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The utility of leaf flavonoids as Taxonomic Markers in the subfamily and generic classification of the velloziaceae
BiochemicalSystematicsand Ecology,Vol. 19, No. 6, pp. 483-495, 1991. Printed in Great Britain.
0305-1978/91 $3.00+0.00 © 1991PergamonPressplc.
The Utility of Leaf Flavonoids as Taxonomic Markers in the Subfamily and Generic Classification of the Velloziaceae CHRISTINE A. WILLIAMS,* JEFFREY B. HARBORNE* and NANUZA L. DE MENEZESt *Botany Department, University of Reading, Whiteknights, Reading, RG6 2AS, U.K.; tlnstituto de Bioci~ncias, Universidade de S&o Paulo, Brazil
Key Word Index--Velloziaceae; flavones; 6-hydroxyflavones; flavone; leaf surface flavonoids; chemotaxonomy.
flavonols; flavone C-glycosides; amento-
Abstract--As a result of surveying 99 species of the Velloziaceae, it was found that the major leaf flavonoids are flavonol glycosides (in 68%) and flavone C-glycosides (in 48% of taxa). In the subfamily Vellozioideae, flavones such as luteolin occur in the free state together with flavone C-glycosides and this combination of characters distinguishes this subfamily from the Barbacenioideae. The genus Pleurostima is unique in producing two new 6-hydroxyflavone glycosides identified as the 7-apiosides of 6-hydroxyluteolin and 6-hydroxy-8-roethoxyluteolin. The genus Barbacenia is distinguished by the regular presence of isorhamnetin and occasional presence of quercetin 3-methyl ether. The only other plants where isorhamnetin is frequent are the Madagascan Xerophyta species, which also produce quercetin 3-methyl ether, luteolin and some unidentified constituents, but lack flavone C-glycosides. Otherwise Vellozia and Xerophyta species have similar flavonoid patterns, including the presence of 5-deoxykaempferol and the anthocyanin, petunidin 3,5-diglucoside. Nanuza plicata is distinguished from all other genera by the presence of the biflavonoid, amentoflavone.
Introduction The Velloziaceae, a small family of some 270 species, has its centre of diversity in Brazil but is well represented in eastern Africa by species of the genus Xerophyta. There has been much dispute as to the delimitation of genera and subfamilies. In a recent classification based largely on floral morphology and leaf anatomy, Menezes [1] divided the family into two subfamilies, the Vellozioideae and Barbacenioideae and recognized six genera. In this system the subfamily Vellozioideae is split into two genera, Vellozia (flowers with nine or more stamens and no corona) and Xerophyta (flowers with six stamens and no corona), while the four genera of Barbacenioideae contain species whose flowers have six stamens and a corona. This system differs from that of Smith and Ayensu [2, 3] in the inclusion of Barbaceniopsis, Nanuza, Talbotia and Brazilian Vellozia species in Xerophyta and their transfer, with the African Xerophyta species, from the Barbacenioideae to the Vellozioideae. Thus, only Barbacenia species are retained in the Barbacenioideae and these have been divided into four genera by the reinstatement of Pleurostirna [4], the creation of two new genera Aylthonia [5] and Burlemarxia [1] and the retention of the remaining taxa within Barbacenia. In the present paper, results are presented according to a modified version of Menezes' system [4] in which only valid names have been used and cross reference given to the classification of Smith and Ayensu [2, 3]. The names of genera and sections used here follow Smith and Ayensu [2, 3] except for the genus Barbacenia in which the four segregate genera of Menezes [1] are recognized. The subfamily concepts are, necessarily, qualified as "sensu Menezes" or "sensu Smith and Ayensu" according to the context. Table 4 compares the two published classifications with each other and a grouping of the taxa based on the flavonoid results of this study. The results of a recent leaf wax alkane survey of 110 species of Velloziaceae [6] (Received 17 January 1991) 483
484
C.A. WILLIAMS ETAL.
support the re-establishment of Pleurostirna and the formation of Burlemarxia but do not distinguish between Aylthonia and Barbacenia or lend support for the recognition of Velloziasect. Xerophytoides as a section of Xerophyta. Our previous identification of the biflavonoid, amentoflavone [7], in the family suggested that a leaf flavonoid survey of the Velloziaceae could provide further useful chemical characters to help in the classification of this family. Materials and Methods P/antmater/a/. Both herbarium and air-dried plant material was received from various sources, details of which are given in Table 1. Plant identifications were carried out by one of us (NLdeM). Extraction and detection of /eaf surface f/avono/ds. Leaf material was covered for ca 1 rain with CHCI3, The decanted CHCI3 extract was concentrated and run on silica gel TLC in toluene:HOAc (4:1) against quercetin 3-methyl ether as a marker. If mobile dark UV absorbing compounds were present, surface flavonoids were recorded in the survey. Three compounds from Ve//oz/a evera/d//co-chromatographed, respectively with a mixture of quercetin 3-methyl ether, 3,7-dimethyl ether and 3,7,4'-trimethyl ether markers in BAW, forestal, 50% HOAc, CAW (2:1) and toluene: HOAc (4:1 ). Similarly, a surface compound from Ve//oz/ascopar/a co-chromatographed with a sample of 5-deoxykaempferol in the same solvent systems. More detailed identification of several new surface flavonoids from two other Ve//oziaspecies are being reported separately. /dendfication of/eaf f/avono/ds. The CHCI3 washed leaf material of each sample was extracted in 80% hot MeOH and the concentrated extracts developed by 2D PC in n-BAW and 15% HOAc, to compare flavonoid glycosidic patterns. The same extracts were tested for flavonoid sulphates by electrophoresis on 3MM Whatman paper at pH 2.2 (HOAc-HCO2H buffer) for 2 h at 40 V cm -~. A lack of dark UV absorbing mobile constituents showed that fiavonoid sulphates were absent from the Velloziaceae surveyed. Flavonoid constituents in the 80% MeOH extract were separated from cinnamic acid and other blue UV fluorescent phenolic compounds by either preparative 2D PC in BAW and 15% HOAc or by preparative 1D PC in BAW or 15% HOAc. The combined flavonoid glycoside spots or bands were eluted with 80% MeOH and the concentrated extracts subjected to acid hydrolysis with 2N HCI for 40 rain. Flavonoid aglycones were identified using standard procedures and by TLC comparison with authentic markers. Flavone C-glycosides were confirmed by 4 h acid hydrolysis, extraction into/so-amyl alcohol and TLC on cellulose against vitexin in BAW and H20. Orientin and iso-orientin in Ve//oz/astreptophy//a and V/i/ac/na were identified by co-PC and UV data and their O-glycosides by Rf and UV data and acid hydrolysis. Free flavonoid aglycones present in the direct leaf extracts, which run near the solvent-front in BAW and are barely mobile in 15% HOAc, were eluted separately from the flavonoid glycosides and compared directly by coTLC with marker compounds without previous acid hydrolysis. Luteolin 5-methyl ether from the leaf sheath of Xerophyta ey/esi/co-chromatographed with a marker isolated earlier from Juncus effusus [8]. A flavanone from the same extract gave a UV spectrum with ~.MeOH max 287 nm (alkaline spectral max at 298 and 325 nm) and Rr (×100) values on silica gel TLC of toluene:HOAc (4:1) 0, toluene:ethyl formate:HCO2H (5:4:1) 2 and toluene:pyridine:HCO2H (100:20:7) 0 and on PC:BAW 100, 50% HOAc 100, Forestal 100 and CAW (2:1) 100. It did not co-chromatograph with naringenin or eriodictyol and was not studied further. /dent/fication of 6-hydroxy/uteo//n 7- apios/de (1) and p/eurostim/n 7-apios/de (2) from Pleurostima purpurea. The two glycosides were separated from an 80% MeOH leaf extract by preparative PC in (1) 15% HOAc, (2) BAW and (3) 50% HOAc, compound 2 was further purified by HPLC on a spherisorb 50DS2 column, 25 cm× 8 mm i.d. Solvent A 100% H20, B MeOH-H20-HOAc, 18:1:1. Initial conditions 50% A, 50% B. Gradient over 15 rain to 5% A, 95% B and back to initial conditions after 16 rain. Flow speed 2 ml min -1 RRt 9.52 (1 = 9.22), Rf (×100) values of I and 2 (compared with 6-hydroxyluteolin 7-glucoside): BAW 39, 51(22), Foresta143, 63 (48); 50% HOAc 43, 64 (37) and CAW (1:1) 41, 55 (30). UV spectral data: 1 ;~a, MeOH 283, 347; +NaOAc 264, 362; -I-H3BO3 262, 284, 359 and 2 MeOH 260, 278, 351 ; +NaOAc 266, 358 and +H3BO3 266, 264 nm. Acid hydrolysis of I with 2N HCI for 1 h gave 6-hydroxyluteolin (co-TLC and UV spectral data) and apiose, identified by co-PC with an authentic specimen obtained from acid hydrolysis of apiin from Ap/um graveo/ensseed; P~ TBPW 20, BEW 19, Phenol 140 and BAW 19. Similar acid hydrolysis of 2 gave pleurostimin (6-hydroxy-8-methoxyluteolin) (3) and apiose. Rfvalues of 3 compared with 6-hydroxyluteolin: BAW 63 (47), Forestal 64 (42), 50% HOAc 47 (27) and CAW (2:1 ) 31 (12). UV spectral data for 3: ~ a , MeOH 256, 279, 353; +NaOAc 225, 263, 371 and +H3BO3 265, 377. FAB MS gave a Mrof 332 with strong ions at 176 and 184. This Mr suggests a hexahydroxyflavonewith one methyl group. As 3, like 6-hydroxyluteolin, is dark absorbing in UV light and remains unchanged by NH3 this suggests that there is a 6-hydroxyl with an extra hydroxyl at the 8 or 5'-positions. The positive spectral shifts with sodium acetate and with borate show that the 7, 3' and 4' hydroxyls are free, so that the methyl group could be located at the 6- or 8-position. However, the published UV spectral data for 5,7,8,3',4'-pentahydroxy-6-methoxyflavone [9] differ from those for 3. Furthermore, the ElMS of pleurostimin gave a molecular ion at 332 (100%) (C~6H1208requires 332) with fragments at 317 (M-15) (8%), 289 (M-43) (38%) and at 137 (238); this latter fragment corresponds to a 3,4-dihydroxyphenol B-ring, which eliminates an alternative structure with a trihydroxyl B-ring. The location of the methoxyl at the 8- position of the A-ring is also apparent from the relative intensity of the [M-15] ion (8%) compared to that of 6-methoxyluteolin (M-15 ion 59% of the molecular
C-glycosides
Species
+ +
+ + +
-------
ex Schult.
B fulva Goeth & Henr,
Barbacenia aft. globata Goeth & Henr. B. involucrata L. B. Smith B. rubro-virens Mart. +
--
+
--
(+)
--
(+)
+
Km
+
+
--
+
+
+
+
+
Isorh
m
m
m
Others
+
--
+
---
B umbrosa L. B. Smith & Ayensu +
+
+
---
+ +
+7 --
B. pulverulenta L. B. Smith & Ayensu B. riedeliana Goeth. & Henr.
& Ayensu
B gardneriSeubert B. graminifolia L. B. Smith B. paranaensis L. B. Smith
+
--
(+)
+
--
--
+ +
--
+
--
--
+
--
B. coronata P. F. Ravenna e x L. B. Smith
A. macrantha (Lem.) N. Menezes "~4ylthonia" sp. Barbacenia b/anchet~" Goeth. & Henr.
N. Menezes
Ayithonia b/ackii L. B. Smith A. gen~'anoides (Goeth & Henr.)
--
--
--
+
--
--
--
+
+
+
3
5
+
1
1
3
3
- -
3
--
--
5
2 +
Tricin --
Anthocyanin Qu3ME - -
--
6477
Harley et al. SPF 36077 CFCR
Anderson et aL 35123 Brazil K K
12288 (SPF)
11243a (SPF)
17379 Brazil
57H892/6 L. B. Smith
11251 (SPF)
Hatchbach 30183
412-7904208 Minas Gerais Brazil
Harley etal. 22512 Brazil
Harley et al. 18659 Brazil
Harley et al. 20623 Brazil
11596 (SPF)
11243 (SPF)
5312
11242 (SPF)
11581 (SPF)
11232 (SPF)
12329 (SPF)
Minas Gerais Brazil
Irwin et al. 22457
Maguire et al. 49116
11233 (SPF)
11586 (SPF)
11228 (SPF)
number
Collection
CFCR
CFSC
K
CFSC
H
K
K
K
K
CFSC
CFSC
CFCR
CFSC
CFSC
CFSC
CFCR
K
K
CFSC
CFSC
---
CFSC
Source
--
flavonoids
Surface
Anthocyanin Qu3ME - -
--
--
--
(+)
--
--
Anthocyanin
--
(+)
m
m
m
--
+
+
+
(+)
Aylthonia Menezes (including eight Barbacenia species considered as Aylthonia by Menezes but not yet validated as such)
+
+
+
---
Qu
Barbacenia bishopii L. B. Smith & Ayensu B. conicos#gma Goeth & Henr. B. flava Mart.
Barbacenioideae sensu Menezes [1] Barbacenia sensu Menezes
Flavone
Section
Genus
Subfamily*
TABLE 1. A LEAF FLAVONOID SURVEY OF THE VELLOZIACEAE
c}
<
-I
03
z O
>~
. -. --.
R fanniei N. Menezes R gounelliana (Beauv.) N. Menezes P. irwiniana (L. B. Smith) N. Menezes
P. pabstiana (L. B. Smith & Ayensu)
Pleurostima
N. Menezes
R inclinata (Goeth. and Henr.) N. Menezes R Iongi$capa (Goeth.) N. Menezes R nuda L. B. Smith & Ayensu P, plantaginea (L. B. Smith)
Ayensu) N. Menezes
Pluereuros#~na andersonii (L. B. Smith &
section Grazielae
-----
--
R spectabilis (L. B. Smith & Ayensu)
N. Menezes
. . --
.
R purpurea (Hook.) Raf .
N. Menezes
N.
Menezes R delicatula (L. B. Smith & Ayensu) N. Menezes
---
--
--
.
.
.
.
Flavone C-glycosides
Pleurostima caricina (Goeth. & Henr.)
Pleurostima Raf. Section Pleurostima
N. Menezes & Semir
B. rodriguesii N. Menezes & Semir B. spiralis (L. B. Smith & Ayensu)
N. Menezes & Semir
Burlemarxia pungens
Burlemarxia N. Menezes & Semir
Section Species
Genus
Subfamily*
TABLE 1--CONTINUED
.
÷ + + 4-
+
+
+ +
4-
+ 4-
+
+
.
.
.
.
Qu
. .
.
.
.
+ + + +
--
--
+ (+)
+
+ +
(+)
(+)
Km
-----
+?
--
---
--
---
--
--
Isorh
Anthocyanin Anthocyanin Anthocyanin Anthocyanin
--
60H Lu 60H Lu
60H Lu
60H Lu 6,8-DiOH Lu 8-ME
60H Lu -_
6,8-DiOH Lu 8-Me Unknown DK/DK free Lu
60H Lu
Anthocyanin Anthocyanin
_
_
Others
3 1 3 1?
--
---
--
--
--
-3
--
--
---
--
--
Surface flavonoids
CFCR CFCR CFSC CFSC
K
K Menezes
12322 (SPF) 12280 (SPF) 11614 (SPF) 11209 (SPF)
Ralter et aL 3833 Brazil
H.E. Strang 698, Brazil 722 (SPF)
635 (SPF)
A.C. Brade 17019 Brazil A.C. Brade 19774 Brazil
K K Menezes
670 (SPF) Harley 20350 Brazil
323 Rio de Janeiro, Brazil 12292 (SPF)
D. Sucre 2481 Braga
10574 47320 12286 (SPF) 12295 (SPF)
8833 40990
Collection number
Menezes K
CFCR
K
CFCR SPF CFCR CFCR
CFCR SPF
Source
m ~,
~.
c~ " ~"
--
+
--
.
.
(+) (+) --
+ + +
-.
+ + +
V. leptopetala Goeth. & Henr. --
+
--
--
V. glochidea Pohl V. hemisphaerica S e u b e r t V. incurvata M a r t .
.
.
V. glauca Pohl (leaf)
(stem)
+ .
.
.
--
+
--
V. glabra Mikan
--
--
+
V. flavicans M a r t . e x Schultes f.
--
--
.
--
--
--
--
+
+
--
--
--
--
--
--
.
--
.
--
--
--
--
.
--
(+)
+ +
(+)
--
+
--
+
+
+
+
--
+
+
+
--
V. epidendroides var. major L. B. S m i t h V. everaldii N. M e n e z e s (ined.)
V. coronata L. B. S m i t h V. declinans Goeth. & Henr. V. epidendroides M a r t . ex S c h u l t e s f. V. aft. epidendroides Mart. ex S c h u l t e s f.
V. caruncularis M a r t . e x S e u b e r t V. compacta M a r t . ex Schultes f .
Vellozia alata L. B. S m i t h V. aloifolia M a r t . V. bahiana L. B. S m i t h & A y e n s u V. breviscapa Mart. ex Schultes f.
Section Vellozia
Vellozia sensu S m i t h & A y e n s u
Vellozioideae
Nanuza plicata (Mart.) L. B. S m i t h & A y e n s u +
.
.
Nanuza L. B. S m i t h & A y e n s u (included in Xerophyta s e c t i o n Talbotia b y Menezes)
Barbaceniopsis humahuaquensis N o h e r
.
.
.
.
.
--
--
--
--
--
--
+
--
--
--
--
--
--
--
--
(+)
--
--
.
.
.
.
.
.
.
DK/DK unknown
DK/Y unknown
DK/DK unknowns
DK/Y &
2 DK/DK unknowns
Free Lu
DK/Y unknown
--
3
--
--
--
--
diME & triME
DK/DK unknown DK/relY unknown
Qu 3ME
--
--
--
2
5
--
1
--
--
--
1
Free Lu
DK/DK unknown
DK/DK unknowns
Free Lu, 2
DK/DK unknown
Free Lu
DK/DK unknown
DK/DK unknown
DK/DK unknown
B/Y glycoside
DK/DK unknown
DK/DK unknown
Free Lu Q u M E
Amentoflavone
DK/DK unknown
Barbaceniopsis L. B. S m i t h ( i n c l u d e d in Xemphyta section Xerophyta b y M e n e z e s a n d in B a r b a c e n i o i d e a e of S m i t h & A y e n s u )
Vellozioideae sensu Menezes [1 ]
CFSC
K
K
K
K
CFSC
K
K
CFCR
K
CFSC
K
CFSC
CFSC
CFSC
CFSC
K
K
H
K
CFSC
RNG
K
A n d e r s o n 10263 Brazil
11210 (SPF)
S. A m e r .
W . J . Burchell 7031 E. Trop.
Harley et al. 19681 Brazil
I r w i n et al. 34397 Brazil
W.R.
11231 (SPF)
I r w i n et al. 8073 Brazil
1 + 8 9 2 / 6 2 1 3 Brazil
12287 (SPF)
A n d e r s o n et al. 36200 Brazil
11250 (SPF)
I r w i n et al. 22173 Brazil
11579 (SPF)
11256 (SPF)
11256 (SPF)
2869 (SPF)
Philcox & F r e e m a n 4721 Brazil
Irwin et aL 27930 Brazil
L . B . S m i t h & A y e n s u Brazil
I r w i n et al. 22373 Brazil
11618 (SPF)
O. G u i m a r a e s Brazil
G. Hatchback 47086 &
S.A. Renvoise 3436 A r g e n t i n a
<
E~ ol
O Z
(+)
+
+ +
--
--
---
--
---
Km
_
_
_ --
--
_ (+)
Isorh
Vellozia abiedna Mart.
(+) --
+ +
_
(+)
Vellozia section Xerophytoides L. B. Smith & Ayensu (considered as Xerophyta section Talbotia by Menezes)
V. dawsonia L. B. Smith V. tubi#ora (A. Rich.) H.B.K.
Vellozia section Radia (A. Richard) L. B. Smith & Ayensu
--
---
+ -F +
--
+
V. verruculosa Mart.
(+) (+)
+ +
V. sdpitata L. B. Smith & Ayensu V. streptophylla L. B. Smith V. taxifolia (Mart. ex Schultes f.) Mart. ex Seubert V. variabilis Mart ex Schultes f.
(+)?
+ +
(+)
Qu
+
+
+
+
C-glycosides
Flavone
V. pusilla Pohl
V. phalocarpa Pohl
nanuzae L. B. Smith & Ayensu
v. nivea L. B. Smith & Ayensu
lilacina L. B. Smith
Genus Section Species
Sub~mily*
TABLE 1--CONTINUED
Surface
Free Lu
Anthocyanin Free Lu Free Lu
DK/DK unknown DK/Y unknown
DK/DK unknown DK/DK unknown DK/DK unknown
Qu3ME Free (Lu) DK/DK unknown
3
2
nd
K K
CFCR
K MO
Irwin et al. 2177 Brazil
Irwin et al. 20672 Brazil
12277 (SPF)
Irwin etal. 22173 Brazil R. Liesner 17243
Miss D. Smith 334 Brazil
W. R. Anderson 8414 Brazil Parani et al. 1503 Brazil Irwin et al. 9809 Brazil
Irwin et al. 20016 Brazil
K
Ynes Mexia 5873 Brazil 4317 (SPF)
11617 (SPF) 12300 (SPF)
K CFCR
11202 (SPF)
11598 (SPF)
11236 (SPF)
10371 (SPF)
Collection number
CFSC CFCR
CFSC
DK/DK unknown DK/DK unknowns Anthocyanin Free Lu DK/DK & DK/Y unknowns Qu3ME
CFSC
CFSC
CFSC
Source
2DK/DK & 3 other unknowns Free Lu
5 3
flavonoids
2 DK/DK unknowns Free Lu
2 DK/DK unknowns Petunidin Free Lu,
Anthocyanin Free Lu,
Others
+ +
+ +
V. scoparia Goeth. & Henr.
V. aft. scoparia Goeth. & Henr.
V. tragacantha Mart. ex. Schult
~erophyta" giuliettiae N. Menezes +
+
+
+
+
--
+
+
+
+
m
m
--
Free Lu
Anthocyanin
Free Lu
Anthocyanin
5-DeoxyKm
Free Lu
Anthocyanin
Free Lu
Anthocyanin
DK/Y free aglycone
Anthocyanin
Free Lu
Anthocyanin
--
3
--
6+5-DeoxyKm
4+5-DeoxyKm
--
4
CFCR
CFCR
CFSC
CFCR
CFSC
CFCR
T. Edwards 6.3.89
12289 (SPF)
12318 (SPF)
11597 (SPF)
12303 (SPF)
11208 (SPF)
12290 (SPF)
--
X. dasylirioides Baker var, pectinata
(+)
--
--
X. pinifolia Lam. var. villosa (H. Perr.)
X. equisetoides Baker X, equisetoides Baker var. trichophylla (Baker) L. B. Smith . .
+ -t-
Xerophyta section Barbacenioides L. B. Smith and Ayensu
(+)
+
--
H. Perr.
+
--
+
+
X. dasylirioides Baker var. (new?) X. eglandulosa H. Perr. vat. eglandulosa X. eglandulosa H. Perr. var. (new?)
(Baker) H, Perr.
--
X. dasylirioides Baker
Xerophyta Juss. section Xerophytoides (Madagascan species)
.
.
m
.
.
.
.
(+)
(+)
+
+
+
+
.
.
(new) above
X. eglandulosa var.
as in
DK/DK unknown
X. pinifolia)
(one same as in
DK/DK unknowns
Qu3ME 3
DK/DK unknown
Free Lu, Qu3ME
(Qu3ME)
DK/DK unknowns
Free Lu Qu3ME 2
DK/DK unknowns
Qu3ME 2
--
--
--
--
2
2
K
K
MB
MB
MB
MB
MB
MB
Mason District S. Province
W.J. Eggeling 6391 No. 1951
G.F. de Witte 2879 Zaire
167 (SPF)
170 (SPF)
175 (SPF)
170a (SPF)
176 (SPF)
173 (SPF)
Xerophyta Juss. (All the following African species are included by Menezes in her Xerophyta section Xerophyta but Smith & Ayensu divide them into three sections and place them in the Barbacenioideae
Talbo~'a elegans Bay. --
+
V. minima Pohl
sp. nov. ined.
+
V. laevis L. B. Smith
C-glycosides +?
Species
X. eylesfi (Greves) N. Menezes (leaf)
.
+ + + + +
--+ + --
X. schnitzleinia (Hochst.) Baker
. . --
(+) --
.
.
.
.
. .
.
.
.
--
---
---
--
-.
--
.
.
. .
.
.
Isorh
.
.
.
.
. .
.
.
Surface flavonoids
Procyanidin
DK/DK unknown DK/DK unknown
6 DK/DK unknowns DK/DK unknown
2 DK/DK
DK/DK unknown
DK/DK unknown
DK/DK unknown DK/DK unknown
4
---
---
--
--
--
1 4
Lu 5 M e Lu flavanone - B/Y unknown +
Others
K K K
K K
K K
K K
K
K K
K K
K K
K
K
K
K
Source
W. Africa
J . K . Jackson 1807, Sudan MerxmL~ller & Giess 30477 H. & H.E. Wanntorp No. 171
Thulin et al. 3605, Ethiopia Mrs J. Ash 2116
Tanzania Flora of Tanzania Terr. no. 1971B Mrs Jonquil Ash 16.4.76 Ethiopia
Ethiopia Christina Skarp Botswana W . J . Eggeling Nov. 1951,
S.W. Arabia M . G . Gilbert & T. G. Jefford 4537
P.R.O. Bally 11046 Somalia J . J . Lavranos 14/28.8.62
Spjut & Ensor 2790 Kenya Schmith 6043 Zaire
Natal, S. Africa Verdcourt & Polhill 2697 Kenya Napper 1641 Kenya
Moll & Nell 5505
L.J. Brass 16144 Malawi
O. West 6132 Zimbabwe O. West 6132 Zimbabwe
number
Collection
for Kew; Qu = quercetin; Km = kaempferol; Isorh = isorhamnetin; Lu = luteolin; 60H Lu = 6-hydroxyluteolin; Chrys = chrysoeriol; pleurostimin = 6-hydroxy-8-methoxyluteolin; DK/DK = dark to dark in UV light with and without ammonia vapour; Qu3ME = quercetin 3-methyl ether; diME = dimethyl ether; triME = trimethyl ether; B/Y = blue to yellow in U V + N H 3. The numbers of surface flavonoids found per sample are indicated in the appropriate column (+indicates general presence).
*Subfamiliy classificaton sensu Menezes [1 ], genera and sections according to Smith & Ayensu [2, 3] with cross reference to the proposed system of Menezes. Key: K = material from the Herbarium, Royal Botanic Gardens, Kew; M o = material from Missouri Botanic Garden; H = material from Howard University, Washington, USA; RNG = material from Reading University Herbarium; CFSC = Collection Flora of Serra do Cipb, Brazil; CFCR = Collection Flora of "Campos rupestres", Brazil; SPF = Herbarium of University of S&o Paulo, Brazil; M B = David Du Puy Collection
X. schnitzleinia (Hochst.) Baker var. somalensis X. simulans L. B. Smith & Ayensu X. squarrosa Baker X. viscosa Baker
+ --
.
-(+)
+ +
+
-
X. scabrida (Pax) Th. Dur. & Schinz
-
. --
nd
--
---
(+) +
.
.
.
.
.
--
X. humilis (Baker) Th. Dur. & Schinz
.
.
Km
+
nd .
X. acuminata (Baker) N. Menezes X. arabica (Baker) N. Menezes nd
(+) .
(+) +
Xerophyta section VellozioidesL. B. Smith & Ayensu
X. zambiana L. B. Smith & Ayensu
+ +
+
.
+ +
.
+
X. kirkii (Hemsl.) L. B. Smith & Ayensu X. retinen/is Baker
X. spekei Baker
.
.
.
Qu
(sheath)
.
Flavone
Section
Genus
Subfamily*
TABLE 1--CONTINUED
c~
P P ~_ rr-
o
LEAF FLAVONOIDSiN THE VELLOZlACEAE
491
ion). The absence of NaOAc shifts in the glycosides 1 and 2 suggest that the apiose moeity is attached at the 7-hydroxyl in both compounds. Anthocyanin identificadon. The pigments were extracted from dried leaf of Pleurosdma plantaginea and Vellozia streptophylla with methanolic HCI (after preliminary extraction with methanolic acetic acid and electrophoresis showed that zwitterionic anthocyanins were absent) and the concentrated extracts were separated by preparative PC in BAW and 15% HOAc. Petunidin 3,5-diglucoside was identified as the major pigment of both plants from the spectrum (;k~a` 275, 335 nm, A440/A535ratio 11%), acid hydrolysis to give petunidin and glucose (identified after co-chromatography in four solvents and separation from added galactose), H202 oxidation to give glucose and TLC co-chromatography in four solvents with an authentic marker. Delphinidin 3,5-diglucoside was identified as a minor pigment in P. plantaginea by similar procedures.
Results The results of a leaf flavonoid survey of 99 species of the Velloziaceae are presented in Table 1. The data refer to flavonoid aglycones detected after acid hydrolysis of partially purified 80% methanolic leaf extracts. The aglycones were characterized by TLC comparison of Rfvalues and colour reactions in UV light with authentic markers. These findings were confirmed by 2D PC of direct leaf extracts and more detailed examination of the flavonoid profile of selected species. The major leaf flavonoids were found to be the flavonol quercetin (in 68% of species) and flavone C-glycosides (in 48% of taxa). Other flavonols: kaempferol (in 26% of the sample) and isorhamnetin (in 18%) were less frequent leaf constituents. In a more detailed analysis of the leaf tissue of Vellozia streptophylla and V. lilacina, the well known glycoflavones orientin and iso-orientin were identified in both plants. In V streptophylla a number of flavone C-glycoside-O-glycosides, orientin and isoorientin 7-O-glucoside, an iso-orientin 7-O-diglucoside and an unusual iso-orientin 7-O-apioside were also detected. Two rarer components, 6-hydroxyluteolin and 6-hydroxy-8-methoxyluteolin (pleurostimin) were characterized as their 7-apiosides in Pleurostirna caricina (see Materials and Methods). This is the first report of pleurostimin and of both glycosides in nature. 6-Hydroxyluteolin was detected in a further four and pleurostimin in one other Pleurostima species of the 13 surveyed. The biflavonoid, amentoflavone, previously characterized from Nanuza plicata [7], did not appear to be present in the other 98 taxa of this survey. Leaf surface washings were taken from all plant samples, before the internal flavonoids were examined, and some 36% of the species were discovered to contain prenylated flavonols in the waxes. These compounds are currently being investigated and will be reported on in a separate paper. The "internal flavonoid" fraction, besides containing the glycosides described above, also has a number of non-glycosidic components as well. Five of these were identified readily as well known substances, namely luteolin (in 21 species of the plants surveyed), quercetin 3-methyl ether (in 10 species) luteolin 5-methyl ether (in one species, Xerophyta eylesii), tricin (in one species, Barbacenia riedeliana) and 5-deoxykaempferol (in one species, Vellozia aft. scoparia). Numerous further lipophilic components of high Rf values in butanolic solvents which appeared dark-absorbing in UV light, unchanged by ammonia treatment, were also detected (see Table 1) but could not be further identified. Anthocyanins were detected, from 2D chromatograms of direct leaf extracts, in some 19% of the taxa (Table 1). From Rfvalues and colour reactions, the same major pigment appeared to be present throughout the family. This was isolated from Pleurostima plantaginea and Vellozia streptophylla and identified as petunidin 3,5-diglucoside. It was accompanied by small amounts of delphinidin 3,5-diglucoside in the former plant. Petunidin 3,5-diglucoside is a relatively rare pigment within the monocotyledons and its regular occurrence in the Velloziaceae would appear to be a distinctive feature of the family. The flavonoid data (Table 1) are clearly related to subfamily and generic divisions within the family. Thus, the Barbacenioideae sensu Menezes [1] is distinguished from the Vellozioideae by the accumulation of flavonol glycosides and by the absence of both
C.A. WILLIAMS E T A L
492
flavone C-glycosides and free flavones such as luteolin. Although the flavonol quercetin is ubiquitous in Barbacenia, Aylthonia and Burlemarxia species, isorhamnetin is restricted to Barbacenia and three species of Barbacenia that are considered to be in Aylthonia by Menezes. Kaempferol is a characteristic constituent of both Aylthonia sensu strictu and Burlemarxia but was found also in more than 50% of Barbacenia species. Quercetin and kaempferol are frequent constituents of the genus Pleurostima (in 70% and 50% of species, respectively). However, Pleurostima section Pleurostirna is distinguished from Pleurostima section Grazielae and all other Velloziaceae by the presence of 6-hydroxyluteolin and pleurostimin. The flavonoid results for subfamily Vellozioideae are summarized in Table 2. Here taxa have been arranged according to Smith and Ayensu [2, 3], because some of the genera and sections of the Vellozioideae in Menezes' system have not yet been validated. From these data it can be seen that glycoflavones are the major leaf components of all three sections of Velloziaand of Xerophyta section Barbacenioides, and are trace constituents in Nanuza. Otherwise they are present in Xerophyta section Vellozioides (in 33% of species) but are absent from Barbaceniopsis, Talbotia and Xerophyta section Xerophytoides, where they are replaced as the main leaf components by flavonols. Xerophyta section Xerophytoides is further distinguished by the presence of isorhamnetin in all the taxa surveyed, together with quercetin 3-methyl ether in 83% of species. Flavonols are also common constituents of Vellozia section Vellozia (in 48% of taxa) and Velloziasection Xerophytoides (in 86% of the sample) but were not found in the two species examined from Vellozia section Radia. The latter section is further distinguished by the accumulation of free luteolin (in 86% of taxa) and the anthocyanin petunidin 3,5-diglucoside in all the species examined. Free luteolin is also characteristic of Vellozia section Vellozia (in 34% of species). Surface flavonoids were most frequent in Velloziasection Xerophytoides but occurred also in all other groups except Velloziasection Radia and Nanuza. The latter is distinct from all other members of the subfamily in producing amentoflavone as a major leaf constituent.
TABLE 2. A SUMMARY OF THE LEAF FLAVONOIDS IN THE SUBFAMILYVELLOZIOIDEAE* (PERCENTAGE FREQUENCIES)
Flavone C-glycosides Qu Km Isorh Free Lu Qu3ME Anthocyanin 5-Deoxykm DK/DK unknowns DK/Y unknowns Procyanidin Surface flavonoids Lu5ME+ffavanone Amentoflavone
V* (63)
VV (29)
VR (2)
VX (7)
XX (6)
XB (7)
70 55 6 13 30 13 16 2 47 9 2 34 2 0
86 48 0 10 34 10 10 0 72 7 0 38 O 0
100 0 0 0 0 O 0 0 50 50 0 0 0 0
100 86 43 0 86 0 100 14 O 0 0 71 0 0
0 100 0 100 33 83 0 0 83 0 0 33 0 0
100 14 0 0 0 0 0 0 14 0 0 28 14 0
XV (9) 33 78 11 0 0 0 0 0 45 0 11 11 0 0
T (1)
B (1)
N (1)
-100 100 0 O 0 0 0 O 0 0 O 0 0
0 100 O 6 0 0 O 0 100 0 0 0 0 0
100 0 0 0 0 0 0 0 0 O 0 0 0 100
*Subfamily classification according to Menezes [1], Authorities for sectional classification are given in Table 1. Key: V = Vellozioideae; VV = Vellozia section Vellozia; VR = Vellozia section Radia; VX = Vellozia section Xerophytoides; XX = Xerophyta section Xerophyta; XB = Xerophyta section Barbacenioideae; XV = Xerophyta section Vellozioideae; T = Talbotia; B = Barbaceniopsis; N = Nanuza; Qu = quercetin, K m = kaempferol; Isorh = isorhamnetin; Lu = luteolin; Qu3ME = quercetin 3-methyl ether; 5-deoxykm = 5-deoxykaempferol; Lu5ME = luteotin 5-methyl ether; DK/DK = dark to dark in UV light+NH3; DK/Y = dark to yellow in UV-PNH3. Figures in parentheses refer to number of species surveyed.
LEAF FLAVONOIDS IN THE VELLOZIACEAE
493
Discussion A summary of the leaf flavonoids found in the Velloziaceae is given in Table 3. As in Table 1 the results are listed according to a combination of the two current systems of family classification by Menezes [1] and Smith and Ayensu [2, 3], which are compared in Table 4. Eight conclusions may be drawn from the chemical evidence in support or otherwise of these two systems. Thus, (1) the Barbacenioideae sensu Menezes is distinguished from the Vellozioideae by the absence of flavone C-glycosides. (2) Pleurostima section Grazielae is unique in producing 6-hydroxyflavones. This suggests an evolutionary advanced position for the genus Pleurostima within the family. (3) Isorhamnetin is characteristic of all Barbacenia species sensu strictu except B. aft. globata and is also present in three species considered as Aylthonia by Menezes, namely: B. gardneri, B. paranaensis and one specimen of B. riedeliana. The latter sample is also distinguished from these and all other Velloziaceae by containing tricin in the free state. (4) Isorhamnetin is absent from Aylthonia s.s., Burlemarxia and five of the Barbacenia species considered as Aylthonia by Menezes: B. blancheti~ B. coronata, B. umbrosa, B. graminifolia and B. pulverulenta. The latter two species are distinct in producing quercetin 3-methyl ether, but Aylthonia and Burlemarxia cannot otherwise be separated using the flavonoid data. (5) Biflavone production in angiosperms is recognized as an ancient character and therefore the formation of the biflavone, arnentoflavone, in Nanuza indicates that it is chemically the most "primitive" member of the family [7]. The presence in this genus of glycoflavones suggests that it is more closely related to the Vellozia than to Xerophyta. (6) Within Xerophyta the Madagascan species (section Xerophytoides) are distinct in their leaf pattern (quercetin, isorhamnetin, quercetin 3-methyl ether) and are chemically better placed in the Barbacenioideae together with Barbaceniopsis, Talbotia and two other "flavonol only" species of Xerophyta section Vellozioides : X. schnitzleinia and X. viscosa. However, the predominance of flavone C-glycosides in Xerophyta section Barbacenioides and in X. scabrida, X. simulans and X. squarrosa in Xerophta section Vellozioides suggests that these plants may be chemically more closely allied with Vellozia than with members of the Barbacenioideae. (7) Some species of Vellozia section Xerophytoides which Menezes considers as belonging to Xerophyta section Talbotia, cannot be distinguished by the flavonoid evidence from other Vellozia taxa. (8) Within Vellozia, section Vellozia is unusual in the large number of species which produce surface flavonoids and section Radia is distinguished by the absence of flavonols. Otherwise all three Vellozia sections have similar flavonoid patterns. From these observations it is possible to construct a phylogenetic classification of the Velloziaceae based solely on flavonoid characters (Table 4). A cladistic analysis of the Velloziaceae based on morphological and anatomical characters [10] is in agreement with the chemical data and suggests that the genus Vellozia occupies a primitive position in the family and that Nanuza is an independent primitive clade in a large monophyletic sister group of the genus Vellozia. There is also agreement between the cladistic analysis and chemical data in the separation of Talbotia from the genus Xerophyta (sensu Smith and Ayensu, Table 4) but not for its placement in the Barbacenioideae. The cladistic study supports the retention of Barbaceniopsis as an independent group which is closer to Madagascan Xerophyta species (in section Xerophytoides) and Xerophyta section Vellozioides than to Xerophyta section Barbacenioides. The results of a leaf alkane survey of 110 Velloziaceae species [6] also agree with the flavonoid data in supporting Menezes' circumscription of the Barbacenioideae and her view that Pleurostima occupies an intermediate position between the two subfamilies. Similarly, the Brazilian Xerophyta species (i.e. sensu Menezes) cannot be separated from African Xerophyta taxa on alkane data. However, the alkane evidence, unlike the
Xerophytoides
Vellozioides
section
section
9
7
6
1
7
2
29
1?
1
--
--
3
7
7
2
25
--
--
--
--
--
--
Flavone C-glycosides
1
.
7
1
6
1
6
14
.
5
4
3
7
8
4
Qu
.
.
. 1
1
3
--
--
.
4
3
3
5
2
4
.
Flavonols Km
.
.
.
3
.
.
6
--
.
3
1?
6
--
.
.
.
.
.
Isorh
.
.
.
.
.
.
5
.
--
--
--
.
--
.
.
--
.
.
.
60H Lu
.
.
.
.
.
.
.
.
1
2
6
11
--
--
.
.
.
.
.
.
.
3
5
--
--
--
2
.
.
.
Flavones "Free" Lu Qu3ME
2
.
7
3
4
--
--
.
2
1
.
.
Anthocyanin
1
1
4
1
5
--
1
20
--
--
--
--
.
DK/DK unknowns
1
2
2 1
2
5
Flavanone (1) Procyanidin (1)
--
11
--
1
4
--
4
2
Surface flavonoids
Lu5ME (1)
--
5-Deoxykm
--
--
Amentoflavone (1)
--
--
--
--
"Free" tricin (1)
--
Others
*Subfamily classification according to Menezes [1]. tGenedc and sectional classification according to Smith and Ayensu [2, 3]. Key: Qu = quercetin, K m = kaempferol; Isorh = isorhamnetin; 6OH Lu = 6-hydroxyluteolin; Lu = luteolin; Qu3ME = quercetin 3-methyl ether; DK/DK = dark to dark in UV tight+NH 3. Figures refer to number of species with positive records in all cases.
Xerophytoides
Barbacenioides
section
Xerophyta
Talbotia
section
Vellozia
Radia
section section
Vellozia
1
1
Barbaceniopsis
Nanuza
5
8
3
7
8
4
section G r a z i e l a e Vellozioideaet
section
Pleurostima
strictu
"Aylthonia'Menezes)
Pleurostima
s.s
Barbacenia
Burlemarxia
(=
sensu
Barbacenia
Aylthonia
Barbacenioideae
Subfamily genus*
No. of species examined
TABLE 3. A SUMMARY OF THE LEAF FLAVONOIDS OF THE VELLOZIACEAE
o~
~>
~>
495
LEAF FLAVONOIDS IN THE VELLOZIACEAE TABLE 4. COMPARISONOF CLASSIFICATION SYSTEMS OF THE VELLOZIACEAE Smith and Ayensu (2,3] Vellozioideae Vellozia 1. section Vellozia 2. section Radia 3. section Xerophytoides 4. Nanuza
Barbacenioideae 5. Talbotia Xerophyta 6. section Xerophytoides 7. section Vellozioides 8. section Barbacenioides 9. Barbaceniopsis 10. Barbacenia
Menezes [1]
This paper
Vellozia 1. section Vellozia 2. section Radia Xerophyta 3-5. section Talbotia 6-9. section Xerophyta
4. Nanuza Vellozia 1. section Vellozia 2. section Radia 3. section Xerophytoides Xerophyta 8. section Barbacenioides 7. section Vellozioides
10. Burlemarxia 10. Pleurostima section Pleurostima section Grazielae
Xerophyta 6. section Xerophytoides 5. Talbotia 10. Pleurosdma 9. Barbaceniopsis
1O. Barbacenia
10. Aylthon~ 10. Burlemarxia 10. Barbacen~
10. Aylthonia
flavonoid data, does not distinguish between support the recognition of Burlemarxia.
Aylthonia
s.s a n d B a r b a c e n i a , b u t d o e s
Acknowledgements--Wethank
Dr Simon Mayo, Royal Botanic Gardens, Kew for assistance with the taxonomic aspects of this work and we are grateful to Mr J. Eagles, Food Research institute, Norwich for MS determinations.
References 1. Menezes, N. L. de (1980) in Petaloid Monocotyledons: HoFdcultural and BotanicaI Research Linn. Soc. Syrup. Ser. 8, 117. 2. Smith, L. B, and Ayensu, E. S. (1974) KewBull. 29, 181. 3. Smith, L. B. and Ayensu, E. S. (1976) Smithson. Contribu. Bot. 30, 1. 4. Menezes, N. L. de (1980) Revta bras. BoL 3, 37. 5. Menezes, N. L. de (1971) Ci~nc. Cult. 23, 421. 6. Salatino, M. L. F., Salatino, A., Menezes, N. L. de and Mello-Silva, R. de (1989) Phytochemistry28, 1105. 7. Williams, C. A., Harborne, J. B. and Tomas-Barberan, F. A. (1987) Phytochemistry26, 2553. 8. Williams, C. A. and Harborne, J. B. (1975) Biochem. SysL Ecol. 3, 181. 9. Proksch, P., Budzikiewicz, H., Tariowitz, B. D. and Smith, D. M. (1984) Phytochemistry 23, 679. 10. Menezes, N. L. de, Mello-Silva, R. de and Mayo, S. J. (1992) KewBull. (in press).
0305-1978/91 $3.00+0.00 © 1991PergamonPressplc.
The Utility of Leaf Flavonoids as Taxonomic Markers in the Subfamily and Generic Classification of the Velloziaceae CHRISTINE A. WILLIAMS,* JEFFREY B. HARBORNE* and NANUZA L. DE MENEZESt *Botany Department, University of Reading, Whiteknights, Reading, RG6 2AS, U.K.; tlnstituto de Bioci~ncias, Universidade de S&o Paulo, Brazil
Key Word Index--Velloziaceae; flavones; 6-hydroxyflavones; flavone; leaf surface flavonoids; chemotaxonomy.
flavonols; flavone C-glycosides; amento-
Abstract--As a result of surveying 99 species of the Velloziaceae, it was found that the major leaf flavonoids are flavonol glycosides (in 68%) and flavone C-glycosides (in 48% of taxa). In the subfamily Vellozioideae, flavones such as luteolin occur in the free state together with flavone C-glycosides and this combination of characters distinguishes this subfamily from the Barbacenioideae. The genus Pleurostima is unique in producing two new 6-hydroxyflavone glycosides identified as the 7-apiosides of 6-hydroxyluteolin and 6-hydroxy-8-roethoxyluteolin. The genus Barbacenia is distinguished by the regular presence of isorhamnetin and occasional presence of quercetin 3-methyl ether. The only other plants where isorhamnetin is frequent are the Madagascan Xerophyta species, which also produce quercetin 3-methyl ether, luteolin and some unidentified constituents, but lack flavone C-glycosides. Otherwise Vellozia and Xerophyta species have similar flavonoid patterns, including the presence of 5-deoxykaempferol and the anthocyanin, petunidin 3,5-diglucoside. Nanuza plicata is distinguished from all other genera by the presence of the biflavonoid, amentoflavone.
Introduction The Velloziaceae, a small family of some 270 species, has its centre of diversity in Brazil but is well represented in eastern Africa by species of the genus Xerophyta. There has been much dispute as to the delimitation of genera and subfamilies. In a recent classification based largely on floral morphology and leaf anatomy, Menezes [1] divided the family into two subfamilies, the Vellozioideae and Barbacenioideae and recognized six genera. In this system the subfamily Vellozioideae is split into two genera, Vellozia (flowers with nine or more stamens and no corona) and Xerophyta (flowers with six stamens and no corona), while the four genera of Barbacenioideae contain species whose flowers have six stamens and a corona. This system differs from that of Smith and Ayensu [2, 3] in the inclusion of Barbaceniopsis, Nanuza, Talbotia and Brazilian Vellozia species in Xerophyta and their transfer, with the African Xerophyta species, from the Barbacenioideae to the Vellozioideae. Thus, only Barbacenia species are retained in the Barbacenioideae and these have been divided into four genera by the reinstatement of Pleurostirna [4], the creation of two new genera Aylthonia [5] and Burlemarxia [1] and the retention of the remaining taxa within Barbacenia. In the present paper, results are presented according to a modified version of Menezes' system [4] in which only valid names have been used and cross reference given to the classification of Smith and Ayensu [2, 3]. The names of genera and sections used here follow Smith and Ayensu [2, 3] except for the genus Barbacenia in which the four segregate genera of Menezes [1] are recognized. The subfamily concepts are, necessarily, qualified as "sensu Menezes" or "sensu Smith and Ayensu" according to the context. Table 4 compares the two published classifications with each other and a grouping of the taxa based on the flavonoid results of this study. The results of a recent leaf wax alkane survey of 110 species of Velloziaceae [6] (Received 17 January 1991) 483
484
C.A. WILLIAMS ETAL.
support the re-establishment of Pleurostirna and the formation of Burlemarxia but do not distinguish between Aylthonia and Barbacenia or lend support for the recognition of Velloziasect. Xerophytoides as a section of Xerophyta. Our previous identification of the biflavonoid, amentoflavone [7], in the family suggested that a leaf flavonoid survey of the Velloziaceae could provide further useful chemical characters to help in the classification of this family. Materials and Methods P/antmater/a/. Both herbarium and air-dried plant material was received from various sources, details of which are given in Table 1. Plant identifications were carried out by one of us (NLdeM). Extraction and detection of /eaf surface f/avono/ds. Leaf material was covered for ca 1 rain with CHCI3, The decanted CHCI3 extract was concentrated and run on silica gel TLC in toluene:HOAc (4:1) against quercetin 3-methyl ether as a marker. If mobile dark UV absorbing compounds were present, surface flavonoids were recorded in the survey. Three compounds from Ve//oz/a evera/d//co-chromatographed, respectively with a mixture of quercetin 3-methyl ether, 3,7-dimethyl ether and 3,7,4'-trimethyl ether markers in BAW, forestal, 50% HOAc, CAW (2:1) and toluene: HOAc (4:1 ). Similarly, a surface compound from Ve//oz/ascopar/a co-chromatographed with a sample of 5-deoxykaempferol in the same solvent systems. More detailed identification of several new surface flavonoids from two other Ve//oziaspecies are being reported separately. /dendfication of/eaf f/avono/ds. The CHCI3 washed leaf material of each sample was extracted in 80% hot MeOH and the concentrated extracts developed by 2D PC in n-BAW and 15% HOAc, to compare flavonoid glycosidic patterns. The same extracts were tested for flavonoid sulphates by electrophoresis on 3MM Whatman paper at pH 2.2 (HOAc-HCO2H buffer) for 2 h at 40 V cm -~. A lack of dark UV absorbing mobile constituents showed that fiavonoid sulphates were absent from the Velloziaceae surveyed. Flavonoid constituents in the 80% MeOH extract were separated from cinnamic acid and other blue UV fluorescent phenolic compounds by either preparative 2D PC in BAW and 15% HOAc or by preparative 1D PC in BAW or 15% HOAc. The combined flavonoid glycoside spots or bands were eluted with 80% MeOH and the concentrated extracts subjected to acid hydrolysis with 2N HCI for 40 rain. Flavonoid aglycones were identified using standard procedures and by TLC comparison with authentic markers. Flavone C-glycosides were confirmed by 4 h acid hydrolysis, extraction into/so-amyl alcohol and TLC on cellulose against vitexin in BAW and H20. Orientin and iso-orientin in Ve//oz/astreptophy//a and V/i/ac/na were identified by co-PC and UV data and their O-glycosides by Rf and UV data and acid hydrolysis. Free flavonoid aglycones present in the direct leaf extracts, which run near the solvent-front in BAW and are barely mobile in 15% HOAc, were eluted separately from the flavonoid glycosides and compared directly by coTLC with marker compounds without previous acid hydrolysis. Luteolin 5-methyl ether from the leaf sheath of Xerophyta ey/esi/co-chromatographed with a marker isolated earlier from Juncus effusus [8]. A flavanone from the same extract gave a UV spectrum with ~.MeOH max 287 nm (alkaline spectral max at 298 and 325 nm) and Rr (×100) values on silica gel TLC of toluene:HOAc (4:1) 0, toluene:ethyl formate:HCO2H (5:4:1) 2 and toluene:pyridine:HCO2H (100:20:7) 0 and on PC:BAW 100, 50% HOAc 100, Forestal 100 and CAW (2:1) 100. It did not co-chromatograph with naringenin or eriodictyol and was not studied further. /dent/fication of 6-hydroxy/uteo//n 7- apios/de (1) and p/eurostim/n 7-apios/de (2) from Pleurostima purpurea. The two glycosides were separated from an 80% MeOH leaf extract by preparative PC in (1) 15% HOAc, (2) BAW and (3) 50% HOAc, compound 2 was further purified by HPLC on a spherisorb 50DS2 column, 25 cm× 8 mm i.d. Solvent A 100% H20, B MeOH-H20-HOAc, 18:1:1. Initial conditions 50% A, 50% B. Gradient over 15 rain to 5% A, 95% B and back to initial conditions after 16 rain. Flow speed 2 ml min -1 RRt 9.52 (1 = 9.22), Rf (×100) values of I and 2 (compared with 6-hydroxyluteolin 7-glucoside): BAW 39, 51(22), Foresta143, 63 (48); 50% HOAc 43, 64 (37) and CAW (1:1) 41, 55 (30). UV spectral data: 1 ;~a, MeOH 283, 347; +NaOAc 264, 362; -I-H3BO3 262, 284, 359 and 2 MeOH 260, 278, 351 ; +NaOAc 266, 358 and +H3BO3 266, 264 nm. Acid hydrolysis of I with 2N HCI for 1 h gave 6-hydroxyluteolin (co-TLC and UV spectral data) and apiose, identified by co-PC with an authentic specimen obtained from acid hydrolysis of apiin from Ap/um graveo/ensseed; P~ TBPW 20, BEW 19, Phenol 140 and BAW 19. Similar acid hydrolysis of 2 gave pleurostimin (6-hydroxy-8-methoxyluteolin) (3) and apiose. Rfvalues of 3 compared with 6-hydroxyluteolin: BAW 63 (47), Forestal 64 (42), 50% HOAc 47 (27) and CAW (2:1 ) 31 (12). UV spectral data for 3: ~ a , MeOH 256, 279, 353; +NaOAc 225, 263, 371 and +H3BO3 265, 377. FAB MS gave a Mrof 332 with strong ions at 176 and 184. This Mr suggests a hexahydroxyflavonewith one methyl group. As 3, like 6-hydroxyluteolin, is dark absorbing in UV light and remains unchanged by NH3 this suggests that there is a 6-hydroxyl with an extra hydroxyl at the 8 or 5'-positions. The positive spectral shifts with sodium acetate and with borate show that the 7, 3' and 4' hydroxyls are free, so that the methyl group could be located at the 6- or 8-position. However, the published UV spectral data for 5,7,8,3',4'-pentahydroxy-6-methoxyflavone [9] differ from those for 3. Furthermore, the ElMS of pleurostimin gave a molecular ion at 332 (100%) (C~6H1208requires 332) with fragments at 317 (M-15) (8%), 289 (M-43) (38%) and at 137 (238); this latter fragment corresponds to a 3,4-dihydroxyphenol B-ring, which eliminates an alternative structure with a trihydroxyl B-ring. The location of the methoxyl at the 8- position of the A-ring is also apparent from the relative intensity of the [M-15] ion (8%) compared to that of 6-methoxyluteolin (M-15 ion 59% of the molecular
C-glycosides
Species
+ +
+ + +
-------
ex Schult.
B fulva Goeth & Henr,
Barbacenia aft. globata Goeth & Henr. B. involucrata L. B. Smith B. rubro-virens Mart. +
--
+
--
(+)
--
(+)
+
Km
+
+
--
+
+
+
+
+
Isorh
m
m
m
Others
+
--
+
---
B umbrosa L. B. Smith & Ayensu +
+
+
---
+ +
+7 --
B. pulverulenta L. B. Smith & Ayensu B. riedeliana Goeth. & Henr.
& Ayensu
B gardneriSeubert B. graminifolia L. B. Smith B. paranaensis L. B. Smith
+
--
(+)
+
--
--
+ +
--
+
--
--
+
--
B. coronata P. F. Ravenna e x L. B. Smith
A. macrantha (Lem.) N. Menezes "~4ylthonia" sp. Barbacenia b/anchet~" Goeth. & Henr.
N. Menezes
Ayithonia b/ackii L. B. Smith A. gen~'anoides (Goeth & Henr.)
--
--
--
+
--
--
--
+
+
+
3
5
+
1
1
3
3
- -
3
--
--
5
2 +
Tricin --
Anthocyanin Qu3ME - -
--
6477
Harley et al. SPF 36077 CFCR
Anderson et aL 35123 Brazil K K
12288 (SPF)
11243a (SPF)
17379 Brazil
57H892/6 L. B. Smith
11251 (SPF)
Hatchbach 30183
412-7904208 Minas Gerais Brazil
Harley etal. 22512 Brazil
Harley et al. 18659 Brazil
Harley et al. 20623 Brazil
11596 (SPF)
11243 (SPF)
5312
11242 (SPF)
11581 (SPF)
11232 (SPF)
12329 (SPF)
Minas Gerais Brazil
Irwin et al. 22457
Maguire et al. 49116
11233 (SPF)
11586 (SPF)
11228 (SPF)
number
Collection
CFCR
CFSC
K
CFSC
H
K
K
K
K
CFSC
CFSC
CFCR
CFSC
CFSC
CFSC
CFCR
K
K
CFSC
CFSC
---
CFSC
Source
--
flavonoids
Surface
Anthocyanin Qu3ME - -
--
--
--
(+)
--
--
Anthocyanin
--
(+)
m
m
m
--
+
+
+
(+)
Aylthonia Menezes (including eight Barbacenia species considered as Aylthonia by Menezes but not yet validated as such)
+
+
+
---
Qu
Barbacenia bishopii L. B. Smith & Ayensu B. conicos#gma Goeth & Henr. B. flava Mart.
Barbacenioideae sensu Menezes [1] Barbacenia sensu Menezes
Flavone
Section
Genus
Subfamily*
TABLE 1. A LEAF FLAVONOID SURVEY OF THE VELLOZIACEAE
c}
<
-I
03
z O
>~
. -. --.
R fanniei N. Menezes R gounelliana (Beauv.) N. Menezes P. irwiniana (L. B. Smith) N. Menezes
P. pabstiana (L. B. Smith & Ayensu)
Pleurostima
N. Menezes
R inclinata (Goeth. and Henr.) N. Menezes R Iongi$capa (Goeth.) N. Menezes R nuda L. B. Smith & Ayensu P, plantaginea (L. B. Smith)
Ayensu) N. Menezes
Pluereuros#~na andersonii (L. B. Smith &
section Grazielae
-----
--
R spectabilis (L. B. Smith & Ayensu)
N. Menezes
. . --
.
R purpurea (Hook.) Raf .
N. Menezes
N.
Menezes R delicatula (L. B. Smith & Ayensu) N. Menezes
---
--
--
.
.
.
.
Flavone C-glycosides
Pleurostima caricina (Goeth. & Henr.)
Pleurostima Raf. Section Pleurostima
N. Menezes & Semir
B. rodriguesii N. Menezes & Semir B. spiralis (L. B. Smith & Ayensu)
N. Menezes & Semir
Burlemarxia pungens
Burlemarxia N. Menezes & Semir
Section Species
Genus
Subfamily*
TABLE 1--CONTINUED
.
÷ + + 4-
+
+
+ +
4-
+ 4-
+
+
.
.
.
.
Qu
. .
.
.
.
+ + + +
--
--
+ (+)
+
+ +
(+)
(+)
Km
-----
+?
--
---
--
---
--
--
Isorh
Anthocyanin Anthocyanin Anthocyanin Anthocyanin
--
60H Lu 60H Lu
60H Lu
60H Lu 6,8-DiOH Lu 8-ME
60H Lu -_
6,8-DiOH Lu 8-Me Unknown DK/DK free Lu
60H Lu
Anthocyanin Anthocyanin
_
_
Others
3 1 3 1?
--
---
--
--
--
-3
--
--
---
--
--
Surface flavonoids
CFCR CFCR CFSC CFSC
K
K Menezes
12322 (SPF) 12280 (SPF) 11614 (SPF) 11209 (SPF)
Ralter et aL 3833 Brazil
H.E. Strang 698, Brazil 722 (SPF)
635 (SPF)
A.C. Brade 17019 Brazil A.C. Brade 19774 Brazil
K K Menezes
670 (SPF) Harley 20350 Brazil
323 Rio de Janeiro, Brazil 12292 (SPF)
D. Sucre 2481 Braga
10574 47320 12286 (SPF) 12295 (SPF)
8833 40990
Collection number
Menezes K
CFCR
K
CFCR SPF CFCR CFCR
CFCR SPF
Source
m ~,
~.
c~ " ~"
--
+
--
.
.
(+) (+) --
+ + +
-.
+ + +
V. leptopetala Goeth. & Henr. --
+
--
--
V. glochidea Pohl V. hemisphaerica S e u b e r t V. incurvata M a r t .
.
.
V. glauca Pohl (leaf)
(stem)
+ .
.
.
--
+
--
V. glabra Mikan
--
--
+
V. flavicans M a r t . e x Schultes f.
--
--
.
--
--
--
--
+
+
--
--
--
--
--
--
.
--
.
--
--
--
--
.
--
(+)
+ +
(+)
--
+
--
+
+
+
+
--
+
+
+
--
V. epidendroides var. major L. B. S m i t h V. everaldii N. M e n e z e s (ined.)
V. coronata L. B. S m i t h V. declinans Goeth. & Henr. V. epidendroides M a r t . ex S c h u l t e s f. V. aft. epidendroides Mart. ex S c h u l t e s f.
V. caruncularis M a r t . e x S e u b e r t V. compacta M a r t . ex Schultes f .
Vellozia alata L. B. S m i t h V. aloifolia M a r t . V. bahiana L. B. S m i t h & A y e n s u V. breviscapa Mart. ex Schultes f.
Section Vellozia
Vellozia sensu S m i t h & A y e n s u
Vellozioideae
Nanuza plicata (Mart.) L. B. S m i t h & A y e n s u +
.
.
Nanuza L. B. S m i t h & A y e n s u (included in Xerophyta s e c t i o n Talbotia b y Menezes)
Barbaceniopsis humahuaquensis N o h e r
.
.
.
.
.
--
--
--
--
--
--
+
--
--
--
--
--
--
--
--
(+)
--
--
.
.
.
.
.
.
.
DK/DK unknown
DK/Y unknown
DK/DK unknowns
DK/Y &
2 DK/DK unknowns
Free Lu
DK/Y unknown
--
3
--
--
--
--
diME & triME
DK/DK unknown DK/relY unknown
Qu 3ME
--
--
--
2
5
--
1
--
--
--
1
Free Lu
DK/DK unknown
DK/DK unknowns
Free Lu, 2
DK/DK unknown
Free Lu
DK/DK unknown
DK/DK unknown
DK/DK unknown
B/Y glycoside
DK/DK unknown
DK/DK unknown
Free Lu Q u M E
Amentoflavone
DK/DK unknown
Barbaceniopsis L. B. S m i t h ( i n c l u d e d in Xemphyta section Xerophyta b y M e n e z e s a n d in B a r b a c e n i o i d e a e of S m i t h & A y e n s u )
Vellozioideae sensu Menezes [1 ]
CFSC
K
K
K
K
CFSC
K
K
CFCR
K
CFSC
K
CFSC
CFSC
CFSC
CFSC
K
K
H
K
CFSC
RNG
K
A n d e r s o n 10263 Brazil
11210 (SPF)
S. A m e r .
W . J . Burchell 7031 E. Trop.
Harley et al. 19681 Brazil
I r w i n et al. 34397 Brazil
W.R.
11231 (SPF)
I r w i n et al. 8073 Brazil
1 + 8 9 2 / 6 2 1 3 Brazil
12287 (SPF)
A n d e r s o n et al. 36200 Brazil
11250 (SPF)
I r w i n et al. 22173 Brazil
11579 (SPF)
11256 (SPF)
11256 (SPF)
2869 (SPF)
Philcox & F r e e m a n 4721 Brazil
Irwin et aL 27930 Brazil
L . B . S m i t h & A y e n s u Brazil
I r w i n et al. 22373 Brazil
11618 (SPF)
O. G u i m a r a e s Brazil
G. Hatchback 47086 &
S.A. Renvoise 3436 A r g e n t i n a
<
E~ ol
O Z
(+)
+
+ +
--
--
---
--
---
Km
_
_
_ --
--
_ (+)
Isorh
Vellozia abiedna Mart.
(+) --
+ +
_
(+)
Vellozia section Xerophytoides L. B. Smith & Ayensu (considered as Xerophyta section Talbotia by Menezes)
V. dawsonia L. B. Smith V. tubi#ora (A. Rich.) H.B.K.
Vellozia section Radia (A. Richard) L. B. Smith & Ayensu
--
---
+ -F +
--
+
V. verruculosa Mart.
(+) (+)
+ +
V. sdpitata L. B. Smith & Ayensu V. streptophylla L. B. Smith V. taxifolia (Mart. ex Schultes f.) Mart. ex Seubert V. variabilis Mart ex Schultes f.
(+)?
+ +
(+)
Qu
+
+
+
+
C-glycosides
Flavone
V. pusilla Pohl
V. phalocarpa Pohl
nanuzae L. B. Smith & Ayensu
v. nivea L. B. Smith & Ayensu
lilacina L. B. Smith
Genus Section Species
Sub~mily*
TABLE 1--CONTINUED
Surface
Free Lu
Anthocyanin Free Lu Free Lu
DK/DK unknown DK/Y unknown
DK/DK unknown DK/DK unknown DK/DK unknown
Qu3ME Free (Lu) DK/DK unknown
3
2
nd
K K
CFCR
K MO
Irwin et al. 2177 Brazil
Irwin et al. 20672 Brazil
12277 (SPF)
Irwin etal. 22173 Brazil R. Liesner 17243
Miss D. Smith 334 Brazil
W. R. Anderson 8414 Brazil Parani et al. 1503 Brazil Irwin et al. 9809 Brazil
Irwin et al. 20016 Brazil
K
Ynes Mexia 5873 Brazil 4317 (SPF)
11617 (SPF) 12300 (SPF)
K CFCR
11202 (SPF)
11598 (SPF)
11236 (SPF)
10371 (SPF)
Collection number
CFSC CFCR
CFSC
DK/DK unknown DK/DK unknowns Anthocyanin Free Lu DK/DK & DK/Y unknowns Qu3ME
CFSC
CFSC
CFSC
Source
2DK/DK & 3 other unknowns Free Lu
5 3
flavonoids
2 DK/DK unknowns Free Lu
2 DK/DK unknowns Petunidin Free Lu,
Anthocyanin Free Lu,
Others
+ +
+ +
V. scoparia Goeth. & Henr.
V. aft. scoparia Goeth. & Henr.
V. tragacantha Mart. ex. Schult
~erophyta" giuliettiae N. Menezes +
+
+
+
+
--
+
+
+
+
m
m
--
Free Lu
Anthocyanin
Free Lu
Anthocyanin
5-DeoxyKm
Free Lu
Anthocyanin
Free Lu
Anthocyanin
DK/Y free aglycone
Anthocyanin
Free Lu
Anthocyanin
--
3
--
6+5-DeoxyKm
4+5-DeoxyKm
--
4
CFCR
CFCR
CFSC
CFCR
CFSC
CFCR
T. Edwards 6.3.89
12289 (SPF)
12318 (SPF)
11597 (SPF)
12303 (SPF)
11208 (SPF)
12290 (SPF)
--
X. dasylirioides Baker var, pectinata
(+)
--
--
X. pinifolia Lam. var. villosa (H. Perr.)
X. equisetoides Baker X, equisetoides Baker var. trichophylla (Baker) L. B. Smith . .
+ -t-
Xerophyta section Barbacenioides L. B. Smith and Ayensu
(+)
+
--
H. Perr.
+
--
+
+
X. dasylirioides Baker var. (new?) X. eglandulosa H. Perr. vat. eglandulosa X. eglandulosa H. Perr. var. (new?)
(Baker) H, Perr.
--
X. dasylirioides Baker
Xerophyta Juss. section Xerophytoides (Madagascan species)
.
.
m
.
.
.
.
(+)
(+)
+
+
+
+
.
.
(new) above
X. eglandulosa var.
as in
DK/DK unknown
X. pinifolia)
(one same as in
DK/DK unknowns
Qu3ME 3
DK/DK unknown
Free Lu, Qu3ME
(Qu3ME)
DK/DK unknowns
Free Lu Qu3ME 2
DK/DK unknowns
Qu3ME 2
--
--
--
--
2
2
K
K
MB
MB
MB
MB
MB
MB
Mason District S. Province
W.J. Eggeling 6391 No. 1951
G.F. de Witte 2879 Zaire
167 (SPF)
170 (SPF)
175 (SPF)
170a (SPF)
176 (SPF)
173 (SPF)
Xerophyta Juss. (All the following African species are included by Menezes in her Xerophyta section Xerophyta but Smith & Ayensu divide them into three sections and place them in the Barbacenioideae
Talbo~'a elegans Bay. --
+
V. minima Pohl
sp. nov. ined.
+
V. laevis L. B. Smith
C-glycosides +?
Species
X. eylesfi (Greves) N. Menezes (leaf)
.
+ + + + +
--+ + --
X. schnitzleinia (Hochst.) Baker
. . --
(+) --
.
.
.
.
. .
.
.
.
--
---
---
--
-.
--
.
.
. .
.
.
Isorh
.
.
.
.
. .
.
.
Surface flavonoids
Procyanidin
DK/DK unknown DK/DK unknown
6 DK/DK unknowns DK/DK unknown
2 DK/DK
DK/DK unknown
DK/DK unknown
DK/DK unknown DK/DK unknown
4
---
---
--
--
--
1 4
Lu 5 M e Lu flavanone - B/Y unknown +
Others
K K K
K K
K K
K K
K
K K
K K
K K
K
K
K
K
Source
W. Africa
J . K . Jackson 1807, Sudan MerxmL~ller & Giess 30477 H. & H.E. Wanntorp No. 171
Thulin et al. 3605, Ethiopia Mrs J. Ash 2116
Tanzania Flora of Tanzania Terr. no. 1971B Mrs Jonquil Ash 16.4.76 Ethiopia
Ethiopia Christina Skarp Botswana W . J . Eggeling Nov. 1951,
S.W. Arabia M . G . Gilbert & T. G. Jefford 4537
P.R.O. Bally 11046 Somalia J . J . Lavranos 14/28.8.62
Spjut & Ensor 2790 Kenya Schmith 6043 Zaire
Natal, S. Africa Verdcourt & Polhill 2697 Kenya Napper 1641 Kenya
Moll & Nell 5505
L.J. Brass 16144 Malawi
O. West 6132 Zimbabwe O. West 6132 Zimbabwe
number
Collection
for Kew; Qu = quercetin; Km = kaempferol; Isorh = isorhamnetin; Lu = luteolin; 60H Lu = 6-hydroxyluteolin; Chrys = chrysoeriol; pleurostimin = 6-hydroxy-8-methoxyluteolin; DK/DK = dark to dark in UV light with and without ammonia vapour; Qu3ME = quercetin 3-methyl ether; diME = dimethyl ether; triME = trimethyl ether; B/Y = blue to yellow in U V + N H 3. The numbers of surface flavonoids found per sample are indicated in the appropriate column (+indicates general presence).
*Subfamiliy classificaton sensu Menezes [1 ], genera and sections according to Smith & Ayensu [2, 3] with cross reference to the proposed system of Menezes. Key: K = material from the Herbarium, Royal Botanic Gardens, Kew; M o = material from Missouri Botanic Garden; H = material from Howard University, Washington, USA; RNG = material from Reading University Herbarium; CFSC = Collection Flora of Serra do Cipb, Brazil; CFCR = Collection Flora of "Campos rupestres", Brazil; SPF = Herbarium of University of S&o Paulo, Brazil; M B = David Du Puy Collection
X. schnitzleinia (Hochst.) Baker var. somalensis X. simulans L. B. Smith & Ayensu X. squarrosa Baker X. viscosa Baker
+ --
.
-(+)
+ +
+
-
X. scabrida (Pax) Th. Dur. & Schinz
-
. --
nd
--
---
(+) +
.
.
.
.
.
--
X. humilis (Baker) Th. Dur. & Schinz
.
.
Km
+
nd .
X. acuminata (Baker) N. Menezes X. arabica (Baker) N. Menezes nd
(+) .
(+) +
Xerophyta section VellozioidesL. B. Smith & Ayensu
X. zambiana L. B. Smith & Ayensu
+ +
+
.
+ +
.
+
X. kirkii (Hemsl.) L. B. Smith & Ayensu X. retinen/is Baker
X. spekei Baker
.
.
.
Qu
(sheath)
.
Flavone
Section
Genus
Subfamily*
TABLE 1--CONTINUED
c~
P P ~_ rr-
o
LEAF FLAVONOIDSiN THE VELLOZlACEAE
491
ion). The absence of NaOAc shifts in the glycosides 1 and 2 suggest that the apiose moeity is attached at the 7-hydroxyl in both compounds. Anthocyanin identificadon. The pigments were extracted from dried leaf of Pleurosdma plantaginea and Vellozia streptophylla with methanolic HCI (after preliminary extraction with methanolic acetic acid and electrophoresis showed that zwitterionic anthocyanins were absent) and the concentrated extracts were separated by preparative PC in BAW and 15% HOAc. Petunidin 3,5-diglucoside was identified as the major pigment of both plants from the spectrum (;k~a` 275, 335 nm, A440/A535ratio 11%), acid hydrolysis to give petunidin and glucose (identified after co-chromatography in four solvents and separation from added galactose), H202 oxidation to give glucose and TLC co-chromatography in four solvents with an authentic marker. Delphinidin 3,5-diglucoside was identified as a minor pigment in P. plantaginea by similar procedures.
Results The results of a leaf flavonoid survey of 99 species of the Velloziaceae are presented in Table 1. The data refer to flavonoid aglycones detected after acid hydrolysis of partially purified 80% methanolic leaf extracts. The aglycones were characterized by TLC comparison of Rfvalues and colour reactions in UV light with authentic markers. These findings were confirmed by 2D PC of direct leaf extracts and more detailed examination of the flavonoid profile of selected species. The major leaf flavonoids were found to be the flavonol quercetin (in 68% of species) and flavone C-glycosides (in 48% of taxa). Other flavonols: kaempferol (in 26% of the sample) and isorhamnetin (in 18%) were less frequent leaf constituents. In a more detailed analysis of the leaf tissue of Vellozia streptophylla and V. lilacina, the well known glycoflavones orientin and iso-orientin were identified in both plants. In V streptophylla a number of flavone C-glycoside-O-glycosides, orientin and isoorientin 7-O-glucoside, an iso-orientin 7-O-diglucoside and an unusual iso-orientin 7-O-apioside were also detected. Two rarer components, 6-hydroxyluteolin and 6-hydroxy-8-methoxyluteolin (pleurostimin) were characterized as their 7-apiosides in Pleurostirna caricina (see Materials and Methods). This is the first report of pleurostimin and of both glycosides in nature. 6-Hydroxyluteolin was detected in a further four and pleurostimin in one other Pleurostima species of the 13 surveyed. The biflavonoid, amentoflavone, previously characterized from Nanuza plicata [7], did not appear to be present in the other 98 taxa of this survey. Leaf surface washings were taken from all plant samples, before the internal flavonoids were examined, and some 36% of the species were discovered to contain prenylated flavonols in the waxes. These compounds are currently being investigated and will be reported on in a separate paper. The "internal flavonoid" fraction, besides containing the glycosides described above, also has a number of non-glycosidic components as well. Five of these were identified readily as well known substances, namely luteolin (in 21 species of the plants surveyed), quercetin 3-methyl ether (in 10 species) luteolin 5-methyl ether (in one species, Xerophyta eylesii), tricin (in one species, Barbacenia riedeliana) and 5-deoxykaempferol (in one species, Vellozia aft. scoparia). Numerous further lipophilic components of high Rf values in butanolic solvents which appeared dark-absorbing in UV light, unchanged by ammonia treatment, were also detected (see Table 1) but could not be further identified. Anthocyanins were detected, from 2D chromatograms of direct leaf extracts, in some 19% of the taxa (Table 1). From Rfvalues and colour reactions, the same major pigment appeared to be present throughout the family. This was isolated from Pleurostima plantaginea and Vellozia streptophylla and identified as petunidin 3,5-diglucoside. It was accompanied by small amounts of delphinidin 3,5-diglucoside in the former plant. Petunidin 3,5-diglucoside is a relatively rare pigment within the monocotyledons and its regular occurrence in the Velloziaceae would appear to be a distinctive feature of the family. The flavonoid data (Table 1) are clearly related to subfamily and generic divisions within the family. Thus, the Barbacenioideae sensu Menezes [1] is distinguished from the Vellozioideae by the accumulation of flavonol glycosides and by the absence of both
C.A. WILLIAMS E T A L
492
flavone C-glycosides and free flavones such as luteolin. Although the flavonol quercetin is ubiquitous in Barbacenia, Aylthonia and Burlemarxia species, isorhamnetin is restricted to Barbacenia and three species of Barbacenia that are considered to be in Aylthonia by Menezes. Kaempferol is a characteristic constituent of both Aylthonia sensu strictu and Burlemarxia but was found also in more than 50% of Barbacenia species. Quercetin and kaempferol are frequent constituents of the genus Pleurostima (in 70% and 50% of species, respectively). However, Pleurostima section Pleurostirna is distinguished from Pleurostima section Grazielae and all other Velloziaceae by the presence of 6-hydroxyluteolin and pleurostimin. The flavonoid results for subfamily Vellozioideae are summarized in Table 2. Here taxa have been arranged according to Smith and Ayensu [2, 3], because some of the genera and sections of the Vellozioideae in Menezes' system have not yet been validated. From these data it can be seen that glycoflavones are the major leaf components of all three sections of Velloziaand of Xerophyta section Barbacenioides, and are trace constituents in Nanuza. Otherwise they are present in Xerophyta section Vellozioides (in 33% of species) but are absent from Barbaceniopsis, Talbotia and Xerophyta section Xerophytoides, where they are replaced as the main leaf components by flavonols. Xerophyta section Xerophytoides is further distinguished by the presence of isorhamnetin in all the taxa surveyed, together with quercetin 3-methyl ether in 83% of species. Flavonols are also common constituents of Vellozia section Vellozia (in 48% of taxa) and Velloziasection Xerophytoides (in 86% of the sample) but were not found in the two species examined from Vellozia section Radia. The latter section is further distinguished by the accumulation of free luteolin (in 86% of taxa) and the anthocyanin petunidin 3,5-diglucoside in all the species examined. Free luteolin is also characteristic of Vellozia section Vellozia (in 34% of species). Surface flavonoids were most frequent in Velloziasection Xerophytoides but occurred also in all other groups except Velloziasection Radia and Nanuza. The latter is distinct from all other members of the subfamily in producing amentoflavone as a major leaf constituent.
TABLE 2. A SUMMARY OF THE LEAF FLAVONOIDS IN THE SUBFAMILYVELLOZIOIDEAE* (PERCENTAGE FREQUENCIES)
Flavone C-glycosides Qu Km Isorh Free Lu Qu3ME Anthocyanin 5-Deoxykm DK/DK unknowns DK/Y unknowns Procyanidin Surface flavonoids Lu5ME+ffavanone Amentoflavone
V* (63)
VV (29)
VR (2)
VX (7)
XX (6)
XB (7)
70 55 6 13 30 13 16 2 47 9 2 34 2 0
86 48 0 10 34 10 10 0 72 7 0 38 O 0
100 0 0 0 0 O 0 0 50 50 0 0 0 0
100 86 43 0 86 0 100 14 O 0 0 71 0 0
0 100 0 100 33 83 0 0 83 0 0 33 0 0
100 14 0 0 0 0 0 0 14 0 0 28 14 0
XV (9) 33 78 11 0 0 0 0 0 45 0 11 11 0 0
T (1)
B (1)
N (1)
-100 100 0 O 0 0 0 O 0 0 O 0 0
0 100 O 6 0 0 O 0 100 0 0 0 0 0
100 0 0 0 0 0 0 0 0 O 0 0 0 100
*Subfamily classification according to Menezes [1], Authorities for sectional classification are given in Table 1. Key: V = Vellozioideae; VV = Vellozia section Vellozia; VR = Vellozia section Radia; VX = Vellozia section Xerophytoides; XX = Xerophyta section Xerophyta; XB = Xerophyta section Barbacenioideae; XV = Xerophyta section Vellozioideae; T = Talbotia; B = Barbaceniopsis; N = Nanuza; Qu = quercetin, K m = kaempferol; Isorh = isorhamnetin; Lu = luteolin; Qu3ME = quercetin 3-methyl ether; 5-deoxykm = 5-deoxykaempferol; Lu5ME = luteotin 5-methyl ether; DK/DK = dark to dark in UV light+NH3; DK/Y = dark to yellow in UV-PNH3. Figures in parentheses refer to number of species surveyed.
LEAF FLAVONOIDS IN THE VELLOZIACEAE
493
Discussion A summary of the leaf flavonoids found in the Velloziaceae is given in Table 3. As in Table 1 the results are listed according to a combination of the two current systems of family classification by Menezes [1] and Smith and Ayensu [2, 3], which are compared in Table 4. Eight conclusions may be drawn from the chemical evidence in support or otherwise of these two systems. Thus, (1) the Barbacenioideae sensu Menezes is distinguished from the Vellozioideae by the absence of flavone C-glycosides. (2) Pleurostima section Grazielae is unique in producing 6-hydroxyflavones. This suggests an evolutionary advanced position for the genus Pleurostima within the family. (3) Isorhamnetin is characteristic of all Barbacenia species sensu strictu except B. aft. globata and is also present in three species considered as Aylthonia by Menezes, namely: B. gardneri, B. paranaensis and one specimen of B. riedeliana. The latter sample is also distinguished from these and all other Velloziaceae by containing tricin in the free state. (4) Isorhamnetin is absent from Aylthonia s.s., Burlemarxia and five of the Barbacenia species considered as Aylthonia by Menezes: B. blancheti~ B. coronata, B. umbrosa, B. graminifolia and B. pulverulenta. The latter two species are distinct in producing quercetin 3-methyl ether, but Aylthonia and Burlemarxia cannot otherwise be separated using the flavonoid data. (5) Biflavone production in angiosperms is recognized as an ancient character and therefore the formation of the biflavone, arnentoflavone, in Nanuza indicates that it is chemically the most "primitive" member of the family [7]. The presence in this genus of glycoflavones suggests that it is more closely related to the Vellozia than to Xerophyta. (6) Within Xerophyta the Madagascan species (section Xerophytoides) are distinct in their leaf pattern (quercetin, isorhamnetin, quercetin 3-methyl ether) and are chemically better placed in the Barbacenioideae together with Barbaceniopsis, Talbotia and two other "flavonol only" species of Xerophyta section Vellozioides : X. schnitzleinia and X. viscosa. However, the predominance of flavone C-glycosides in Xerophyta section Barbacenioides and in X. scabrida, X. simulans and X. squarrosa in Xerophta section Vellozioides suggests that these plants may be chemically more closely allied with Vellozia than with members of the Barbacenioideae. (7) Some species of Vellozia section Xerophytoides which Menezes considers as belonging to Xerophyta section Talbotia, cannot be distinguished by the flavonoid evidence from other Vellozia taxa. (8) Within Vellozia, section Vellozia is unusual in the large number of species which produce surface flavonoids and section Radia is distinguished by the absence of flavonols. Otherwise all three Vellozia sections have similar flavonoid patterns. From these observations it is possible to construct a phylogenetic classification of the Velloziaceae based solely on flavonoid characters (Table 4). A cladistic analysis of the Velloziaceae based on morphological and anatomical characters [10] is in agreement with the chemical data and suggests that the genus Vellozia occupies a primitive position in the family and that Nanuza is an independent primitive clade in a large monophyletic sister group of the genus Vellozia. There is also agreement between the cladistic analysis and chemical data in the separation of Talbotia from the genus Xerophyta (sensu Smith and Ayensu, Table 4) but not for its placement in the Barbacenioideae. The cladistic study supports the retention of Barbaceniopsis as an independent group which is closer to Madagascan Xerophyta species (in section Xerophytoides) and Xerophyta section Vellozioides than to Xerophyta section Barbacenioides. The results of a leaf alkane survey of 110 Velloziaceae species [6] also agree with the flavonoid data in supporting Menezes' circumscription of the Barbacenioideae and her view that Pleurostima occupies an intermediate position between the two subfamilies. Similarly, the Brazilian Xerophyta species (i.e. sensu Menezes) cannot be separated from African Xerophyta taxa on alkane data. However, the alkane evidence, unlike the
Xerophytoides
Vellozioides
section
section
9
7
6
1
7
2
29
1?
1
--
--
3
7
7
2
25
--
--
--
--
--
--
Flavone C-glycosides
1
.
7
1
6
1
6
14
.
5
4
3
7
8
4
Qu
.
.
. 1
1
3
--
--
.
4
3
3
5
2
4
.
Flavonols Km
.
.
.
3
.
.
6
--
.
3
1?
6
--
.
.
.
.
.
Isorh
.
.
.
.
.
.
5
.
--
--
--
.
--
.
.
--
.
.
.
60H Lu
.
.
.
.
.
.
.
.
1
2
6
11
--
--
.
.
.
.
.
.
.
3
5
--
--
--
2
.
.
.
Flavones "Free" Lu Qu3ME
2
.
7
3
4
--
--
.
2
1
.
.
Anthocyanin
1
1
4
1
5
--
1
20
--
--
--
--
.
DK/DK unknowns
1
2
2 1
2
5
Flavanone (1) Procyanidin (1)
--
11
--
1
4
--
4
2
Surface flavonoids
Lu5ME (1)
--
5-Deoxykm
--
--
Amentoflavone (1)
--
--
--
--
"Free" tricin (1)
--
Others
*Subfamily classification according to Menezes [1]. tGenedc and sectional classification according to Smith and Ayensu [2, 3]. Key: Qu = quercetin, K m = kaempferol; Isorh = isorhamnetin; 6OH Lu = 6-hydroxyluteolin; Lu = luteolin; Qu3ME = quercetin 3-methyl ether; DK/DK = dark to dark in UV tight+NH 3. Figures refer to number of species with positive records in all cases.
Xerophytoides
Barbacenioides
section
Xerophyta
Talbotia
section
Vellozia
Radia
section section
Vellozia
1
1
Barbaceniopsis
Nanuza
5
8
3
7
8
4
section G r a z i e l a e Vellozioideaet
section
Pleurostima
strictu
"Aylthonia'Menezes)
Pleurostima
s.s
Barbacenia
Burlemarxia
(=
sensu
Barbacenia
Aylthonia
Barbacenioideae
Subfamily genus*
No. of species examined
TABLE 3. A SUMMARY OF THE LEAF FLAVONOIDS OF THE VELLOZIACEAE
o~
~>
~>
495
LEAF FLAVONOIDS IN THE VELLOZIACEAE TABLE 4. COMPARISONOF CLASSIFICATION SYSTEMS OF THE VELLOZIACEAE Smith and Ayensu (2,3] Vellozioideae Vellozia 1. section Vellozia 2. section Radia 3. section Xerophytoides 4. Nanuza
Barbacenioideae 5. Talbotia Xerophyta 6. section Xerophytoides 7. section Vellozioides 8. section Barbacenioides 9. Barbaceniopsis 10. Barbacenia
Menezes [1]
This paper
Vellozia 1. section Vellozia 2. section Radia Xerophyta 3-5. section Talbotia 6-9. section Xerophyta
4. Nanuza Vellozia 1. section Vellozia 2. section Radia 3. section Xerophytoides Xerophyta 8. section Barbacenioides 7. section Vellozioides
10. Burlemarxia 10. Pleurostima section Pleurostima section Grazielae
Xerophyta 6. section Xerophytoides 5. Talbotia 10. Pleurosdma 9. Barbaceniopsis
1O. Barbacenia
10. Aylthon~ 10. Burlemarxia 10. Barbacen~
10. Aylthonia
flavonoid data, does not distinguish between support the recognition of Burlemarxia.
Aylthonia
s.s a n d B a r b a c e n i a , b u t d o e s
Acknowledgements--Wethank
Dr Simon Mayo, Royal Botanic Gardens, Kew for assistance with the taxonomic aspects of this work and we are grateful to Mr J. Eagles, Food Research institute, Norwich for MS determinations.
References 1. Menezes, N. L. de (1980) in Petaloid Monocotyledons: HoFdcultural and BotanicaI Research Linn. Soc. Syrup. Ser. 8, 117. 2. Smith, L. B, and Ayensu, E. S. (1974) KewBull. 29, 181. 3. Smith, L. B. and Ayensu, E. S. (1976) Smithson. Contribu. Bot. 30, 1. 4. Menezes, N. L. de (1980) Revta bras. BoL 3, 37. 5. Menezes, N. L. de (1971) Ci~nc. Cult. 23, 421. 6. Salatino, M. L. F., Salatino, A., Menezes, N. L. de and Mello-Silva, R. de (1989) Phytochemistry28, 1105. 7. Williams, C. A., Harborne, J. B. and Tomas-Barberan, F. A. (1987) Phytochemistry26, 2553. 8. Williams, C. A. and Harborne, J. B. (1975) Biochem. SysL Ecol. 3, 181. 9. Proksch, P., Budzikiewicz, H., Tariowitz, B. D. and Smith, D. M. (1984) Phytochemistry 23, 679. 10. Menezes, N. L. de, Mello-Silva, R. de and Mayo, S. J. (1992) KewBull. (in press).