.8/103.3 72.9 74.3 74.3 70.2 19.1119.5 56.0 56.8

Spectra were run in pyridine-d, at 400 MHz. Couplmg constants are given in parentheses.

of material

of 1 and 2

zJ

“J

164.9

106.2

158.217

106.2, 112.1

163.5

129.1, 163.5, 164.9 115.4, 124.5

112.1

163.5, 164.6 73.5, 102.9

76.6 74.3

70.2

74.3 166.4 163.5

Chemotaxonomy of the orchids from the subtribe Omithocephalinae Compound

1 was identified also in four other Zyg-

estates species: Z. alleniana, Z. lunata, Z. pellucida and Z. pustulata. However, from Z. grandiflora and Z. multijlora

two further apigenin 7,4’-dimethyl ether C-glycoside-Orhamnosides (3 and 4, respectively) were isolated, which differed in R, both from 1 and each other before and after acid treatment but otherwise appeared to be very similar structures. It is possible that the 6-C-glucose may occur in a different configuration and/or with a different linkage in these compounds or another isomeric hexose sugar such as galactose may be present. Apigenin 7,4’-dimethyl ether 6-C-glycosides appear to be good taxonomic markers for all the Zygostates species studied. However, Z. pustulata, which is placed in subgenus Dungsiella can be distinguished from the other six taxa of subgenus Zygostates by the additional presence of an acylated apigenin 7-methyl ether C-glycoside (5). By contrast, the five Ornithocephalus species studied were more heterogeneous in their chemistry. Thus, 0. gladiatus and 0. lankesteri were distinguished from the other three taxa (but not each other) by the absence of leaf flavonoids and presence of two possible phenanthrene derivatives (see above and Experimental). In 0. myrticola an apigenin C-glycoside (6) was isolated, which had a molecular weight of 416 suggesting that it might be an apigenin C-rhamnoside. However, the chromatographic mobility of 6 compared with an authentic marker of apigenin 6 C-rhamnoside (RZs 36, 28 in 15% HOAc, respectively) indicated that the C-linked sugar was not rhamnose and could possibly be 6-deoxyglucose or another 6-deoxy sugar. On acid treatment an isomer with higher R, was detected in BAW and 15% acetic acid suggesting that 6 may be the 8-C-isomer, apigenin 8-C-6deoxyglycoside. The three other glycosides isolated from 0. myrticola were all apigenin 7-methyl ether derivatives (7-9). Compound 7 is tentatively identified as an apigenin 7-methyl ether C-glycoside-0-glucoside, 8 as an apigenin 7-methyl ether 0-rhamnoside and 9 as an acylated apigenin 7-methyl ether C-glycoside-0-rhamnoside. Compound 6 was found also in 0. kruegeri together with yet another apigenin 7,4’-dimethyl ether C-glycoside (lo), which had higher R,s than the 6-isomers of acid treated 1, 3 and 4 in BAW, 15% HOAc and water. Compound 10 was present also in 0. bicornis. There was insufficient leaf tissue to complete the identification of compounds 7-10. The representative species from the genera Chytroglossa, Phymatidium and Rauhiella all had distinctive flavonoid profiles and could be distinguished from each other and all the Zygostates and Ornithoeephalus species studied, although two further apigenin 7,4’-dimethyl ether C-glycosides and two apigenin 7-methyl ether Cglycosides (11-14) were identified in P. falcifolium. Compound 12 was identified as an apigenin 7,4’-dimethyl ether C-glucoside (or C-galactoside), which is probably the I-isomer since an isomer with higher R, in BAW appeared after acid treatment (6-8-isomerization). However, neither isomer co-chromatographed with acid treated 1 suggesting some difference in the structure or configuration of the C-sugar. A structure with the same R,s as 11, apigenin 7,4’-dimethyl ether C-glycoside-O-

1049

arabinoside, was also detected in C. marileoniae thus linking Phymatidium and Chytroglossa with Zygostates and Ornithocephalus. Rauhiella silvana, on the other hand, was distinguished from all the other genera studied in the Ornithocephalinae by the presence of isovitexin and isovitexin 0-xylosylglucoside. DISCUSSION

Undoubtedly, the most important outcome of the present flavonoid survey of orchids from the Ornithocephalinae is the discovery of rare glycoflavone 7,4’-dimethyl ethers which have previously only been reported in a very small number of plants. Also, although flavone Cglycosides are known to be characteristic constituents of the Orchidaceae, the large variety of apigenin 7,4’-dimethyl ether and 7-monomethyl ether C-glycosides present in members of this subtribe is very unusual and indeed these compounds are quite rare in angiosperms. Only six apigenin 7,4’-dimethyl ether C-glycosides have been found previously. These are apigenin 7,4’-dimethyl ether 6- and 8-C-glucosides (embigenin and isoembigenin), isoembigenin 2”-0-glucoside and 2”-0-rhamnoside which have been reported from Siphonoglossa Oerst. (Acanthaceae) [S, 93 and the 6-C-arabinoside and its 2”0-glucoside, which have been characterized in leaves of Asterostigma riedelianum Kuntze (Araceae) [lo]. Methylated flavonoids are considered to be advanced chemical characters [ 111, suggesting that Zygostates, Ornithocephalus, Chytroglossa and Phymatidium may be highly evolved genera, which is in agreement with evidence from their complex floral and vegetative morphology. The presence of isovitexin and absence of methylated C-glycosylflavones in Rauhiella clearly separate this genus from other members of the Ornithocephalinae studied, although, as already stated here, it shares several other features with Chytroglossa. However, it would be necessary to examine further species of Rauhiella and Chytroglossa to confirm this chemical difference. The remaining genera are all linked by the presence of apigenin 7,4’-dimethyl ether C-glycosides but can be distinguished from each other by the occurrence of different isomers or different O-sugars. Thus, Zygosdates species produce mainly apigenin 7,4’-dimethyl ether 6-C-glycosides; Orniahocephalus species a unique apigenin 8-methylpentoside and possibly apigenin 7-methyl ether 8-C-glycosides; and Phymatidium falcifolium apigenin 7,4’-dimethyl ether and 7-monomethyl ether C-glucoside-0-arabinosides. At the generic level it is interesting that the present flavonoid data, i.e. the presence of the apigenin 7,4’dimethyl ether 6-C-glycosides, corroborates evidence from floral morphological analyses, supporting the inclusion of the genus Dipteranthus Barb. Rodr. in the synonymy of Zygostates. Thus, it is significant that Z. pellucida, Z. grandijlora and Z. pustulata, which have been recently treated in Dipteranthus [12], all have apigenin 7,4’-dimethyl ether 6-C-glycosides as their leaf flavonoids. Zygostates pustulata, now placed in subgenus Dungsiella, differs from the six species of Zygostates subgenus Zygostates surveyed in the occurrence of the

C. A.

1050

WILLIAMS et al.

acylated apigenin 7-methyl ether C-glycoside 5, thus supporting its placement in a different subgenus. However, further research is required to determine the Aavonoid profile of the other species in subgenus ~ungsiell~. As already mentioned here, subtribe Oncidiinae has been considered as a probable sister group of the Ornithocephalinae. However, the four On~idium species so far surveyed [4] possess a rather distinct Aavonoid profile, providing no evidence for such a relationship. They Iack flavone C-glycosides and have some new and unusual 6hydroxyflavone glycosides as their only leaf flavonoids. Nevertheless, when compared with the size of the Oncidiinae, 77 genera and 1232 species [ZJ the number of species of the Oncidiinae investigated is obviously not representative. Further work to examine the leaf tiavonoids of the major clades of the Oncidiinae might contribute to a better understanding of relationships within and outside this subtribe. EXPERIMENTAL Plant materiel. Most of the plant mate~al was collected and verified by one of us (A.L.T.B.) but specimens from a number of herbaria were also used for which

Table

4.

details are given in Table 1. Voucher specimens have been lodged in various herbarium as cited in Table 1. COSY-45, COSY-LR, TOSY, HC-COBI and HMBC spectra were run on a Bruker AMX-400 NMR spectrometer using standard microprograms. Identl~catio~ of leu~~u~onoids. Direct 80% MeOH leaf extracts of each species were run two dimensionally on Whatman No. 1 paper in (1) BAW and 15% HOAc and (2) BAW and Hz0 against rutin as a marker. Acid treated (2 M HCI at 100” for 40 min) leaf extractswere run two dimensionally in the same solvents. Flavonoid glycosides were isolated by multiple ZDPC on 3 MM paper and further purified by 1DPG in 30% HOAc and H,O in most cases. R, and some HPLC data for glycosides 1-16 are given in Table 4, R,s of l-15 after acid treatment in Table 5 and UV spectral data for l-16 in Table 6. Characterization of apigenin 7,~~imethyl ether 6-Cglucose-~-0-rh~noside (1) and apigenin 4’-methyl ether 6-C”glucoside-2”-O-rhamnoside~om Zygostates cornuta. R,, HPLC and UV spectral data are given in Tables 4-6.

The UV absorbance of 1 plus 2 in MeOH at 274,331 nm was characteristic of an apigenin derivative and this was

R, and WPLC data for fiavone C-giycosides found in some orchid species from the subtribe Ornitho~pha~ae R, x 100 on cellulose TLC in

Flavone C-

Colour in

glycosides

W-bNH,

BAW(1)

15% HOAc

N,O

CAW 1: 1

1+2t 3 4 5 6 7 8 9 10 11 12 13 x4 15 16

Dk/Dk Dk/Dk Dk/Dk DkjY DkjY Dk/Y Dk,‘Y Dk/Y Dk/Dk Dk/Dk Dk/Dk Dk/Y Dk/Y Dk/Y DkjY

62 79 80 85 73 86 83 84 89 72 57 57 45 68 58

93 86 75 82 36 62 83 81 61 81 19 74 17 39 66

67 62 55 56 11 23 67 91 19 62 04 54 03 11 43

92 79 92 93 71 93 92 90 91 91 75 61 58 38

1

DkjY

-

35

06

-

Apigenin 6C-Rha$ Isovitexin$

Dk/Y Dk/Y

-

28 39

-_

-

11

58

~methylat~

68

HPLC BAW (2) R&G

78 88 75 -

24.39 21.85 26.13

86

21.37

-

^__ 86,92*

80,90

.-

90 71

Key: BAW (1) = n-butanol:acetic acid:water, 4: 1:5 (top layer); BAW (2) = CAW =chloroform: acetic acid:water, 1: 1:O.l; Dk =dark; Y =yeItow. *There was insurgent material to separate the two compounds detected in BAW(2). tcompounds 1 and 2 could not be separated by TLC or HPLC but could be distinguished by ‘H NMR.

SAuthentic marker included for R, comparison. $HPLC was carried out on a Waters 600 System using a Waters Bondapak Phenyl Cl8 column (3.9 mm id. x 30 cm) with the following conditions: A 2% HOAc, B MeOH-HOAc-H~O (18: 1:

Chemotaxonomy

of the orchids from the subtribe Ornithocephalinae

1051

Table 5. R, values for flavone C-glycosides 1-16 after acid treatment Isomerized flavone Cglycosides

Colour in UV + NH,

1+2 3 4 5 6 I 8 9 10 11*

12 13 14 15 16

R, x 100 on cellulose in

BAW

15% HOAc

H,O

CAW 1:l

BAW (2)

Dk/Dk Dk/Dk Dk/Dk Dk/DY

19 83 83 98

14, 54 17,45 14,38 22, 50

11 07 06 09

93 74 91 91

13,88 91 83 -

Dk/DY Dk/DY Dk/DY Dk/DY Dk/Dk Dk/Dk Dk/Dk Dk/Y Dk/Y Dk/Y Dk/Y

78,97 81 ‘JO,85 96 64,89 61.71 61,14 45 45 41,68 41,68

21,42 13 :!2, 52 07 21,61 22, 38, 54, 62 20 16 15 14, 39 14,39

06, 12 01 03, 16 04 05, 19 03, 14 02, 15 03, 14 02,15 02,ll 02, 11

76 83 83,93 87 91 91 63 64 21, 58 21, 58

82,95

*Contained two apigenin 7,4’-dimethyl ether C-glycoside-0-glycosides which gave four spots (two sets of isomers) in 15% HOAc after acid hydrolysis. Key: BAW (l)=n-butanol:acetic acid: water, 4: 1: 5 (top layer); BAW (2)= butan-2-01: acetic acid:water, 70:5:25; CAW =chloroform:acetic acid:water 1: 1:O.l; Dk=dark; Y =yellow.

Table 6. W spectral data for flavone C-glycosides 1-16 Flavone Cglycoside

UV &II,, MeOH

+ NaOAc

+ H,BO,

+ NaOH

+ AlCl,

+ AlClJHCl

1+2* 3 4 5 6 7 8 9

214, 331 214,332 213, 332 271,332 210,334 269, 330 270, 332 271, 322 212,330 271, 324 271, 324 212, 326 271,325 212, 330 271,331

274, 321 214,335 213, 332 212, 335 280,388 269,334 210, 355 271, 326 211,335 271, 325 271, 325 272, 350 271,348 280,386 280,378

214, 333 214, 335 213,332 272,331 213, 345 269,345 270, 346 271, 330 212,331 271, 335 271, 327 212, 338 271, 330 214, 330 273,343

300,380, dec. 282, 336, 391 299,380 dec. 283,390 395 279 383 282, 368 dec. 286 dec. 281, 381 282,311 219, 382 265,212,383 398 391

350 214, 303,342 384 341,386 344, 388 339, 387

347 280,302, 344 383 338,384 340,386 338, 385

272, 304,335,370 212 304,331,310 272, 304, 338, 368 272,304,340,388 280, 346, 380, 278, 346

271, 303, 331, 370 212, 304, 337, 383 276, 303, 338, 382 273, 303, 339, 386 278, 343, 380, 217,343

10

11 12 13 14 15 16

dec. = Decrease in intensity. *Compounds 1 and 2 could not be separated by TLC or HPLC but were distinguished by ‘H NMK.

supported by negative borate and AlClJHCl shifts. The absence of a NaOAc shift indicated that the 7-hydroxyl was substituted in 1 (the major component) and the dark to dark colour (in UV + NH,) and the lower intensity of band II on addition of NaOH suggested that the 4’hydroxyl was substituted. Acid hydrolysis gave rhamnose and two isomeric aglycones, which still appeared dark to dark in UV+NH, and gave no borate shift suggesting that that they were apigenin 7,4’-dimethyl ether derivatives. This was confirmed by FAB-MS of the two isomers which both gave a molecular ion at 460, C,,H,,OrO requires 460. Compound 1 plus 2 were confirmed as

flavone C-glycosides by their resistance of 4 hr acid hydrolysis. Demethylation gave a product with similar R, to isovitexin in 15% HOAc (35,39 resp.) and Hz0 (06, 11). Compound 2 was detected and characterized by ‘H NMR (see text). Partial identification ofapigenin 7,4‘-dimethyl ether Cglycoside-0-rhamnosides from Z. grandiflora (3) and Z. multiflora (4). Compounds 3 and 4 showed similar colour properties in UV + NHJ, UV absorbance and shift reactions to 1 and to each other. On acid hydrolysis both gave rhamnose and an aglycone (flavone C-glycoside), which gave no NaOAc shift and was still dark to dark in UV

1052

C. A. WILLIAMS

+NH,. However, the aglycones of 1, 3 and 4 showed distinct chromatographic mobilities suggesting that there must be some difference in the form or linkage of the Csugars. As there was insufficient material for FAB-MS or NMR it was not possible to identify the C-sugars in 3 or 4. Partial identijcation of apigenin 8-C-methylpentoside (6)from Ornithocephalus myrticola. R,, HPLC and UV spectral data are given in Tables 4-6. The UV absorbance of 6 in MeOH and negative borate and AlCl,/HCl shifts suggested it was an apigenin derivative and the positive NaOAc shift that the 7-hydroxyl was free. Negative FABMS of 6 gave a deprotonated molecular ion at m/z 415 and positive FAB-MS a positive protonated molecular ion at m/z 417 (C,, H,eO, required 416). Acid hydrolysis gave a mixt. of 2 flavone C-glycoside isomers but no sugar was detected. Compound 6 was resistant to 4 hr acid hydrolysis confirming it was a C-glycosylflavone. On acid treatment an isomer with a higher R, was detected in BAW and 15% HOAc suggesting that 6 may be an S-Cisomer. However, it ran ahead of an authentic marker of apigenin 6-C-rhamnoside in 15% HOAc, confirming that it was not apigenin 8-C-rhamnoside, which would have a lower R, than the 6-isomer in this solvent. Therefore from this data and by analogy with the C-sugar found in 1,6 is tentatively identified as apigenin 8-C-methylpentoside. Partial characterization of 5 and 7-14. Compound 5 was shown to be an apigenin derivative from its MeOH UV spectrum. The absence of a NaOAc shift and its chromatographic mobility and dark to yellow colour in UV + NH, suggested it was a 7-methyl ether (Tables 4 and 6). Acid hydrolysis gave two isomers in 15% HOAc but no sugar was detected. Alkaline hydrolysis gave a product with much lower mobility than 5 in 15% HOAc (R/s 12,82) and H,O (R,s 10,.56) suggesting the presence of an acyl group but neither this nor the C-sugar could be identified from the small amount of plant material available. Compound 5 is suggested to be an acylated apigenin 7-methyl ether C-glycoside. Compounds 7-9 were all shown to be apigenin 7methyl ether C-glycoside derivatives from their R,, UV spectral data and dark to yellow colour in UV + NH,. Acid hydrolysis gave 3 chromatographically different apigenin 7-methyl ether C-glycosides, glucose from 7 and rhamnose from 8 and 9. The high mobility of 9 in H,O suggested it was acylated but the acyl moiety was not identified. The tentative identifications are: apigenin 7methyl ether C-glycoside-X”-O-glucoside (7), apigenin 7methyl ether C-glycoside-X”-0-rhamnoside (8) and acylated apigenin 7-methyl ether C-glycoside-X”-O-rhamnoside (9). Compound 10 appeared to be another apigenin 7,4’dimethyl ether C-glycoside from its R, and UV spectral data chromatographic mobility and dark to dark colour (in UV + NH,). Acid treatment produced an isomer with lower R, than 10 in BAW, 15% HOAc and H,O suggesting that 10 is a 6-C-isomer. R,, UV spectral data and colour properties (Tables 4-6) suggest that 11 and 12 are apigenin 7,4’-dimethyl ether C-glycosides and 13 and 14 are apigenin 7-methyl ether C-glycosides. On acid hydrolysis 11 gave 12 plus

rt 01.

arabinose and 13 gave 14 plus arabinose. Positive FABMS of 12 gave a positive protonated molecular ion at m/z 461 (Cz3H,,0,, requires 460) which is in agreement with the structure apigenin 7,4’-dimethyl ether C-glucoside (or C-galactoside). Acid treatment gave a mixt. of 12 plus an isomer with higher R, in BAW and H,O suggesting that 12 is an S-isomer. The C-sugars of 11,13 and 14 could not be determined from the material available. Partial identifications are: apigenin 7,4’-dimethyl ether S-C-glucosideX”-0-arabinoside (11) and apigenin 7-methyl ether S-Cglycoside (14) and its X”-0-arabinoside (13). Compound 15 was identified by UV spectral data, acid hydrolysis and co-chromatography with an authentic marker as isovitexin. Compound 16 gave isovitexin, xylose and glucose on acid hydrolysis. Its UV spectral data indicated that the 5.7- and 4’-hydroxyls were free so both sugars must be attached to the C-glucose. The presence of a di-O-glycoside was confirmed by the high mobility of 16 in aq. solvents. Compound 16 is therefore either isovitexin X”-0-xylosylglucoside or X”-O-glucosylxyloside. Datafor two unknown phenolic compoundsfrom Omithocephalus lankesteri and 0. gladiatus. Both compounds appeared dark to dark (in UV + NH,) and gave the same UV and R, values. UV 1:::” 250, 273. 316: +NaOAc 250,273,316, +H,BO, 250,273,316, + NaOH 288,375. The spectral data remained the same after acid hydrolysis. R, data on cellulose TLC before (and after hydrolysis): BAW 85 (SS), 15% HOAc 90(44,74), HI0 71(18,39), CAW 93 (100) and 11% MeOH/CHCl, (silica gel TLC) 09, 18 (15, 40, 46). Acknowledgements-The authors are very grateful to Professor Dr H. D. Zinsmeister of the Botany Department, University of Saarlades, Germany for TLC comparision of compound 6 with authentic apigenin 6-Crhamnoside (isofurcatin), vitexin, isovitexin, orientin and iso-orientin. We also thank Mrs Jenny Greenham, Botany Department, University of Reading for the HPLC analyses. One of us (A.L.T.B.) acknowledges financial support from Coordenaogo de Aperfeieoamento de Pessoal de Nivel Superior (CAPES), Brasilia, Brazil and from the Margaret Mee Amazon Trust, Kew, U.K. We thank Dr Phillip Cribb of the Royal Botanic Gardens, Kew for his critical comments. NMR spectra were run at the University of Strathclyde NMR Laboratory. REFERENCES

1. Rasmussen, F. N. (1985) in The Families of the Monocotyledons: Structure, Evolution and Taxonomy (Dahlgren, R. M. T., Clifford, H. T. and Yeo, F., eds), p. 254. Springer, Berlin. 2. Dressler, R. L. (1993) Phylogeny and CIassijication of the Orchid Family. Diocorides Press, Oregon. 3. Chase, M. W. and Pippen, J. (1988) Syst. Bat. 13,313. 4. Willlams. C. A. (1979) Phytochemistry 18, 803. 5. Veerraju, P.. Prakasa Rao, N. S., Japanmohan Rao, L., Jagannadha Rao, K. V. and Mohana Rao, P. R. (1989) Phytochemistry 28. 3031.

Chemotaxonomy of the orchids from the subtrihe Omithocephalinae 6. Bax, A. and Summers, M. S. F. (1986) J. Am. Chem. Sot. 106,2093. 7. Markham, K. R. (1989) Methods in Plant Biochemistry (Harborne, J.B., ed.). Vol. 1, p. 197. Academic Press, London. 8. Hilsenbeck, R. A. and Mabry, T. J. (1983) Phytochemistry 22, 2215. 9. Hilsenbeck, R. A. and Mabry, T. J. (1990) Phytochemistry 29, 2181.

1053

10. Markham, K. R. and Williams, C. A. (1980) Phytochemistry 19, 2789. 11. Harborne, J. B. and Turner, B. L. (1984) Plant Chemosystematics. Academic Press, London. 12. Pabst, G. F. J. and Dungs, F. (1977) Orchidaceae Brasilienses. Band II. Briicke, Kurt Schmersow, Hildesheim.