Journal of Ethnopharmacology,
51
33 (1991) 51-55
Elsevier Scientific Publishers Ireland Ltd.
Isolation and identification of the antibacterial from Helichrysum stoechas
compounds
J.L. Rios”, M.C. Recioa and A. Villarb “Farmacognosia i Farmacodinamia, Deparramento de Farmacologia i FarmacotPcnia. Facultat de Farmacia, Vniversitar de Vakncia. Avda. Blasco Ibdfiez 13, 46010-Vakncia and hFarmacognosia y Farmacodinamia, Departamento de Farmacologia, Fact&ad de Farmaria. Vniversidad Complurense de Madrid, Ciudad Vniversitaria. 28040 Madrid (Spain)
(Accepted November 6, 1990) Fractionation of the dicholoromethane extract of the aerial parts of Helichrysum stoechas yielded seven isolates (l-7), which exhibited varying antimicrobial activity against Gram-positive bacteria. Pure compounds 1-3 have been previously reported in the same species and 4 and 5 were identified as italipyrone and plicatipyrone, previously isolated from H. iralicum and H. plicarum. The two other isolates are helipyrone (6) and homoarenol (7) mixed with related substances. Compound 6e (4,4’-dihydroxy-5,6,5’,6’tetramethyl-3,3’-methylen-di-pyr-2-one) and 6b helipyrone with one methyl substitution, have not been previously reported in other species. Key words: Helichrysum sfoechas: antibacterial activity: pyrone derivatives; phloroglucinois.
Introduction
Methods
Helichrysum stoechas (L.) Moench is a shrub with yellow flowers that grows widely in the Mediterranean area. This species is considered antimicrobial (Rios et al., 1987), antiinflammatory and digestive (Font Quer, 1983). Previous studies on its essential oil have been reported, and several phytochemical compounds have been isolated and identified as hydroxyisopenten-acetophenone and related compounds, triterpenoids, steroids (free and glycosidic forms) and a lipid fraction (Garcia de Quesada et al., 1972; Martin-Panizo et al., 1972). Some phloroglucinol derivatives of this species (dimethylallylitalipyrone and plicatipyrone) have been reported by Hgnsel et al., 1980. Our laboratory recently established the antimicrobial activity of the dichloromethane extract from this species (Rios et al., 1987).
Plant material
Correspondence 10: J.L. Rios Caiidwte. Farmacognosia i Farmacodinamia, Departament de Farmacologia i Farmacotkcnia.
Facultat de Farmacia, Universitat de Vakncia. Avda. Blasco IbPiiez 13, 46010-Vakncia. Spain. 037%8741/$03.50 0 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
The aerial parts of Helichrysum stoechas were collected at flowering in Sierra Perenchiza (Chiva, Vall?ncia, Spain) and a voucher specimen was deposited in the herbarium of the Departamento de Biologia Vegetal, Facultad de Farmacia, Universidad de Valkncia, Spain. Extraction and isolation Dried powdered plant (700 g) was successively extracted by maceration with dichloromethane and methanol. The extracted liquids were concentrated at vacuum and the extracts assayed against different bacteria and a yeast. The active dichloromethane extract was chromatographed on silica gel 60 (Merck) with chloroform/methanol mixtures of increasing polarity. Elution was followed by TLC and the antimicrobial activities of the fractions were assayed by an agar diffusion method (Rios et al., 1988) and the active fractions identified by TLC and bioautographic methods
52
(Rios et al., 1988). The active fractions were chromatographed by column chromatography on silica gel 60H (Merck) with chloroform/methanol as the mobile phase. Compounds were purified by precipitation and preparative-TLC and antimicrobial activities followed by an agar dilution method (Rios et al., 1988). Antimicrobiul
MIC (minimum inhibitory concentration) was determined by serial two-fold dilutions in the same type of agar, with concentrations ranging from 1 to 50 mg/l (Rios et al., 1988). The inoculum was prepared from a fresh overnight broth culture in trypticase soy broth (Difco) and adjusted to an approximate concentration of lo6 colony-forming units/spot. Plates were incubated for 18 h at 35°C. The MIC was detined as the lowest concentration of compounds that inhibited development of visible growth on agar.
testing
The antimicrobial activities were evaluated by the classic agar diffusion procedure using MuellerHinton agar (Difco) (Rios et al., 1988). Samples were diluted in methanol (2.5 mg/l) using 20 ~1 of each sample solution. The Petri dishes were cooled at 4°C for 2 h before incubation at 35.5”C. The
Spectrul
data
Structural elucidations (Fig. 1) were made by UV spectra (Pye-Unicam), IR spectra (Hitachi Perkin Elmer), ‘H-NMR and “C-NMR (Bruker
@-Sitosterol-/3-O-glucosides I: R = glucose
3: 4-Hydroxy-3(isopenten-2-yl)
2: R = glucose-glucose
acetophenone
A0 5: Plicatipyrone
4: ltalipyrone
6s: Helipyrone 6b: R, = -CH,.
RI = R, = -CH,-CH, R, = -CH,-CH,
6~: R, = R, = -CH, Fig.
I.
Isolates from Hdiclwy.surn s/orbs
7a: Homoarenol 7b: Arenol aerial parts
R = -CH,-CHJ
R = -CH,
53
WP 200 and Varian XL 300) EIMS (Mass spectrometer VG 12-250 Massland), EIMS and DCIMS NH3, positive mode (Nermag R103G spectrometer). Compound 6c (4,4’-dihydroxy-5,6,5’,6’-tetramethyl-3,3’methylen-di-pyr-2-one). 13C-NMR: 174.3 (C=O), 110.2, 166.3 (C-OH), 100.2, 154.6, 10.0 (CH,-C), 16.9 (CH3-C-O) and 18.8 (CH2-). ‘H-NMR: s, 1.78 (s, 6H), 2.09 (s, 6H), 3.36 (s, 2H). EIMS m/z (rel. int.): 292 (100) 277 (lo), 259 (6), 249 (lo), 231 (8), 221 (15), 207 (17), 193 (42) 177 (9) 167 (16), 166 (46) 165 (41), 149 (24), 141 (20) 139 (7) 99 (1 l), 77 (9), 65 (5) and 43 (33). For spectral data of helipyrone, see Opitz and Hansel (1970); homoarenol and arenol, see Vrkoc et al. (1970) and for italipyrone and plicatipyrone, see Hansel et al. (1980). Results Isolation and identification
CHCl,/EtOAc (6:4) and two active fractions were obtained. Fraction C was chromatographed on silica gel 60H using CHCls/MeOH (9:l) as the mobile phase, and three compounds were obtained (3, 4 and 5). From fraction E, two isolates (6 and 7) were obtained by column chromatography on silica gel 60H using CHCls/MeOH (96:4). Compounds 1 and 2 were identified as /3-sitoand fl-sitosteryl-3-&Osteryl-3-P-0-glucoside diglucoside by comparison with the spectral data of standard samples. Compound 3 was identified as 4-hydroxy-3(isopentent-2-yl)acetophenone by ‘H-NMR and MS and comparison with spectral data of an authentic sample. Compounds 2 and 3 had previously been identified in this same way (Martin Panizo et al., 1972; Garcia de Quesada et al., 1987). Compounds 4 and 5 were identified as italipyrone and plicatipyrone by comparison of their ‘H-NMR and MS data with bibliographic spectral data (Hansel et al., 1980). These compounds had been previously reported from H. italicum and H. plicatum.
The dichloromethane extract was the most active one in the antimicrobial screen, with an MIC of 250, 125 and 250 mg/l against Staphylococcus aureus, Mycobacterium phlei and Candida albicans, respectively (equivalent to 4.0, 2.0 and 4.0 g of dried plant/liter of culture medium). Bioautography of the active extract showed that the active compounds were probably phenolic compounds (substances with positive reaction to phenolic reagents in TLC). The extract was chromatographed and the fractions, eluted with mixtures of chloroform/ethyl acetate (95:5), showed antimicrobial activity against Gram-positive bacteria when the agar diffusion method was used. The anti-Candida activity disappeared when the total extract was fractionated (MIC > 100 mg/l). Bioautography of the three active fractions (F4, FS and Fb) showed that there were several different active principles present (compounds with R,, between 0.20 and 0.50 in 9: 1 CHCI,/EtOAc + 0.01% AcOH). The active fractions were concentrated and a precipitate was obtained. Two compounds were then separated from it and identified as &sitosteryl glycosides (1 and 2). The concentrated fraction was chromatographed on silica gel 60, eluted with
Isolates 6 and 7 were a mixture of related compounds. The MS of 6 (DC1 NHs+ and EIMS) showed a mixture of three compounds at a ratio of 1:6:12. A study of the 13C-NMR spectrum and MS made it possible for us to identify these as helipyrone (6a) and two related compounds. Compound 6a has been previously reported from H. italicum (Opitz et al, 1970) but 6c and 6b have not been reported to date. Isolate 7 is a mixture (1:8) of homoarenol (7a) and arenol (7b), compounds previously isolated from H. arenarium (Vrkoc et al., 1970). Thus, only 6b and 6c are new naturally occurring compounds. Antimicrobial
testing
The total chloroform extract had an MIC of 250 mg/l against Staphylococcus aureus and Candida albicans, and 125 mg/l against Mycobacterium phlei. After fractionation of the extract, the activity against Candida albicans disappeared. All the isolated compounds were assayed against representative Gram-positive and Gram-negative bacteria. None inhibited the growth of Gram-negative bacteria at 100 mg/l. All the isolates were active against Gram-positive bacteria when assayed
54 TABLE
I
ANTIMICROBIAL
ACTIVITY
OF ISOLATES MIC
Siuphylococcus
uurcus
S. c~pidiwnidis Mycohucrcriunt
phki
Eschc~richiu coli Klchsic4lu pncunwniuc~ Pseudomonus urruginow
Sulmonellu I.vplliniurium Cut&/u ulbicuns
STOECHAS
(mgl)
I Bucillus suhtilis
FROM HELICHRYSUM
2
3
>50 >50
>50 >50
>50 >50 >I00 >I00 > 100
>50 >50 >I00 >I00 >I00
>I00
>I00
>50
>50
using the agar diffusion method. The activity was assayed at concentrations of 50, 25, 12.5, 6.25 and 3.125 mg/l. The results obtained are summarized in Table 1. Discussion We have isolated the principles responsible for the antimicrobial activity of Hefichrysum stoechas previously reported (Rios et al, 1987). We have isolated five pure principles previously identified in related species. Two other isolates observed in this study are mixtures of helipyrone (6) and homoarenol (7) with related compounds. While homoarenol and helipyrone have been previously reported in other species, in H. sroechas they are only minor compounds. Compound 6c has not been reported previously in other species and is here characterized. Compound 6b has been identified by comparison of EM of the three substances. The antimicrobial activity of these compounds is reported here and the antimicrobial activity of H. stoechas is confirmed. Some researchers have described the antimicrobial activity of related compounds (Dekker et al, 1983; Tomas-Barberan et al., 1990) against yeast-like fungi and Gramnegative bacteria, but in the case of H. stoechas there was no activity observed against Gram-
4
5
6
7
>50
3
6
6
12
>50
6
12
6
25
>50
6
12
6
25
>50
3
6
6
> 100
> 100
> 100
> 100
>I00
>I00
>I00
>I00
> 100
> 100
>I00
> 100
> 100
> 100
> 100
> 100
> 100
> 100
>I00
>lOO
>50
>50
>50
I2
>50
>50
negative bacteria. There was only activity against Mycobacterium phlei and Gram-positive bacteria and only when the assayed compound was an alpha-pyrone derivative. The acetophenone derivative and P-sitosteryl derivatives showed only slight activity against Gram-positive bacteria when they were assayed by an agar diffusion method at higher concentrations. The relationship between polarity or number of hydroxyl groups and antimicrobial activity could not be confirmed, because 4 (3 hydroxyl groups) was most active with 6 (2 hydroxyl groups), more active than 5 (4 hydroxyl groups) and 7 (4 hydroxyl groups) least active. Acknowledgement This work was supported by Fundacion maceutica Avenzoar from Sevilla (Spain).
Far-
References Dekker.
T.G..
Fourie,
T.G..
Snyckers,
F.O.
Schyf. C.J. (1983) Studies of South African Part 2: Caespitin. timicrobial
a new phloroglucinol
properties from Helichr.wm~
A.fiicun Journul of Chcw1isrr.v 36. I 14-l Font Quer,
P. (1983)
derivative
Garcia de Quesada. T., Rodriguez.
Der
with an-
cuespiti/ium.
South
16.
Lus plun~us mdicinules.
renovado, 8th edn., Ed. Labor,
and Van
medicinal plants.
Barcelona,
El Dioscoricles pp. 783-785.
B. and Valverde.
S.
( 1972)
55 The constituents
of Helichrysum stoechas. Phyrochemistry
11,446449. Hansel, R., Cybulski. E.M., Qtbukcu. B.. Merich, A.H., Bohlmann, F. and Zdero, C. (1980) Neue pyron-derivate aus Helichrysum-arten. Phytochemisfry 19, 639-644. Martin-Panizo, F., Rodriguez, B., Valverde, S. and MartinLomas, M. (1972) Glucosidos esteroidales de1 Helichrysum stoechas (L.) DC. Anales de Quimica 68, 21 l-214. Opitz, L. and Hansel, R. (1970) Helipyron, ein methylen-bistriacetsiiurelacton aus Helichrysum italicurn. Tetrahedron Letters 59, 3369-3370.
Rios, J.L., Recio, M.C. and Villar, A. (1987) Antimicrobial activity of selected plants employed in the Spanish Mediterranean area. Journal of Ethnopharmacology 21, 139-I 52.
Rios, J.L., Recio, M.C. and Villar, A. (1988) Screening methods for natural products with antimicrobial activity: A review of the literature. Journal of Ethnopharmacology 23, 127-149.
Tomas-Barberan. F., Iniesta-Sanmartin, E.. Tomas-Lorente. F. and Rumbero, A. (1990) Antimicrobial phenolic compounds from three Spanish Helichrysum species. Phyrochemistry 29, 1093-1095.
Vrkoc, J., Doleya, L., Sedmera, P., Vasickowa, S. and Sorm, F. (1970) The structure of arenol and homoarenol, o-pyrone derivatives from Helichrysum arenarium (L.) Moench. Teirahedron Letters 59, 247-250.
51
33 (1991) 51-55
Elsevier Scientific Publishers Ireland Ltd.
Isolation and identification of the antibacterial from Helichrysum stoechas
compounds
J.L. Rios”, M.C. Recioa and A. Villarb “Farmacognosia i Farmacodinamia, Deparramento de Farmacologia i FarmacotPcnia. Facultat de Farmacia, Vniversitar de Vakncia. Avda. Blasco Ibdfiez 13, 46010-Vakncia and hFarmacognosia y Farmacodinamia, Departamento de Farmacologia, Fact&ad de Farmaria. Vniversidad Complurense de Madrid, Ciudad Vniversitaria. 28040 Madrid (Spain)
(Accepted November 6, 1990) Fractionation of the dicholoromethane extract of the aerial parts of Helichrysum stoechas yielded seven isolates (l-7), which exhibited varying antimicrobial activity against Gram-positive bacteria. Pure compounds 1-3 have been previously reported in the same species and 4 and 5 were identified as italipyrone and plicatipyrone, previously isolated from H. iralicum and H. plicarum. The two other isolates are helipyrone (6) and homoarenol (7) mixed with related substances. Compound 6e (4,4’-dihydroxy-5,6,5’,6’tetramethyl-3,3’-methylen-di-pyr-2-one) and 6b helipyrone with one methyl substitution, have not been previously reported in other species. Key words: Helichrysum sfoechas: antibacterial activity: pyrone derivatives; phloroglucinois.
Introduction
Methods
Helichrysum stoechas (L.) Moench is a shrub with yellow flowers that grows widely in the Mediterranean area. This species is considered antimicrobial (Rios et al., 1987), antiinflammatory and digestive (Font Quer, 1983). Previous studies on its essential oil have been reported, and several phytochemical compounds have been isolated and identified as hydroxyisopenten-acetophenone and related compounds, triterpenoids, steroids (free and glycosidic forms) and a lipid fraction (Garcia de Quesada et al., 1972; Martin-Panizo et al., 1972). Some phloroglucinol derivatives of this species (dimethylallylitalipyrone and plicatipyrone) have been reported by Hgnsel et al., 1980. Our laboratory recently established the antimicrobial activity of the dichloromethane extract from this species (Rios et al., 1987).
Plant material
Correspondence 10: J.L. Rios Caiidwte. Farmacognosia i Farmacodinamia, Departament de Farmacologia i Farmacotkcnia.
Facultat de Farmacia, Universitat de Vakncia. Avda. Blasco IbPiiez 13, 46010-Vakncia. Spain. 037%8741/$03.50 0 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
The aerial parts of Helichrysum stoechas were collected at flowering in Sierra Perenchiza (Chiva, Vall?ncia, Spain) and a voucher specimen was deposited in the herbarium of the Departamento de Biologia Vegetal, Facultad de Farmacia, Universidad de Valkncia, Spain. Extraction and isolation Dried powdered plant (700 g) was successively extracted by maceration with dichloromethane and methanol. The extracted liquids were concentrated at vacuum and the extracts assayed against different bacteria and a yeast. The active dichloromethane extract was chromatographed on silica gel 60 (Merck) with chloroform/methanol mixtures of increasing polarity. Elution was followed by TLC and the antimicrobial activities of the fractions were assayed by an agar diffusion method (Rios et al., 1988) and the active fractions identified by TLC and bioautographic methods
52
(Rios et al., 1988). The active fractions were chromatographed by column chromatography on silica gel 60H (Merck) with chloroform/methanol as the mobile phase. Compounds were purified by precipitation and preparative-TLC and antimicrobial activities followed by an agar dilution method (Rios et al., 1988). Antimicrobiul
MIC (minimum inhibitory concentration) was determined by serial two-fold dilutions in the same type of agar, with concentrations ranging from 1 to 50 mg/l (Rios et al., 1988). The inoculum was prepared from a fresh overnight broth culture in trypticase soy broth (Difco) and adjusted to an approximate concentration of lo6 colony-forming units/spot. Plates were incubated for 18 h at 35°C. The MIC was detined as the lowest concentration of compounds that inhibited development of visible growth on agar.
testing
The antimicrobial activities were evaluated by the classic agar diffusion procedure using MuellerHinton agar (Difco) (Rios et al., 1988). Samples were diluted in methanol (2.5 mg/l) using 20 ~1 of each sample solution. The Petri dishes were cooled at 4°C for 2 h before incubation at 35.5”C. The
Spectrul
data
Structural elucidations (Fig. 1) were made by UV spectra (Pye-Unicam), IR spectra (Hitachi Perkin Elmer), ‘H-NMR and “C-NMR (Bruker
@-Sitosterol-/3-O-glucosides I: R = glucose
3: 4-Hydroxy-3(isopenten-2-yl)
2: R = glucose-glucose
acetophenone
A0 5: Plicatipyrone
4: ltalipyrone
6s: Helipyrone 6b: R, = -CH,.
RI = R, = -CH,-CH, R, = -CH,-CH,
6~: R, = R, = -CH, Fig.
I.
Isolates from Hdiclwy.surn s/orbs
7a: Homoarenol 7b: Arenol aerial parts
R = -CH,-CHJ
R = -CH,
53
WP 200 and Varian XL 300) EIMS (Mass spectrometer VG 12-250 Massland), EIMS and DCIMS NH3, positive mode (Nermag R103G spectrometer). Compound 6c (4,4’-dihydroxy-5,6,5’,6’-tetramethyl-3,3’methylen-di-pyr-2-one). 13C-NMR: 174.3 (C=O), 110.2, 166.3 (C-OH), 100.2, 154.6, 10.0 (CH,-C), 16.9 (CH3-C-O) and 18.8 (CH2-). ‘H-NMR: s, 1.78 (s, 6H), 2.09 (s, 6H), 3.36 (s, 2H). EIMS m/z (rel. int.): 292 (100) 277 (lo), 259 (6), 249 (lo), 231 (8), 221 (15), 207 (17), 193 (42) 177 (9) 167 (16), 166 (46) 165 (41), 149 (24), 141 (20) 139 (7) 99 (1 l), 77 (9), 65 (5) and 43 (33). For spectral data of helipyrone, see Opitz and Hansel (1970); homoarenol and arenol, see Vrkoc et al. (1970) and for italipyrone and plicatipyrone, see Hansel et al. (1980). Results Isolation and identification
CHCl,/EtOAc (6:4) and two active fractions were obtained. Fraction C was chromatographed on silica gel 60H using CHCls/MeOH (9:l) as the mobile phase, and three compounds were obtained (3, 4 and 5). From fraction E, two isolates (6 and 7) were obtained by column chromatography on silica gel 60H using CHCls/MeOH (96:4). Compounds 1 and 2 were identified as /3-sitoand fl-sitosteryl-3-&Osteryl-3-P-0-glucoside diglucoside by comparison with the spectral data of standard samples. Compound 3 was identified as 4-hydroxy-3(isopentent-2-yl)acetophenone by ‘H-NMR and MS and comparison with spectral data of an authentic sample. Compounds 2 and 3 had previously been identified in this same way (Martin Panizo et al., 1972; Garcia de Quesada et al., 1987). Compounds 4 and 5 were identified as italipyrone and plicatipyrone by comparison of their ‘H-NMR and MS data with bibliographic spectral data (Hansel et al., 1980). These compounds had been previously reported from H. italicum and H. plicatum.
The dichloromethane extract was the most active one in the antimicrobial screen, with an MIC of 250, 125 and 250 mg/l against Staphylococcus aureus, Mycobacterium phlei and Candida albicans, respectively (equivalent to 4.0, 2.0 and 4.0 g of dried plant/liter of culture medium). Bioautography of the active extract showed that the active compounds were probably phenolic compounds (substances with positive reaction to phenolic reagents in TLC). The extract was chromatographed and the fractions, eluted with mixtures of chloroform/ethyl acetate (95:5), showed antimicrobial activity against Gram-positive bacteria when the agar diffusion method was used. The anti-Candida activity disappeared when the total extract was fractionated (MIC > 100 mg/l). Bioautography of the three active fractions (F4, FS and Fb) showed that there were several different active principles present (compounds with R,, between 0.20 and 0.50 in 9: 1 CHCI,/EtOAc + 0.01% AcOH). The active fractions were concentrated and a precipitate was obtained. Two compounds were then separated from it and identified as &sitosteryl glycosides (1 and 2). The concentrated fraction was chromatographed on silica gel 60, eluted with
Isolates 6 and 7 were a mixture of related compounds. The MS of 6 (DC1 NHs+ and EIMS) showed a mixture of three compounds at a ratio of 1:6:12. A study of the 13C-NMR spectrum and MS made it possible for us to identify these as helipyrone (6a) and two related compounds. Compound 6a has been previously reported from H. italicum (Opitz et al, 1970) but 6c and 6b have not been reported to date. Isolate 7 is a mixture (1:8) of homoarenol (7a) and arenol (7b), compounds previously isolated from H. arenarium (Vrkoc et al., 1970). Thus, only 6b and 6c are new naturally occurring compounds. Antimicrobial
testing
The total chloroform extract had an MIC of 250 mg/l against Staphylococcus aureus and Candida albicans, and 125 mg/l against Mycobacterium phlei. After fractionation of the extract, the activity against Candida albicans disappeared. All the isolated compounds were assayed against representative Gram-positive and Gram-negative bacteria. None inhibited the growth of Gram-negative bacteria at 100 mg/l. All the isolates were active against Gram-positive bacteria when assayed
54 TABLE
I
ANTIMICROBIAL
ACTIVITY
OF ISOLATES MIC
Siuphylococcus
uurcus
S. c~pidiwnidis Mycohucrcriunt
phki
Eschc~richiu coli Klchsic4lu pncunwniuc~ Pseudomonus urruginow
Sulmonellu I.vplliniurium Cut&/u ulbicuns
STOECHAS
(mgl)
I Bucillus suhtilis
FROM HELICHRYSUM
2
3
>50 >50
>50 >50
>50 >50 >I00 >I00 > 100
>50 >50 >I00 >I00 >I00
>I00
>I00
>50
>50
using the agar diffusion method. The activity was assayed at concentrations of 50, 25, 12.5, 6.25 and 3.125 mg/l. The results obtained are summarized in Table 1. Discussion We have isolated the principles responsible for the antimicrobial activity of Hefichrysum stoechas previously reported (Rios et al, 1987). We have isolated five pure principles previously identified in related species. Two other isolates observed in this study are mixtures of helipyrone (6) and homoarenol (7) with related compounds. While homoarenol and helipyrone have been previously reported in other species, in H. sroechas they are only minor compounds. Compound 6c has not been reported previously in other species and is here characterized. Compound 6b has been identified by comparison of EM of the three substances. The antimicrobial activity of these compounds is reported here and the antimicrobial activity of H. stoechas is confirmed. Some researchers have described the antimicrobial activity of related compounds (Dekker et al, 1983; Tomas-Barberan et al., 1990) against yeast-like fungi and Gramnegative bacteria, but in the case of H. stoechas there was no activity observed against Gram-
4
5
6
7
>50
3
6
6
12
>50
6
12
6
25
>50
6
12
6
25
>50
3
6
6
> 100
> 100
> 100
> 100
>I00
>I00
>I00
>I00
> 100
> 100
>I00
> 100
> 100
> 100
> 100
> 100
> 100
> 100
>I00
>lOO
>50
>50
>50
I2
>50
>50
negative bacteria. There was only activity against Mycobacterium phlei and Gram-positive bacteria and only when the assayed compound was an alpha-pyrone derivative. The acetophenone derivative and P-sitosteryl derivatives showed only slight activity against Gram-positive bacteria when they were assayed by an agar diffusion method at higher concentrations. The relationship between polarity or number of hydroxyl groups and antimicrobial activity could not be confirmed, because 4 (3 hydroxyl groups) was most active with 6 (2 hydroxyl groups), more active than 5 (4 hydroxyl groups) and 7 (4 hydroxyl groups) least active. Acknowledgement This work was supported by Fundacion maceutica Avenzoar from Sevilla (Spain).
Far-
References Dekker.
T.G..
Fourie,
T.G..
Snyckers,
F.O.
Schyf. C.J. (1983) Studies of South African Part 2: Caespitin. timicrobial
a new phloroglucinol
properties from Helichr.wm~
A.fiicun Journul of Chcw1isrr.v 36. I 14-l Font Quer,
P. (1983)
derivative
Garcia de Quesada. T., Rodriguez.
Der
with an-
cuespiti/ium.
South
16.
Lus plun~us mdicinules.
renovado, 8th edn., Ed. Labor,
and Van
medicinal plants.
Barcelona,
El Dioscoricles pp. 783-785.
B. and Valverde.
S.
( 1972)
55 The constituents
of Helichrysum stoechas. Phyrochemistry
11,446449. Hansel, R., Cybulski. E.M., Qtbukcu. B.. Merich, A.H., Bohlmann, F. and Zdero, C. (1980) Neue pyron-derivate aus Helichrysum-arten. Phytochemisfry 19, 639-644. Martin-Panizo, F., Rodriguez, B., Valverde, S. and MartinLomas, M. (1972) Glucosidos esteroidales de1 Helichrysum stoechas (L.) DC. Anales de Quimica 68, 21 l-214. Opitz, L. and Hansel, R. (1970) Helipyron, ein methylen-bistriacetsiiurelacton aus Helichrysum italicurn. Tetrahedron Letters 59, 3369-3370.
Rios, J.L., Recio, M.C. and Villar, A. (1987) Antimicrobial activity of selected plants employed in the Spanish Mediterranean area. Journal of Ethnopharmacology 21, 139-I 52.
Rios, J.L., Recio, M.C. and Villar, A. (1988) Screening methods for natural products with antimicrobial activity: A review of the literature. Journal of Ethnopharmacology 23, 127-149.
Tomas-Barberan. F., Iniesta-Sanmartin, E.. Tomas-Lorente. F. and Rumbero, A. (1990) Antimicrobial phenolic compounds from three Spanish Helichrysum species. Phyrochemistry 29, 1093-1095.
Vrkoc, J., Doleya, L., Sedmera, P., Vasickowa, S. and Sorm, F. (1970) The structure of arenol and homoarenol, o-pyrone derivatives from Helichrysum arenarium (L.) Moench. Teirahedron Letters 59, 247-250.
