187
Journal of Ethnopharmacology, 33 (1991) 187-191 Elsevier Scientific Publishers Ireland Ltd.
The antimicrobial activity of the essential oil from Achilles fragran tissima S. Barela, R. Segalb and J. Yashphe” UDepartment of Bacteriology, Faculty of Medicine and hDepartmenr of Natural Products, School of Pharmacy The Hebrew University. Jerusalem (Israel) (Accepted
August
14. 1990)
Essential oil from AchiNea fragranrissima exerted a bactericidic effect on several gram positive and gram negative bacterial strains, as well as on Candida a/&cans. The oil was fractionated on sillica gel columns by a gradient of ether in petrol ether (3O”C~“C). Two fractions which contained less polar compounds were active against C. alhicans only. The fractions which contained more polar compounds inhibited the growth of all the microorganisms tested. One of these compounds was identified as terpinen4-01. Commercial terpinen-4-01
had a similar
Key words: antimicrobial
antimicrobial
activity;
essential
activity oil; Achilles fragranfi.ssima.
Introduction
flowering season (first half of June), and were identified by Dr. Avinoam Danin from the Department of Botany, The Hebrew University, It was dried at room Jerusalem, Israel. temperature, and its leaves, flowers and stalks were ground in a blender. The essential oil was extracted from the ground plant material by steam distilation. The upper phase, which contained the oil, was separated from the lower one and dried with CaCl*. After filtration, it was stored at -10°C in the dark.
Various species of Achilles have been used in folk remedies for treatment of many illnesses (Duke 1987). The best known of these plants is A. milltfolium (Chandler, 1982). A. fragrantissima is a common plant in southern Israel and is used by the Beduins in the Negev (Southern Israel) for the preparation of antidiuretic drinks and for the treatment of stomach ailments (Flitman et al., 1983). In the present paper we examined the antimicrobial activity of the essential oil which was extracted from A. fragrantissima and tried to isolate the compound(s) which exerted this activity.
Microbial strains and growth conditions The strains used were: Escherichia coli K-12, Stahylococcus aureus, Salmonella typhosa, Shigella sonnei, Streptococcus hemolyticus, Klebsiella pneumoniae. Pseudomonas aeruginosa, Streptococcus faecalis and Candida albicans. The bacterial strains were grown on brain heart infusion broth (Difco) at 37°C harvested at the logarithmic phase of growth (AbOO O.D. 0.5), and suspended in saline. C. albicans was grown at 30°C on YEPD medium which contained 0.25% yeast extract, 1% peptone and 4% dextrose pH 6.8. For the preparation of solid media 1.5% agar-agar (Difco) was added to the aforementioned liquid media.
Materials and Methods Extraction of the essential oil Achilles fragrantissima (Forsk) Sch. Bip. plants were collected from the Negev area of Isreal at its
Correspondence to: J. Yashphe, Department Faculty of Medicine, The Hebrew University,
0378-8741/$03.50 0 1991 Elsevier Published and Printed in Ireland
Scientific
of Bacteriology, Jerusalem, Israel.
Publishers
Ireland
Ltd
188
Agar diffusion test A suspension of the tested microorganism (0.1 ml of lo* cells per ml) was spread on the solid media plates. Paper disks (6 mm diameter) which contained 10 ~1 of the essential oil were placed on the inoculated plates. These plates, after staying at 5°C for 2 h, were incubated over night at 37°C for detecting growth of the bacterial strains or at 30°C for detecting growth of the Candida. The diameters of the inhibition zones around the paper disks were measured. Estimation
of the minimal
inhibitory
Gas chromatography
(CC)
and mass specfrograph~
(MS) A combined GC-MS instrument (LKB 2091) was used for the identification of the components of the most active fraction. The specification of the column in the instrument were: 2 m x 0.2 cm, S.S. lot No. 3-59 3% OV-17 on chromosorb WHP. The essential oil was injected at 55°C and after 2 min the temperature was raised at a rate of 5°C per min to 200°C. The patterns obtained were compared to published ones and to those obtained for standard compounds.
concentration
(MIC) These estimations were carried out in the liquid media which contained decreasing amounts of the essential oil. For better dispersion of the oil in the growth medium, it was mixed with 10% Tween 80, and added to the medium to a final concentration of 1% Tween 80. After mixing vigorously, 0.1 ml of the cell suspension (lo8 cells per ml) was added, and the cultures were incubated at the appropriate temperatures for 24 h. Growth was estimated by measuring the turbidity of the culture at 600 nm. Control experiments have shown that 1% Tween 80 did not inhibit growth. Estimation of the number of the viable cells The estimation was done by diluting the cultures at the end of the experiment with saline and spreading 0.1 ml on the solid media. After 24 h at the appropriate temperatures, the number of colonies on each plate was counted, and the number of the viable cells in the cultures was calculated. Fractionation of the essential oil The fractionation was carried out on columns of sillica gel 60 mesh 230 with a gradient of ether in petrolether (3O”C--4O”C). Fractions of 20 ml were collected, and the composition of each was examined on sillica gel TLC plates with petroletheriether (75:25) as the developer. The compounds on the TLC plates were detected by spraying with a mixture of anisaldehyde (2.1 ml) and phosphomolibdic acid in 5% methanol (5 ml), and heating at 150°C. Fractions which showed similar patterns were pooled and the solvents were evaporated by heating on a water bath and by flushing through nitrogen.
Results Examination of the antimicrobial activity of the oil by the agar diffusion method Table 1 shows that the essential oil extracted from A. fragrantissima inhibited the growth of the microorganisms tested. It was especially active against Staph. aureus, Strep. hemolyticus, Sal. typhosa and C. albicans. Determination of the minimal inhibitory concentration (MIC) The range in which total inhibition of growth was observed extended from the concentration of 0.85 to about 2 mg essential oil per ml (Table 2). The lowest MIC values were obtained for C. albicans, Staph. aureus and Sal. typhosa. The
TABLE
I
THE EFFECT OF A. FRAGRANTISSIMA ESSENTIAL OIL ON THE GROWTH OF VARIOUS MICROORGANISMS Microorganism
Inhibition
E. coli Sal. typhosu
22 30
Shig. sonnei Ps. arruginosu Kleb. pneumoniae Strep. feacalis Strep. hemolyticus Staph. aureus C. ulbicans
I5 17 12 20 32 35 35
diameter
“The agar diffusion method used is described under and Methods. bIncludes the diameters of the paper disk (6 mm). ‘Each disk contained 8.5 mg essential oil.
(mm)“,c
Materials
189
TABLE
2
MINIMAL INHIBITORY CONCENTRATION THE A. FRAGRANTLSSIMA OIL FOR MICROORGANISMS Minimal
Microorganism
(MIC) OF VARIOUS
oil concentration”
(mgml)
bactericidic effect on all the microorganims tested. C. albicans appeared to be the most sensitive organism. A complete loss of its viability was observed at the concentration of 1 mg per ml. For the other strains concentrations of 2-3 mg per ml were required to obtain similar results.
E. coli K-12 Sal. typhosa
1.80 1.00
Fractionation of the essential oil and examination the antibacterial activity of the fraction
Shig. sonnai Ps. aeruginosa Kleb. pneumoniae Strep. faecalis
1.80 1.80 1.90 1.50 1.10 I .20 0.85
The essential oil was fractionated by a silica1 gel column into 8 fractions, as indicated in Fig. 2. Table 3 shows that the less polar fractions which were eluted first (Fractions l-5) did not inhibit the growth of the bacterial strains. However, the growth of C. albicans was inhibited by fractions 2 and 3. The polar (probably alcoholic) fractions 6, 7, 8 inhibited all the bacterial strains while C. albicans was inhibited only by fractions 6 and 7. Fraction number 7 possessed the highest antimicrobial activity. This fraction contained about 15% of the essential oil, Analysis of this fraction by gas chromatography revealed five compounds. One of them was identified by gas chromatography and mass spectrography as terpinen-4-01. The other components of this fraction appeared to be oxydized monoterpens. Table 4 confirms the antimicrobial activity of commercial pure terpinen4-01.
Strep. hemolyticus Staph. aureus C. albicans “The minimal growth.
concentration
which
gave
100% inhibition
of
highest values were obtained for Kleb. pneumonia, Ps. aeruginosa, E. coli and Shig. sonnei. Examination
Figure
of the bactericidic
effect of the oil
1 shows that the essential oil had a
of
Discussion
1 2 3 ACHILLEA OIL (mg/ml)
4
Fig. 1. The effect of A. fiagrantissima oil on the viability of The cells were grown to the various microorganisms. logarithmic phase, incubated with essential oil at 37°C for 30 mitt, and the number of viable cells was estimated as described under Materials and Methods. v , Strep. hemolyticus; q , Sal. typhosa; A Shig. sonnei; n . Staph. aureus; o , Strep. feacalis; o , E. co/i: +, Ps. aeruginosa; l , C. albicans.
The data presented indicate that the essential oil from A. fragrantissima inhibits the grwoth of the tested microorganism to various degrees. C. albicans appeared to be the most sensitive organism. Most of the Gram-negative bacterial strains were less sensitive than the Gram-positive ones. This is specially rue for Ps. aeruginosa and Kleb. pneumoniae. The sensitivity of Sal. typhosa was similar to that of the Gram-positive strains. The essential oil had a bactericidic effect on all the strains tested. In most cases the MIC values obtained from optical density measurements were close to the concentrations required for a total loss of viability (Table 2, Fig. 1). However, Sal. typhosa, Strep. faecalis and Staph. aureus required a much higher concentration of essential oil to obtain a full bactericidic effect. The essential oils are a mixture of a number of
Fig. 2. TLC separation of the A. frugrantissima oil and its fractions. The details of the sillica gel fractionation are described under Materials and Methods. The fraction number is indicated on the top. Samples from the essential oil (first row at the right) and its fractions were applied to sillica gel TLC plates and were subjected to ascending chromatography as described under Materials and Methods.
volatile compounds, which are mainly terpenoids (Harborne, 1973). The composition of the essential oil of A. millifolium has been analyzed and studied extensively. Baside mono- and sesquiterpene lac-
TABLE
3
AGAR DIFFUSION TEST OF ACTIVITY OF THE VARIOUS ESSENTIAL OIL
THE ANTIMICROBIAL FRACTIONS OF THE
Fraction No.
zone”.b (mm)
Diameter
of inhibition
tones it contains azulenes and some other compounds (Duke, 1987). Shalaby and Richter (1964) studied the composition of the essential oil from A. fragrantissima, which was collected in Egypt and is actually identical to the plant studied in this paper. They did not find azulene, proazulen, aldehydes or acids, but detected terpene hydrocarbons, esters, ketones, phenols and alcohols. While their
TABLE
4
EFFECT VARIOUS
OF TERPINEN-4-OL” MICROORGANISMS
Sal.
E.
Staph.
C.
typhosa
coli
aweus
albicans
I
N.1.C
N.1.
N.I.
N.I.
2
N.I.
N.I.
N.I.
N.I.
E. coli
38
3
N.I.
N.I.
N.I.
N.I.
Sal. typhosa
47
4
NJ.
N.I.
N.I.
N.I.
Shig. sonnei
45
5
N.I.
N.I.
N.I.
N.I.
Staph. aweus
25
6
22
I7
20
12
C. albicans
T.I.4
7
25
20
21
20
8
12
15
25
N.I.
Microorganism
“Includes the diameter of the paper disk (6 mm). bEach disk contained about 8 mg of the fraction. ‘No inhibition
of growth.
“Analytical blncluding
Inhibition
ON
GROWTH
diameter
OF
(mm)b,c
grade, produced by Aldrich Chemical Co. the diameter of the paper disk (6 mm).
‘Each disk contained 4 mg terpinen-4-01. dGrowth was inhibited all over the plate. resistent mutants were observed.
Some colonies
of
191
alcoholic fraction may correspond to fractions 6, 7, 8 (Fig. l), which contain the more polar comit does not contain terpinen-4-01. pounds, However, the latter is present in the A. millifolium essential oil (Chandler et al., 1982). These fractions, and especially terpinen-4-01 demonstrated a significant antimicrobial activity. The observation that fractions 2 and 3 which contain less polar compounds ihibited only the growth of C. albicans may be of importance. These fractions may contain compounds which act only on eukaryotic cells.
References Chandler, R.F., Hooper, S.N. and Harvey, N.J. (1982) Ethnobotany and phytochemistry of Yarrow, Achilles millifolium, compositae. Economic Botany 36(2), 203-215. Duke, J.A. (1987) Handbook of Medical Herbs. CRC Press, Boca Raton, FL. Flitman, U., Hen, C., Darin, A. and Shmida, A. (1983) Pictorial Flora of Israel. Massada Ltd. Tel-Aviv, p. 204. Harborn, J.B. (1973). Essential oils. In: Phytochemical Methods. Chapman and Hall, London, pp. 92-103. Shalaby, A.F. and Richter, G. (1964). Chromatographic investigation of the essential oil from Achilles fragrantissima. Journal of Pharmaceutical Sciences 53: 1502-l 505.
Journal of Ethnopharmacology, 33 (1991) 187-191 Elsevier Scientific Publishers Ireland Ltd.
The antimicrobial activity of the essential oil from Achilles fragran tissima S. Barela, R. Segalb and J. Yashphe” UDepartment of Bacteriology, Faculty of Medicine and hDepartmenr of Natural Products, School of Pharmacy The Hebrew University. Jerusalem (Israel) (Accepted
August
14. 1990)
Essential oil from AchiNea fragranrissima exerted a bactericidic effect on several gram positive and gram negative bacterial strains, as well as on Candida a/&cans. The oil was fractionated on sillica gel columns by a gradient of ether in petrol ether (3O”C~“C). Two fractions which contained less polar compounds were active against C. alhicans only. The fractions which contained more polar compounds inhibited the growth of all the microorganisms tested. One of these compounds was identified as terpinen4-01. Commercial terpinen-4-01
had a similar
Key words: antimicrobial
antimicrobial
activity;
essential
activity oil; Achilles fragranfi.ssima.
Introduction
flowering season (first half of June), and were identified by Dr. Avinoam Danin from the Department of Botany, The Hebrew University, It was dried at room Jerusalem, Israel. temperature, and its leaves, flowers and stalks were ground in a blender. The essential oil was extracted from the ground plant material by steam distilation. The upper phase, which contained the oil, was separated from the lower one and dried with CaCl*. After filtration, it was stored at -10°C in the dark.
Various species of Achilles have been used in folk remedies for treatment of many illnesses (Duke 1987). The best known of these plants is A. milltfolium (Chandler, 1982). A. fragrantissima is a common plant in southern Israel and is used by the Beduins in the Negev (Southern Israel) for the preparation of antidiuretic drinks and for the treatment of stomach ailments (Flitman et al., 1983). In the present paper we examined the antimicrobial activity of the essential oil which was extracted from A. fragrantissima and tried to isolate the compound(s) which exerted this activity.
Microbial strains and growth conditions The strains used were: Escherichia coli K-12, Stahylococcus aureus, Salmonella typhosa, Shigella sonnei, Streptococcus hemolyticus, Klebsiella pneumoniae. Pseudomonas aeruginosa, Streptococcus faecalis and Candida albicans. The bacterial strains were grown on brain heart infusion broth (Difco) at 37°C harvested at the logarithmic phase of growth (AbOO O.D. 0.5), and suspended in saline. C. albicans was grown at 30°C on YEPD medium which contained 0.25% yeast extract, 1% peptone and 4% dextrose pH 6.8. For the preparation of solid media 1.5% agar-agar (Difco) was added to the aforementioned liquid media.
Materials and Methods Extraction of the essential oil Achilles fragrantissima (Forsk) Sch. Bip. plants were collected from the Negev area of Isreal at its
Correspondence to: J. Yashphe, Department Faculty of Medicine, The Hebrew University,
0378-8741/$03.50 0 1991 Elsevier Published and Printed in Ireland
Scientific
of Bacteriology, Jerusalem, Israel.
Publishers
Ireland
Ltd
188
Agar diffusion test A suspension of the tested microorganism (0.1 ml of lo* cells per ml) was spread on the solid media plates. Paper disks (6 mm diameter) which contained 10 ~1 of the essential oil were placed on the inoculated plates. These plates, after staying at 5°C for 2 h, were incubated over night at 37°C for detecting growth of the bacterial strains or at 30°C for detecting growth of the Candida. The diameters of the inhibition zones around the paper disks were measured. Estimation
of the minimal
inhibitory
Gas chromatography
(CC)
and mass specfrograph~
(MS) A combined GC-MS instrument (LKB 2091) was used for the identification of the components of the most active fraction. The specification of the column in the instrument were: 2 m x 0.2 cm, S.S. lot No. 3-59 3% OV-17 on chromosorb WHP. The essential oil was injected at 55°C and after 2 min the temperature was raised at a rate of 5°C per min to 200°C. The patterns obtained were compared to published ones and to those obtained for standard compounds.
concentration
(MIC) These estimations were carried out in the liquid media which contained decreasing amounts of the essential oil. For better dispersion of the oil in the growth medium, it was mixed with 10% Tween 80, and added to the medium to a final concentration of 1% Tween 80. After mixing vigorously, 0.1 ml of the cell suspension (lo8 cells per ml) was added, and the cultures were incubated at the appropriate temperatures for 24 h. Growth was estimated by measuring the turbidity of the culture at 600 nm. Control experiments have shown that 1% Tween 80 did not inhibit growth. Estimation of the number of the viable cells The estimation was done by diluting the cultures at the end of the experiment with saline and spreading 0.1 ml on the solid media. After 24 h at the appropriate temperatures, the number of colonies on each plate was counted, and the number of the viable cells in the cultures was calculated. Fractionation of the essential oil The fractionation was carried out on columns of sillica gel 60 mesh 230 with a gradient of ether in petrolether (3O”C--4O”C). Fractions of 20 ml were collected, and the composition of each was examined on sillica gel TLC plates with petroletheriether (75:25) as the developer. The compounds on the TLC plates were detected by spraying with a mixture of anisaldehyde (2.1 ml) and phosphomolibdic acid in 5% methanol (5 ml), and heating at 150°C. Fractions which showed similar patterns were pooled and the solvents were evaporated by heating on a water bath and by flushing through nitrogen.
Results Examination of the antimicrobial activity of the oil by the agar diffusion method Table 1 shows that the essential oil extracted from A. fragrantissima inhibited the growth of the microorganisms tested. It was especially active against Staph. aureus, Strep. hemolyticus, Sal. typhosa and C. albicans. Determination of the minimal inhibitory concentration (MIC) The range in which total inhibition of growth was observed extended from the concentration of 0.85 to about 2 mg essential oil per ml (Table 2). The lowest MIC values were obtained for C. albicans, Staph. aureus and Sal. typhosa. The
TABLE
I
THE EFFECT OF A. FRAGRANTISSIMA ESSENTIAL OIL ON THE GROWTH OF VARIOUS MICROORGANISMS Microorganism
Inhibition
E. coli Sal. typhosu
22 30
Shig. sonnei Ps. arruginosu Kleb. pneumoniae Strep. feacalis Strep. hemolyticus Staph. aureus C. ulbicans
I5 17 12 20 32 35 35
diameter
“The agar diffusion method used is described under and Methods. bIncludes the diameters of the paper disk (6 mm). ‘Each disk contained 8.5 mg essential oil.
(mm)“,c
Materials
189
TABLE
2
MINIMAL INHIBITORY CONCENTRATION THE A. FRAGRANTLSSIMA OIL FOR MICROORGANISMS Minimal
Microorganism
(MIC) OF VARIOUS
oil concentration”
(mgml)
bactericidic effect on all the microorganims tested. C. albicans appeared to be the most sensitive organism. A complete loss of its viability was observed at the concentration of 1 mg per ml. For the other strains concentrations of 2-3 mg per ml were required to obtain similar results.
E. coli K-12 Sal. typhosa
1.80 1.00
Fractionation of the essential oil and examination the antibacterial activity of the fraction
Shig. sonnai Ps. aeruginosa Kleb. pneumoniae Strep. faecalis
1.80 1.80 1.90 1.50 1.10 I .20 0.85
The essential oil was fractionated by a silica1 gel column into 8 fractions, as indicated in Fig. 2. Table 3 shows that the less polar fractions which were eluted first (Fractions l-5) did not inhibit the growth of the bacterial strains. However, the growth of C. albicans was inhibited by fractions 2 and 3. The polar (probably alcoholic) fractions 6, 7, 8 inhibited all the bacterial strains while C. albicans was inhibited only by fractions 6 and 7. Fraction number 7 possessed the highest antimicrobial activity. This fraction contained about 15% of the essential oil, Analysis of this fraction by gas chromatography revealed five compounds. One of them was identified by gas chromatography and mass spectrography as terpinen-4-01. The other components of this fraction appeared to be oxydized monoterpens. Table 4 confirms the antimicrobial activity of commercial pure terpinen4-01.
Strep. hemolyticus Staph. aureus C. albicans “The minimal growth.
concentration
which
gave
100% inhibition
of
highest values were obtained for Kleb. pneumonia, Ps. aeruginosa, E. coli and Shig. sonnei. Examination
Figure
of the bactericidic
effect of the oil
1 shows that the essential oil had a
of
Discussion
1 2 3 ACHILLEA OIL (mg/ml)
4
Fig. 1. The effect of A. fiagrantissima oil on the viability of The cells were grown to the various microorganisms. logarithmic phase, incubated with essential oil at 37°C for 30 mitt, and the number of viable cells was estimated as described under Materials and Methods. v , Strep. hemolyticus; q , Sal. typhosa; A Shig. sonnei; n . Staph. aureus; o , Strep. feacalis; o , E. co/i: +, Ps. aeruginosa; l , C. albicans.
The data presented indicate that the essential oil from A. fragrantissima inhibits the grwoth of the tested microorganism to various degrees. C. albicans appeared to be the most sensitive organism. Most of the Gram-negative bacterial strains were less sensitive than the Gram-positive ones. This is specially rue for Ps. aeruginosa and Kleb. pneumoniae. The sensitivity of Sal. typhosa was similar to that of the Gram-positive strains. The essential oil had a bactericidic effect on all the strains tested. In most cases the MIC values obtained from optical density measurements were close to the concentrations required for a total loss of viability (Table 2, Fig. 1). However, Sal. typhosa, Strep. faecalis and Staph. aureus required a much higher concentration of essential oil to obtain a full bactericidic effect. The essential oils are a mixture of a number of
Fig. 2. TLC separation of the A. frugrantissima oil and its fractions. The details of the sillica gel fractionation are described under Materials and Methods. The fraction number is indicated on the top. Samples from the essential oil (first row at the right) and its fractions were applied to sillica gel TLC plates and were subjected to ascending chromatography as described under Materials and Methods.
volatile compounds, which are mainly terpenoids (Harborne, 1973). The composition of the essential oil of A. millifolium has been analyzed and studied extensively. Baside mono- and sesquiterpene lac-
TABLE
3
AGAR DIFFUSION TEST OF ACTIVITY OF THE VARIOUS ESSENTIAL OIL
THE ANTIMICROBIAL FRACTIONS OF THE
Fraction No.
zone”.b (mm)
Diameter
of inhibition
tones it contains azulenes and some other compounds (Duke, 1987). Shalaby and Richter (1964) studied the composition of the essential oil from A. fragrantissima, which was collected in Egypt and is actually identical to the plant studied in this paper. They did not find azulene, proazulen, aldehydes or acids, but detected terpene hydrocarbons, esters, ketones, phenols and alcohols. While their
TABLE
4
EFFECT VARIOUS
OF TERPINEN-4-OL” MICROORGANISMS
Sal.
E.
Staph.
C.
typhosa
coli
aweus
albicans
I
N.1.C
N.1.
N.I.
N.I.
2
N.I.
N.I.
N.I.
N.I.
E. coli
38
3
N.I.
N.I.
N.I.
N.I.
Sal. typhosa
47
4
NJ.
N.I.
N.I.
N.I.
Shig. sonnei
45
5
N.I.
N.I.
N.I.
N.I.
Staph. aweus
25
6
22
I7
20
12
C. albicans
T.I.4
7
25
20
21
20
8
12
15
25
N.I.
Microorganism
“Includes the diameter of the paper disk (6 mm). bEach disk contained about 8 mg of the fraction. ‘No inhibition
of growth.
“Analytical blncluding
Inhibition
ON
GROWTH
diameter
OF
(mm)b,c
grade, produced by Aldrich Chemical Co. the diameter of the paper disk (6 mm).
‘Each disk contained 4 mg terpinen-4-01. dGrowth was inhibited all over the plate. resistent mutants were observed.
Some colonies
of
191
alcoholic fraction may correspond to fractions 6, 7, 8 (Fig. l), which contain the more polar comit does not contain terpinen-4-01. pounds, However, the latter is present in the A. millifolium essential oil (Chandler et al., 1982). These fractions, and especially terpinen-4-01 demonstrated a significant antimicrobial activity. The observation that fractions 2 and 3 which contain less polar compounds ihibited only the growth of C. albicans may be of importance. These fractions may contain compounds which act only on eukaryotic cells.
References Chandler, R.F., Hooper, S.N. and Harvey, N.J. (1982) Ethnobotany and phytochemistry of Yarrow, Achilles millifolium, compositae. Economic Botany 36(2), 203-215. Duke, J.A. (1987) Handbook of Medical Herbs. CRC Press, Boca Raton, FL. Flitman, U., Hen, C., Darin, A. and Shmida, A. (1983) Pictorial Flora of Israel. Massada Ltd. Tel-Aviv, p. 204. Harborn, J.B. (1973). Essential oils. In: Phytochemical Methods. Chapman and Hall, London, pp. 92-103. Shalaby, A.F. and Richter, G. (1964). Chromatographic investigation of the essential oil from Achilles fragrantissima. Journal of Pharmaceutical Sciences 53: 1502-l 505.
