Phytochemimy,Vol. 20, No. 4, pp. 791-794, 1981. 0031-9422/81/040791W4 $02.

00/O
Printed in Great Britain. Q 1981 Pergamon Press Ltd.

6-HYDROXY- AND 6-METHOXYFLAVONOIDS FROM

NEUROLAENALOBATA ANDN. MACROCEPHALA

K. M. KERR, T. J. MABRY and S. YOSER

The Department of Botany, The University of Texas at Austin, TX 78712, U.S.A.

(Received 20 June 1980)

Key Word Index-Neurolaena lobata; N. macrocephala; Asteraceae; Heliantheae; Galinsogineae; 6-

hydroxykaempferol methyl ethers, glucosides and sulfate; quercetagetin methyl ethers, glucosides and sulfate; 6-
hydroxyflavone methyl ethers and glucoside.

Abstract-Twelve flavonoids including one new sulfate were isolated from Neurolaena lobata, and six known flavonoids
were obtained from N. macrocephala. The new compound isolated from N. lobata is 6-hydroxykaempferol 3-methyl
ether 7-sulfate, and the known flavonoids are 6-hydroxykaempferol3,7-dimethyl ether, 6-hydroxykaempferol, 3-methyl
ether 7-glucoside, 6-hydroxykaempferol 7-glucoside, quercetagetin and its 7-glucoside, quercetagetin 3,6- and 3,7-
dimethyl ethers, quercetagetin 3-methyl ether 7-glucoside and 7-sulfate, 6-hydroxyluteolin 3’-methyl ether and 6-
hydroxyluteolin 7-glucoside. The known flavonoids identified from N. macrocephala are quercetagetin 3,6- and 3,1-
dimethyl ethers, quercetagetin 6-methyl ether 7-glucoside, quercetagetin 3,6-dimethyl ether 7-glucoside, quercetagetin
7-glucoside and quercetagetin 3-methyl ether 7-sulfate.

INTRODUCTION methyl ether 7-glucoside (11) [l] and quercetagetin 7-

In a continuation of our biochemical systematic glucoside (12) [ll].
investigation of the genus Neurolaena
The identities of all the known flavonoids were
(Asteraceae-Heliantheae) [l, 21, we report the isolation determined by direct comparison (TLC, UV, NMR) with
and characterization of twelve flavonoids from N. lobata authentic samples previously obtained from N. oaxa-
(L.) R. Br., a widespread tropical weed in southern Mexico cana [l 1. The structure assignment of the new compound
and South America, and six from N. macrocephala Sch.- is discussed separately, and all previously unreported
Bip. ex Hemsl., a taxon endemic to Veracruz, Mexico. One color, TLC, UV, NMR and MS data for all the flavonoids
of the flavonoids from N. lobata, 6-hydroxykaempferol3- are recorded in Tables 1 and 2 or in the Experimental.
methyl ether 7-sulfate, is a new compound. We previously 6-Hydroxykaempferol 3-methyl ether ‘I-sulfate (9)
described fifteen flavonoids from N. oaxacana B. L.
The presence of a 6-hydroxyl group in 9 was first
Turner [I 1, and seven from N. uenturana B. L. Turner [2].
suspected from the purple-brown color of the spot on a
Two sesquiterpene lactones were previously reported
paper chromatogram under UV light which was
from N. lobata [3], and the thymol derivatives of N.
unchanged when exposed to ammonia vapor or sprayed
lobata, N. oaxacana and N. venturana have been described
with NA. The UV band I shift of + 24 nm in AlClJHCl
[41. relative to band I in MeOH indicated a flavonoid with C-6
oxygenation. The absence of a band III, band I at 390 nm
RESULTS in NaOMe vs 400 nm in NaOAc, and no shift of band II in
Leaves of N. lobata and N. macrocephala were both NaOAc suggested an -OR substituent at C-7 [12]. The
extracted with aqueous methanol and the syrup obtained lack of a shift of band I with NaOAc/H,BO, established
after concentrating the extract was partitioned between that there was no ortho-dihydroxyl group in the B-ring.
water and three organic solvents: n-hexane, dichloro- The electrophoretic mobility to the anode (4.5 cm) of this
methane and ethyl acetate. The hexane extract was compound under standard conditions [13] at pH 1.9
discarded. The dichloromethane extract of N. lobata indicated that it was a monosulfated compound. This was
yielded 6-hydroxykaempferol 3,7-dimethyl ether (1) [5], confirmed when sulfatase hydrolysis yielded 6-
quercetagetin 3,7-dimethyl ether (2) [5], quercetagetin hydroxykaempferol 3-methyl ether (TLC comparison
3,6-dimethyl ether (3) [6], 6-hydroxyluteolin 3’-methyl with an authentic sample). The UV spectral findings
ether (4) [7], and 6-hydroxykaempferol 7-glucoside require that the sulfate moiety is at C-7. Thus, the new
(5) [8]. The ethyl acetate extract yielded additional 6- compound is 6-hydroxykaempferol 3-methyl ether 7-
hydroxykaempferol 7-glucoside plus quercetagetin sulfate.
(6) [9], and 6-hydroxyluteolin 7-glucoside (7) [lo]. The The flavonoids isolated from the dichloromethane
water extract yielded the remaining compounds: querce- extract of N. macrocephala were quercetagetin 3,6-
tagetin 3-methyl ether 7-sulfate (8) [ 11, 6-hydroxykaem- dimethyl ether (3) [6] and quercetagetin 3,7-dimethyl
pferol 3-methyl ether 7-sulfate (9) 6-hydroxykaempferol ether (2) [5]. The ethyl acetate extract yielded querceta-
3-methyl ether 7-glucoside (10) [ 11, quercetagetin 3- getin 6-methyl ether 7-glucoside (13) [14], quercetagetin
791
PHYTO
20:4- P
792 K. M. KERR et d.

Table 1. Chromatographic data (R,s x 100 and colors) for flavonoids of N. lohatu*

Cellulose

Paper HOAc TBA n-BAW Polyamide Colors?

_____I_

Compound TBA HOAc 15Y(> 40% BMM RPMM UV IJV’NH, tJV,!NA

6-Hydroxykaempferol
7-glucoside (5) 25 9 5 28 19 24 29 0 PBr PBr PBr
Quercetagetin (6) 20 0.2 2 10 19 31 19 2 P PBr Or
6-Hydroxykaempferol
3-methyl ether
7-sulfate (9) 39 68 56 79 45 48 P PBr PBr
Quercetagetin
3,6-dimethyl
ether
7-glucoside (14) 44 36 34 57 36 40 0 0 P YHr Or

* See ref. [I] for chromatographic data of other flavonoids from N. lobata and solvent key.
t P = purple. PBr = purplish-brown, Or = orange, YBr = yellowish brown; NA = Naturstoff reagenz A in MeOH.

3,6-dimethyl ether 7-glucoside (14) [8], and quercetagetin EXPERIMENTAL

7-glucoside (12) [l 11. Quercetagetin 3-methyl ether 7- Plant material. Leaves and vouchers of N. lobuta were collected
sulfate@) [l ] was obtained from the water fraction. The from along the roadside several km N. of A tzalan at La Calavera,
identities of all these flavonoids were determined by direct Vercruz, Mexico, on 9 March 1979 (voucher specimen K. M.
comparison (TLC, UV) with authentic samples pre- Kerr 124 is deposited in the Lundell Herbarium, The University
viously obtained from N. oaxacana [I 1. The UV and of Texas at Austin). Leaves and vouchers of N. mucrocephala were
chromatographic data for quercetagetin 3,6-dimethyl collected 300m from the entrance to Los Tuxtlas Biological
ether 7-glucoside are reported in Tables 1 and 2.

OH

OH 0

OH 0
1 Rr = Me, R* = H
2 Rr=Me,R’=OH
8 R’ = SO;, R’ = OH 3 R’=H
9 R’=SO,,R’=H 14 R’=glc
10 R’ = glc, R” = H
11 Rr = glc. RL = OH

OR’

OH

5 R’ = glc, R* = H
4 Rr = H, R’ = Me 6 R’ = H, RZ = OH
7 R’ = glc, R2 = H 12 RI = glc, R* = OH
 Table 2. UV data (&,,,, nm) for flavonoids of N. lobata*

Compound MeOH NaOMe AICI, AICIJHCI NaOAc NaOAc/H,BO,

5 374 (sh), 346 (I),? 400 (l), 290 (0.5), 436 (sh), 388 (l), 428 (I), 382 (1.2), 384 (sh), 348 (l), 380 (sh), 386 (l),
276 (0.8), 254 (0.7) 287 (0.9) 267 (1.5) 272 (0.9), 278 (1.5)
256 (0.7), 234 (0.7) 254 (0.8)
6 360 (l), 272 (sh), 390 dec. (l), 444 (l), 280 (0.7) 436 (sh), 396 (I), 388 (1X 376 (l), 292 (sh),
255 (0.7) 304 (3), 244 (4) 270 (1) 260 (0.7) 266 (0.7)
9 366 (1) 294 (sh), 390 (I), 300 (sh), 400 (sh), 364 (I), 398 (sh), 358 (l), 400 (sh),
270 (0.8)$ 270 (0.6) 304 (sh), 278 (1) 302 (sh), 280 (1.5) 344 (l), 268 (0.8) 366 (l), 270 (0.5)
14 350 (I), 292 (sh), 400 (I), 276 (sh), 434 (I), 364 (sh), 400 (sh), 370 (I), 370 (I), 290 (8), 370 (I), 290 (0.8), 264 (1.4)
260 (1.5) 252 (1.3) 326 (sh), 304 (sh), 298 (sh), 260 (1.4)
280 (2) 268 (1)

* See ref. [l] for the UV data of other flavonoids from N. lobata.
t Relative absorptivities are given for each A,,,,, relative to the most intense wavelength peak as (1).
$ MeOHHCl: 338 (1) 280 (0.8).
794 K. M. KERR et al.

Station on 13 March 1979 (voucher specimen K. M. Kerr 136 is in TBA (t-butanol-HOAc-H,O, 3:l:l) on Whatman 3MM
deposited in the Lundell Herbarium, The University of Texas at paper. The flavonoids were eluted with MeOH and purified over
Austin). Sephadex LH-20. The following compounds were obtained:
Extraction, pur$cation and identijication ofjlavonoidsfrom N. quercetagetin 3,6-dimethy! ether (3), 1 mg, and quercetagetin 3,7-
lobata and N. macrocephala. Genera! chromatographic and dimethyl ether (2),2mg. B. EtOAc exrract. After the same
electrophoretic techniques have been previously described [l]. purification procedure of the cont. EtOAc extract the following
Ground leaves of 11’.lohuta (200g) were extracted 3 x with aq. compounds were isolated: quercetagetin 6-methyl ether 7-
MeOH. The combined extracts were coned in IXICUOto lOOm1, glucoside (13), 2mg; quercetagetin 3,6-dimethy! ether 7-
and the aq. concentrate successively extracted with CH,CI, and glucoside (14), 4mg, and quercetagetin 7-glucoside (12), 5 mg. C.
EtOAc. A. CH,C!, extract. The syrup from this extract (5 g) was H,O extract. On purification as above the cone H,O extract gave
passed over Sephadex LH-20, and the flavonoid mixture (0.7g) quercetagetin 6-methyl ether 7-glucoside (13). quercetagetin 7-
obtained from this column was chromatographed over a Polyclar glucoside (12) and a trace of quercetagetin 3-methyl ether 7-
column (4 ic 17cm: 5Og). Elution was initiated with Egger’s sulfate (8).
solvent (CH2CI, -MeOH-~MeCOEt-Me,CO, 20:10:5:1) and
Acknowledgements-This work was supported at The University
the polarity of the solvent was gradually increased by the
of Texas at Austin by the Robert A. Welch Foundation (grant F-
elimination of CH,Cl, (0:lO:S:l). The compounds eluted in the
130), the National Science Foundation (grant DEB 79-02703)
following order: h-hydroxykaempferol 3,7-dimethy! ether (I),
and theNational Institutes ofHealth (grant HD-04488). S.Y. was
1.5 mg: quercetagetin 3,7-dimethy! ether (2), 3mg; quercetagetin
supported by the Summer Science Training Program for High-
3,6-dimethyl ether (3). 1mg; 6-hydroxyluteolin 3’-methyl ether
Ability Secondary School Students.
(4), 1mg; and 6-hydroxykaempferol 7-glucoside (5), 14mg. B.
EtOAc extract. The syrup from this extract (9.2g) was passed
over a Sephadex LH-20 column and the flavonoids were obtained REFERENCES
as a mixture (3g) which was chromatographed over Polyclar 1. Ulubelen, A.. Kerr, K. M. and Mabry. T. J. (1980)
(4 x 32cm: 909). The column was first eluted with Plrl.to~honi,~tr!. 19. 1761.
HzO~-MeOH MeCOEt Me,CO (!3:3:3:!), and the polarity 2. Ulubelen, A., Kerr, K. and Mabry, T. J. (1980) Planta Med.
was decreased by the gradual elimination of H,O. The (in press).
compounds obtained were quercetagetin (6). 5mg, and 6- 3. Manchand, P. S. and Blount, J. F. (197X) J. Org. Chem. 43,
hydroxyluteolin 7-glucoside (7), 8 mg. C. H,O extract. The aq. 4352.
extract concentrate (11.7g) was chromatographed over Polyclar 4. Bohlmann, F., Natu, A. A. and Kerr, K. (1979)
(5 x 35 cm: 150 g) in the same manner as for the EtOAc extract. Phytochemistry 18, 489.
Compounds eluted in the following order: quercetagetin 3- 5. Shen, M. C., Rodriguez, E., Kerr, K. and Mabry, T. J. (1976)
methyl ether 7-sulfate (8), 1 mg; 6-hydroxykaempfero! 3-methyl Phytochemistry 15, 1045.
ether 7-sulfate (9), 4 mg; 6-hydroxykaempfero! 3-methyl ether 7- 6. Dillon, M. O., Mabry, T. J., Besson, E., Bouillant, M. L. and
glucoside (lo), 14mg; quercetagetin 3-methyl ether ‘I-glucoside Chopin, J. (1976) Phytochemistry 15, 1085.
(II), I1 mg: and quercetagetin 7-glucoside (12), 30mg. 7. Taylor, A. 0. and Wong, E. (1965) Tetruhedron Letters 3675.
NMK und .‘MS dutcl fbr N. lobata jlationoids. 6-Hydroxy- 8. Bacon, J. D., Urbatsch, L. E., Bragg, L. H., Mabry, T. J.,
kaempfero! 7-glucoside (5) and quercetagetin (6); MS of 5 Neuman, P. and Jackson, D. W. (1978) Phytochemistry 17,
derivatiaed VI.‘; (re!. int.), M+’ 302 (loo), M - H 301 (40), 1939.
M -~ H,O 286 (50), M - CHO 274 (60), M - COMe 259 (20), 9. Baker, W., Nodzu, R. and Robinson. R. (1929) J. Chem. Sot.
A, 16X (50). A, - 16 152 (60), B, 121 (100). ‘H NMR (90MHz, 74.
CC!,. TMS): ci 3.3 3.8 (5 H, m. 6 glucosyl protons), 4.95 (l-H, d, 10. Barua, A. K., Chadhabarti, P. and Sahya!, P. K. (1969) J.
J = XHz, for H”-l), 6.5 (1 H. s, H-8). 6.8 (2H, d, .I = 9Hz, H-3’ Indian Chem. Sot. 46, 271.
and H-5’). 7.93 (2 H, d, J = 9 Hz, H-2’ and H-6’). Compound 6: 11. Geissman, T. A. and Steelink, C. (1957) _I. Org. Chem. 22,
‘H NMR (9OMHz. CC!,, TMS): 6 6.5 (1 H, s, H-8), 6.85 (1 H, d, 946.
J = 9 Hz, H-5’). 7.65 (I H, c/d, J = 9 and 2 Hr. H-6’), 7.6 (1 H, d, 12. Bacon,J. D.. Mabry.T. J. and Mears.J. A. (1976) Ret:. Latino-
J = 2 Hz. H-2’). am. Quim. 7. X3.
The extraction techniques used for the isolation of flavonoids 13. Al-Khubazi, M. (1977) M. S. Thesis, The University of
from 5Og of dry leaf material of N. macrocephala were similar to Texas.
those already described for N. lobata. A. CHIC!, extract. The 14. Sharma, R. C., Zaman, A. and Kidwai, A. R. (1964) Indian J.
syrup from the CH,C!? extract was chromatographed by 1D PC Chem. 3, X3.