From the CH₂Cl₂ washings of fresh Heliotropium spp. (Cochranea section), flavonoids and aromatic geranyl derivatives have been isolated. Among the flavonoids, flavanones and flavonols are the most common compounds, and only few flavones have been reported. In this communication we report the isolation of several flavonoids from the exudates of H. megalanthum, and H. huascoense. Two of the compounds isolated from H. huascoense are novel flavonoids, belonging to the uncommon groups of penta-O-substituted flavanones and hexa-O-substituted flavonols. Our results show a different external flavonoid composition of H. huascoense from the other Heliotropium spp. so far studied. They are supported by morphological considerations, in special, the presence or absence of glandular trichomes in Heliotropium spp.

KEY WORDS: Heliotropium megalanthum, glandular trichomes, 5-hydroxy-7,3',4',5'-tetramethoxylavanone, 5,3'-dihydroxy-7,4',5'-trimethoxyflavanone, Heliotropium huascoense, non-glandular trichomes, 5-hydroxy-3,7-dimethoxyflavone, (2S, 3S)-3,5-dihydroxy-7-methoxyflavanone.

Del extracto de compuestos superficiales obtenido con CH₂Cl₂ de material fresco de especies de Heliotropium, se han aislado flavonoides y derivados aromáticos geranilados. Entre los flavonoides, los tipos estructurales más comunes corresponden a flavanonas y flavonoles y se han reportado solamente pocas flavonas. En este trabajo se informa el aislamiento de varios flavonoides de los exudados de H. megalanthum y H. huascoense. Dos de los componentes aislados de H. huascoense son flavonoides nuevos, que pertenecen a los grupos poco comunes de flavanonas penta-O-substituidas y flavonoles hexa-O-substituidos. Estos resultados muestran una composición de flavonoides externos en H. huascoense, diferente de las otras especies de Heliotropium estudiadas. Los resultados están reafirmados por consideraciones morfológicas, en especial, la presencia o ausencia de tricomas glandulares en especies de Heliotropium.

PALABRAS CLAVES: Heliotropium megalanthum, tricomas glandulares, 5-hidroxi-7,3'.4'.5'-tetrametoxiflavanona, 5-3'-dihidroxi-7,4',5'-trimetoxiflavanona, 3,5-dihidroxi-7,3',4'5'-tetrametoxiflavona, Heliotropium huascoense, tricomas no glandulares, 5-hidroxi-3,7-dimetoxiflavona, 5-hidroxi-3,7-dimetoxiflavona, (2S, 3S) -3,5-dihidroxi-7-metoxiflavanona.

Section Cochranea (Miers) Reiche of the genus Heliotropium (Boraginaceae), is known only from the coastal hills of northern Chile, and in the south of Perú. This section is of particular ecological interest, because the species grow in arid environment with extreme environmental variation. The only exception is H. stenophyllum that grows in Central Chile between lat. 30° and 33° S¹⁾.

The section Cochranea is represented by 17 species of Heliotropium with a geographic distribution ranging from lat. 15° to 33° S¹⁾. Like many other desert or semidesert plants, most of the Heliotropium spp. produce characteristic resinous exudates secreted by glandular trichomes on the surface of the leaves and stems¹⁾.

Flavanones, flavonols, flavones and aromatic geranyl derivatives have been isolated from the surfaces of Heliotropium spp. these results are presented in Table I.

In this paper we report the flavonoid external composition of H. megalanthum and of H. huascoense, and establish some chemotaxonomic correlations between these species by comparison of their chemistry with that of other previously studied species. In addition the antimicrobial properties of some compounds are presented.

Plant material

Heliotropium megalanthum Johnston (ST-2579) and H. huascoense Johnston (ST-2580) were collected during the flowering season, October 1995, in the north of Vallenar (IV Region, Chile, 28° 45' S, 70° 49' W). Voucher specimens were deposited in the Herbarium of the National History Museusm Santiago, Chile.

Isolation of the external components

The external components of H. megalanthum, nd H. huascoense were obtained by dipping 200 g of each fresh plant in cold CH₂Cl₂ for 15 to 20 s. The extracts were concentrated to partially crystalline residues (2.95 g from H. megalanthum, and 18.2 g from H. huascoense).

External flavonoids from H. huascoense

The resinous exudate of H. huascoense was fractionated by CC on silica gel, using hexane with increasing amounts of EtOAc. The fractions were monitored by TLC on silicagel, using the systems hexane-EtOAc (3:1), CHCl₃ -MeOH (99:1), and CHCl₃ -MeOH (9:1).

Compound 14: 5,7-dihydroxy-3-methoxyflavone (3-methylgalangine) was identified by direct comparison with an authentic sample (FTIR, TLC, HPLC).

Compound 15: 5-hydroxy-3,7-dimethoxyflavone (3,7-dimethylgalangine) was identified by direct comparison with an authentic sample (FTIR, TLC, HPLC).

Compound 5: (2S, 3S)-3,5-dihydroxy-7-methoxyflavanone ((-)-alpinone); crystals from EtOAc, m.p. 184-186°C [a]_D^25° - 28.1° (C: 0.215, CHCl₃); UV (MeOH): l_max 288, 320 (sh) nm; UV (MeOH + NaOAc): l_max 288, 320 (sh) nm; ¹H-NMR (500 MH_z, CDCl₃) d: 3.46 s (1H, OH), 3.80 s (3H, C-7-OMe), 4.54 d (1H, J = 11.9 H_z, H-3), 5.07 d (1H, J = 11.9 H_z, H-2), 6.05 d (1H, J = 2.3 H_z, H-6), 6.11 d (1H, J = 2.3 H_z, H-8), 7.24 s (1H, OH), 7.44 m (3H, H-3', H-4' and H-5'), 7.54 m (2H, H-2' and H-6') and 11.18 s (1H, C-5-OH); EI-MS (m/z, %): 286 (33), 257 (51), 167 (100), 151 (3), 120 (13), 91 (31), 69 (16), 51 (15). HREI-MS: M⁺, 286.087004 (C₁₆H₁₄O₅, requires 286.084125).

TABLE I. Flavonoids and geranyl aromatic derivatives on the surface of Heliotropium spp.

^a1 = H. stenophyllum H.&A.; 2 = H. sinuatum (Miers) Johnston; 3 = H. filifolium (Miers) Reiche; 4 = H. chenopodeaceum var. chenopodeaceum (D.C.) Clos; 5 = H. chenopodeaceum var. ericoideum (Miers)
Reiche; 6 = H. huascoense Johnston; 7 = H. megalanthum Johnston.
* = Present study.

External flavonoids from H. megalanthum

The external components of H. megalanthum were fractioned by CC using the same procedure applied to the resin of H. huascoense.

Compound 10: 5-hydroxy-7,3',4',5'-tetramethoxyflavanone; crystals from EtOAc, m.p. 159-160 °C. [a]_D^25° -33.33° (C: 0.03, MeOH); UV (MeOH): l_max 285, 320 (sh) nm; UV (MeOH + NaOAc), l_max 285, 320 (sh) nm ¹H-NMR (200 MH_z, CDCl₃) d: 2.80 dd (1H, J = 3.12, J = 17.19 H_z), 3.10 dd (1H, J = 13.07, J = 17.19 H_z), 3.82 s (3H, OCH₃), 3.87 s (3H, OCH₃), 3.90 s (6H, 2 OCH₃), 5.35 dd (1H, J = 3.14, J = 13.02 H_z), 6.08 brs (2H, H-6 and H-8), 6.67 s (2H, H-2' and H-6') and 12.02 s (1H, C-5-OH); EI-MS (m/z, %): 361 (M+1, 22), 360 (M⁺, 69), 330 (100), 179 (13), 167 (5), 164 (22), 151 (43), 95 (11). HREI-MS: M⁺, 360.120400 (C₁₉H₂₀O₇, requires 360.120905).

Compound 9: 5,4-dihydroxy-7,3',5'-trimethoxyflavanone; crystals from EtOAc, m.p. 144-146°C; [a]_D^25° -53.66° (C: 0.41, CHCl₃); UV (MeOH): 286, 323 (sh) nm; UV (MeOH + NaOAc): 286, 323 (sh) nm, ¹H-NMR (500 MH_z, CDCl₃) d: 2.79 dd (1H, J = 3.3, J = 17.1 H_z), 3.06 dd (1H, J = 12.6, J = 17.1 H_z), 3.81 s (3H, OCH₃), 3.90 s (3H, OCH₃), 3.91 s (3H, OCH₃), 5.30 dd (1H, J = 3.3, J = 12.6 H_z), 6.06 d (1H, J = 2.1 H_z, H-6 or H-8), 6.08 d (1H, J = 2.1, H-6 or H-8), 6.57 d (1H, J = 1.8 H_z, H-2' or H-6'), 6.69 d (1H, J = 1.8 H_z, H-2' or H-6') and 12.00 s (1H, C.5-OH); EIMS (m/z, %): 347 (M⁺ +1, 43), 346 (M⁺, 100), 345 (22), 246 (6), 193 (14), 180 (16), 168 (8), 167 (78), 166 (7), 165 (23), 137 (7), 69 (6) and 43 (10). HREI-MS: M⁺, 346.102214 (C₁₈H₁₈O₇, requires 346.105255).

Compound 21: 3,5-dihydroxy-7,3',4',5'-tetramethoxyflavone; amorphous powder; UV (MeOH): l_max 264, 340 nm; UV (MeOH + NaOAc): l_max 264, 340 nm; ¹H-NMR (500 MH_z, CDCl₃) d: 3.89 s (6H, 2 x OCH₃), 3.95 s (3H, OCH₃), 4.01 s (3H, OCH₃), 5.94 brs (1H, OH), 6.37 d (1H, J = 2.25 H_z, H-6), 6.45 d (1H, J = 2.25 H_z, H-8), 7.34 d (1H, J = 1.95 H_z, H-6' or H-2'), 7.36 d (1H, J = 1.95 H_z, H-2' or H-6') and 12.57 s (1H, C-5-OH); EI-MS (m/z, %): 374 (M⁺, 100), 373 (23), 359 (42), 331 (16), 174 (3), 176 (11), 166 (1), 167 (13) and 149 (27); HREI-MS: M⁺, 374.098305 (C₁₉H₁₅O₈, requires 374.100168); 359.083748 (C₁₈H₁₅O₅, requires 359.076693); 167.025040 (C₈H₇O₄, requires 167.034440).

Identification of the flavonoids

Compounds 14 and 15 were shown to be 7,5-dihydroxy-3-methoxyflvone (3-methylgalangine), and 5-hydroxy-3,7-dimethoxyflavone (3,7-dimethylgalangine), respectively, by comparison with authentic samples and with spectral data.

Compound 5 shows spectral data and m.p. identical to those reported for (2R, 3R)-3,5-dihydroxy-7-methoxyflavanone ((+)-alpinone)²⁾. The only difference was the negative specific rotation which indicated that compound 5 has the opposite configuration, and therefore was characterized as (2S, 3S)-3,5-dihydroxy-7-methoxyflavanone ((-)-alpinone).

The HREI-MS spectrum for compound 10 shows the molecular formula C₁₉H₂₀O₇. The ¹H-NMR spectrumm shows the presence of a flavanone with four methoxy groups and a C-5-OH. In addition two singlets at 6.08 (2H), and 6.67 (2H) were assigned to H-6, H-8 and H-2', H-6' respectively. Compound 10 therefore was characterized as 5-hydroxy-7,3',4',5'-tetramethoxyflavanone, compound previously isolated from the farinose exudate fronds of Notholeana lemonnii var. lemonnii³⁾.

The HREI-MS spectrum for compound 9 shows the molecular formula C₁₈H₁₈O₇. The ¹H-NMR spectrum shows that 9 was a derivative of compound 10, with only three methoxy groups. The two doublets (J = 2.1) at 6.57 and 6.69, for H-2' and H-6', suggest an asymetrically substituted ring B. The second OH group was assigned to C-3', and therefore compound 9 was characterized as 5,3'-dihydroxy-7,4',5'-trimethoxyflavanone.

The HREI-MS spectrum for compound 21 shows the molecular formula C₁₉H₁₈O₈, and a fragment (A+1)⁺ of molecular formula C₈H₇O₄ consistent with one OH, and one OCH₃ on ring A. The ¹H-NMR spectrum shows the presence of a flavonol with four methoxy groups, and a C-5-OH. Two doublets (J = 2.25) at 6.37 and 6.45 were assigned to H-6 and H-8 respectively. In addition, two other doublets (J = 1.95) at 6.34 and 6.36 for H-2' and H-6', almost a singlet, suggest a symetrically substituted ring B. Therefore compound 21 was characterized as 3,5-dihydroxy-7,3',4',5'-tetramethoxyflavone (7,3',4',5'-tetramethoxymyrecetin).

The compounds 9 and 21 are novel natural flavonoids.

Chemotaxonomic considerations

Heliotropium huascoense bears glandular trichomes similar to those found in H. stenophyllum, H. sinuatum, H. filifolium and H. chenopodiaceum var. chenopodiaceum; by contrast, H. megalanthum, and H. chenopodiaceum var. ericoideum bear non glandular trichomes^1,3,5). These morphological differences are in agreement with the yield of external extracts by those species^4-5). In a previous communication⁵⁾ we found an almost identical external flavonoid composition of H. chenopodiaceum var. chenopodiaceum and H. chenopodiaceum var. ericoideum, which suggests that the presence of glandular trichomes did not determine the external accumulation of the same flavonoids in these two varieties.

This seems not to be the case in this study. The external flavonoids in H. megalanthum are uncommon penta-O-substituted flavanones and hexa-O-substituted flavonols, a pattern of oxygenation not found in other flavonoids isolated from species of Heliotropium so far studied, Table I.

In addition to the aromatic geranyl derivatives isolated from the resinous exudates of H. chenopodiaceum, H. filifolium, and H. huascoense, we have conclusive evidence, obtained by HPLC coupled with UV-vis spectroscopy⁵⁾, that aromatic geranyl derivates are also present in the resinous exudates of H. sinuatum and H. chenopodiaceum var. chenopodiaceum. This result seems to indicate that these geranyl aromatic derivatives accumulate in the glandular trichomes of Heliotropium spp.

It has been shown that surface waxes as well as trichomes, play an important role in the resistance of some plants against insect and microorganisms attack¹¹⁾, since the resinuous exudates, produced by glandular trichomes, and surface waxes constitute the first line of resistance (physical and chemical) of a plant against insect and microorganisms.

In order to advance in the understanding of this phenomenon, the study of the antimicrobial properties of some compounds of Table I (1, 2, 6, 7, 11, 14, 15, 17, and 18) has been undertaken. Compounds 1, 2, 6, 7, 11, and 14 show antibacterial properties, (MIC'S < 500 µg x mL^-1) against Gram-positive and Gram-negative bacteria, using the agar overlay method¹²⁾.

This work was supported by FONDECYT Chile Grant N 1990209, and by DICYT (Universidad de Santiago de Chile).
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*To whom correspondence should be addressed.

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