Antimicrobial screening of essential oils and extracts of some Humulus lupulus L. cultivars C.R. Langezaal, A. Chandra and J.J.C. Scheffer Introduction Various cultivars of hop, H u m u l u s lupulus L., are used in brewing to impart a bitter taste and hop flavour, as well as a characteristic aroma to beer [1]. The glandular hairs (lupulin glands) of the hop cones contain a resinous and a volatile fraction, responsible for the bitter taste and the flavour and aroma of beer, respectively. Hop cones have also been used in traditional medicine since the middle ages, because of the anecdotal evidence of their antimicrobial and antifungal activities [2]. Till now, studies on such activities have been carried out with extracts of two cultivars or with isolated bitter compounds and their derivatives, as was shown by a search t h r o u g h the Chemical Abstracts of the period 1964-1990 [3-7]. No reports on the antimicrobial or antifungal activities of the essential oil isolated from hop cones have appeared. Our aim was to investigate whether extracts of various hop cultivars, obtained by solvent extraction, as well as the corresponding essential oils, obtained by hydrodistillation, showed activities against some micro-organisms. The results obtained for the extract and the oil of each plant are compared as well as the activities observed for the various cultivars.
Methods Materials Eleven H. lupulus L. cultivars, viz. 'Brewers Gold', 'Eroica', 'Galena', 'Hallertau mittelfrfih', 'Hersbrficker sp~it', ' N o r t h e r n Brewer', 'Nugget', 'Olympic', 'Wye Challenger', 'Wye Northdown', 'Wye Target', the variety aureus and a wild type of hop were grown in the pharmacognostical garden near the Gor]aeus Laboratories, Leiden University. The cultivars were supplied by the Landen Tuinbouwschool, Vrij Technisch Instituut, Poperinge, Belgium. The hop cones were col-
Keywords Agar overlay technique Antibiotics Drug screening Essential oil
Humulus lupulus L. C.R. Langezaal, A. Chandra and J. J. C. Scheffer (correspondence): Division of Pharmacognosy, Center for Bio-Pharmaceutical Sciences, Leiden University, Gorlaeus Laboratories, P.O. Box 9502, 2300 RA Leiden, the Netherlands.
14(6) 1992
lected in the first week of September 1990, and dried at room temperature without air circulation, until crisp. Voucher specimens of the plants grown in Leiden have been deposited in the Herbarium Vadense, Agricultural University of Wageningen, the Netherlands. The micro-organisms used for the screening were: Gram-negative bacterium: Escherichia coli (ATCC 8739); Gram-positive bacteria: Bacillus subtilis (ATCC 6633) and Staphylococcus aureus (ATCC 6538); yeast: Candida albicans (ATCC 10231); fungus: Trichophyton mentagrophytes var. interdigitale (CBS 558-66).
Isolation and gas chromatographic analysis of the essential oils The dried cones of each cultivar were submitted to hydrodistillation using the Clevengertype apparatus described in the European Pharmacopoeia [8]. The distillations were performed with 40 g of cones in 2 1 of demineralized water to which approximately 5 g of calcium carbonate were added [9]. The distillation rate was 2.5 ml/min, and the distillation period 2 h. The distilled oil was t a k e n up in n-pentane. Gas chromatographic analyses were carried out on a dual channel gas chromatograph, Packard model 439 (Chrompack BV, Delft, the Netherlands), equipped with two flame ionization detectors each of which was connected with a chromatographic data processor, Chromatopac C-R3A (Shimadzu Corporation, Kyoto, Japan). Gas chromatography conditions were as follows: columns: fused silica, 50 m • 0.23 m m i.d. and 60 m • 0.25 m m i.d., coated with CP-Wax 52cb, film thickness 0.22 ~m (Chrompack BV, Middelburg, the Netherlands), and Durabond DB-1, film thickness 0.25 ttm (J & W Scientific Inc., Rancho Cordova, USA), respectively; oven temperature: programmed, 45-240~ at 3~ and subsequently isothermal at
Langezaal CR, Chandra A, Scheffer JJC. Antimicrobial screening of essential oils and extracts of some Humulus lupulus L. cultivars. Pharm Weekbl [Sci] 1992;14(6):353-6. Abstract The essential oils as well as solvent extracts of 11 hop cultivars, 1 hop variety and a wild type of hop were screened for their antimicrobial activities using the agar overlay technique. The oils were isolated from the cones of the various hop plants by hydrodistillation, the extracts were obtained by soaking the hop cones in chloroform. The oils and the extracts showed activity against the Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) and the fungus (Trichophyton mentagrophytes var. interdigitale), but almost no activity against the Gram-negative bacterium (Escherichia coli) and the yeast (Candida albicans) used in the screening. The peak area percentages of the main volatile components and the contents of the bitter acids of the extracts were determined for all cultivars using chromatographic methods. Accepted August 1992.
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Table 1
Composition of culture media used in the antimicrobial screening Medium*
Composition
CASO broth: CASO agar: ME agar: SAB broth: SAB agar: Sterilization:
15 g CASO in 500 ml demineralized water 15 g CASO + 7.5 g agar in 500 ml demineralized water 15 g malt extract + 7.5 g agar in 500 ml demineralized water 15 g Sabouraud dextrose broth in 500 ml demineralized water 15 g Sabouraud dextrose broth + 7.5 g agar in 500 ml demineralized water 120~ for 20 min at 2 k P a
*CASO: casein-peptone soymeal-peptone; ME: malt extract; SAB: Sabouraud. 240~ for 15 min; t e m p e r a t u r e of injector and detectors: 220~ and 240~ respectively; carrier gas: nitrogen; split sampling technique: ratio 1:100; sample size: 2 t~l of oil dilutions in n-pentane. The percentage composition of the oil samples was computed from the gas chromatographic peak areas without using correction factors. The identity of the eight main components of the oils was assigned by comparison of their retention times on the two columns of different polarities. In earlier studies, the identity of the components of various hop oils was also assigned using gas chromatography-mass spectrometry [10 11].
centrations of the bitter compounds in the extracts were determined as described by Verhagen [1].
Culture and test conditions of antimicrobial screening The medium composition and the culture and test conditions are summarized in the Tables 1 and 2. The bacteria, yeast and fungus were brought into the exponential phase by incubation in liquid medium. After the incubation period, test plates were inoculated with 50 ~1 of the bacterial or 100 ~1 of the fungal suspensions. The antimicrobial and antifungal activities were determined using the agar overlay technique [12]. A filter paper disc (r 6 mm) was Isolation and high pressure liquid chromatographic analysis of the extracts placed in Petri dishes (~ 90 mm) filled with 25 ml Of each cultivar or variety, a 5 g sample of of the appropriate medium and inoculated with coarsely ground cones was soaked in 100 ml of the test organism; per Petri dish one disc was used. chloroform at room t e m p e r a t u r e for 1 h. The ex- The disc was impregnated either with 2.5 ~1 of tract obtained was subsequently filtered and the one of the essential oils or of a solution of one of filtrate concentrated using a rotatory evaporator t h e extracts (20 mg/ml, in ethanol) to be tested, at room t e m p e r a t u r e until evaporation of the sol- or with an antibiotic used in order to check the vent was completed. Of each residue, 50 mg were sensitivity of the micro-organisms. The following dissolved in 9.0 ml methanol to which 1.0 ml of aqueous solutions of antibiotics were used: strepa methanolic solution of the p-nitroanilide of tomycin sulfate (0.5 mg/ml) and chloramphenicol myristic acid (internal standard; 1.32 mg/ml) was (3 mg/ml) for the bacteria, nystatin (50 mg/ml) for added. The resulting solution was analysed by the yeast; griseofulvin (50 mg/ml, in acetone) was high pressure liquid chromatography (HPLC). used for the fungus. After the incubation period The analyses were carried out using an LKB (see test conditions given in Table 2), the di2150 pump, and a UV monitor LKB 2151 ameter of the inhibition zone was measured to (Pharmacia BV, Woerden, the Netherlands) con- the nearest ram, all experiments were performed nected with a chromatographic data processor, in duplicate. Chromatopac C-R3A; for injection, a Rheodyne 7125 injector (Pharmacia) was used. The HPLC R e s u l t s a n d d i s c u s s i o n conditions were as follows; column: Chromsep, Hop extracts are described to possess anti10 cm x 4.6 mm i.d., microsphere C18, particle microbial and antifungal activities, but studies size 3 ~m (Chrompack, Middelburg); eluent: on such activities of hop essential oils were not methanol+water+phosphoric acid (85%) reported until now. Therefore, we tested the es(85+17+0.25), flow rate: 1.5 ml/min; detection: sential oils, obtained by hydrodistillation, of UV light at 314 nm; sample size: 10 ~1. The con- 11 hop cultivars, i variety and a wild type of hop Table 2
Culture methods for the micro-organisms used in the antimicrobial screening Micro-organism
Culture conditions*
Test conditions*
Bacteria Yeast Fungus
18 h at 37~ in CASO broth 18 h at 37~ in CASO broth 7 days at 27~ in SAB broth
24 h at 37~ on CASO agar 24 h at 37~ on ME agar 7 days at 27~ on SAB agar
*CASO: casein-peptone soymeal-peptone; ME: malt extract; SAB: Sabouraud. 354
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14(6) 1992
for their activities against some micro-organisms. The activities were determined u s i n g the a g a r overlay technique, and the inhibition dia m e t e r s observed for the oils are presented in Table 3. Solvent extracts obtained from cones of the same plants were also screened, using the same procedure. The p e a k a r e a percentages of some i m p o r t a n t components of the oils, and the bitter acids contents of the extracts are shown in Table 4. Although the inhibition diameters observed for the essential oils were in all cases but one smaller t h a n those observed for the antibiotics, it
is not justified to conclude t h a t the activities of the oils are thus less. The essential oils showed activity against the Gram-positive bacteria (B. subtilis and S. aureus) and the fungus (T. m e n t a g r o p h y t e s var. interdigitale), but almost no activity against the Gram-negative b a c t e r i u m (E. coli) and the yeast (C. albicans). The chloroform extracts of the hop cones caused large inhibition diameters when tested against the Gram-positive bacteria; some activity against the fungus Was observed, but almost no activities against the Gram-negative b a c t e r i u m and the yeast. Comparable results
Table 3 A v e r a g e i n h i b i t i o n d i a m e t e r s * given in m m ( i n c l u d i n g disc, r 6 mm) observed for hop essential oils and extracts when
using the organisms listed (n = 2) Cultivar/variety/ antibiotic~
Au
BG
Er
Ga
Ha
He
Hw
NB
Nu
•
O1 WC
WN
WT
Ch
St
7
16 24 20
12 20 14
NyGr
Essential oil E. B. S. C. T.
coli subtilis aureus albicans mentagrophytes var. interdigitale
.
-
7
-
-
+ 8
.
_+ 10
10 8
12 21
7
9 10
7 8
7 12
8 9
8 8
_+ 9
7 7
7 10
7
-
+
7
+
+
8
-
+
-
7
7
-
9
8
-
10
17
9
10
15
13
11
18
10
.
.
.
.
.
18
7
22
Solvent extract E. B. S. C. T.
coli subtilis aureus albicans mentagrophytes var. interdigitale
8
8
7
7
7
31 29
24 27
23 22
26 28
31 31
-
-
7
7
7
10
12
7
8
9
-
-
7
8
8
8
+
27 28
24 23
22 24
22 25
20 26
24 27
-
22 25
24 28
-
7
+
7
7
+
7
•
12
7
12
10
11
10
9
8
16 24 20
12 20 14 18 22
* - : no inhibition +: negligible inhibition. Au: var. aureus; BG: 'Brewers Gold'; Er: 'Eroica'; Ga: 'Galena'; Ha: ' H a l l e r t a u mittelfrfih'; He: 'Hersbriicker sp&t'; Hw: hop wild type; NB: ' N o r t h e r n Brewer'; Nu: 'Nugget'; 01: 'Olympic'; WC: 'Wye Challenger'; WN: 'Wye Northdown'; WT: 'Wye Target'; Ch: chloramphenicol; St: streptomycin sulfate; Ny: nystatin; Gr: griseofulvin.
Table 4 S o m e i m p o r t a n t c o m p o n e n t s o f hop essential oils a n d extracts screened for a n t i m i c r o b i a l activities
Cultivar variety*
Au
BG
Er
Ga
Ha
He
Hw
NB
Nu
O1
WC
WN
WT
10.4 12.4 35.0 8.5 1.0 1.8 1.1 1.1
44.0 11.7 26.5 1.1 0.7 1.2 0.2 0.4
65.6 11.3 1.6 2.0 t t t t
31.5 10.2 21.3 1.4 0.9 1.4 0.4 1.0
1.0 10.8 41.3 1.1 2.7 3.0 2.8 5.9
8.2 9.2 28.5 5.4 1.2 1.6 2.0 2.3
5.1 17.6 53.8 0.8 1.4 2.2 0.6 0.7
24.6 13.2 39.1 0.6 0.9 1.5 1.1 1.1
4.4 18.6 38.9 2.6 1.9 3.1 1.2 1.8
25.9 18.3 28.4 2.2 1.2 1.9 1.0 0.8
27.1 9.4 29.9 6.1 0.8 1.3 0.5 0.4
25.9 17.4 34.9 2.0 1.1 1.8 1.0 1.0
42.1 9.5 20.3 1.2 1.4 2.3 0.6 0.4
11.5 23.8
28.4 18.9
30.7 18.6
29.1 26.6
15.8 32.3
13.3 38.0
14.9 20.6
37.2 17.3
25.3 19.8
29.7 15.0
25.8 18.3
25.4 14.2
31.5 17.5
E s s e n t i a l oil~
~-Myrcene /LCaryophyllene ~-Humulene ~-Selinene ~-Cadinene 6-Cadinene /LCaryophyllene oxide a-Humulene oxide S o l v e n t extractt
a-Acids ~-Acids
*Au: var. aureus; BG: 'Brewers Gold'; Er: 'Eroica'; Ga: 'Galena'; Ha: ' H a l l e r t a u mittelfrfih'; He: 'Hersbrficker sp~it'; Hw: hop wild type; NB: ' N o r t h e r n Brewer'; Nu: 'Nugget'; Oh 'Olympic'; WC: 'Wye Challenger'; WN: 'Wye Northdown;' WT: 'Wye Target'. ~Essential oil components are represented by their gas chromatographic p e a k a r e a percentages, bitter acids by their concentrations (% wt/wt) in the extract; t: trace ( < 0.05%). 14(6) 1992
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have been obtained for isolated bitter acids and related compounds or derivatives [5-7]. The extracts of the cones of the cultivar 'Hallertau mittelfriih' and the hop variety a u r e u s showed the highest activity against B. subtilis and S. aureus. However, a clear correlation between the essential oil composition and/or the content of the bitter acids, and the inhibition diameters could not be detected. Table 3 shows t h a t the inhibition diameters observed for the extracts are much larger t h a n those measured for the essential oils; this is especially true for the Gram-positive bacteria. Teuber and Schmalreck [13], using a B. subtilis strain, concluded t h a t the bactericidal activity of hop constituents against Gram-positive bacteria was a result of primary membrane leakage. In a later study, it was stated t h a t the antimicrobial properties are mainly dependent on the hydrophobic parts of the molecules [14]. Although the essential oils tested in our study are rich in hydrophobic compounds, the inhibition diameters observed for the oils were smaller t h a n those of the extracts. This is possibly due to the presence of the bitter acids and flavones [15] in the extracts, which will show a better diffusion in the medium of the Petri dishes. The in most cases r a t h e r small inhibition diameters observed for the oils and extracts when tested against the fungus, m a y be explained by evaporation of essential oil components, and degradation of the hop extract during the long period of incubation (7 days at 27 ~C). Although results obtained with the agar overlay technique should be interpreted with care [16], it can be concluded t h a t hop extracts as well as hop essential oils show antimicrobial activities, especially against the Gram-positive bacteria used in this screening (B. subtilis and S. aureus). Negligible activities against the Gramnegative bacterium (E. coli) and the yeast (C. albicans) were found for both the extracts and the essential oils.
Acknowledgements The authors express their t h a n k s to Mrs. A. Looman for technical assistance, and to the Commission of the European Communities (Brussels) and the Department of Science and Technology (New Delhi, India) for providing a fellowship (to A.C.).
356
References 1 Verhagen LC. Hop analysis. In: Linskens HF, Jackson JF, eds. Modern methods of plant analysis. New series. Vol. 7. Beer analysis. Heidelberg: Springer-Verlag, 1988:67-87. 2 Vanhoucke LC. De folklore van de hop in VlaamsBelgi~ vroeger en nu [The folklore of hop in Flemish Belgium in the past and at present]. Oudenaarde: Drukkerij Sanderus, 1964. 3 Boatwright J. Antimicrobial activity of hop oil emulsion. J Inst Brew 1976;82:334-5. 4 Mizobuchi S, Sato Y. A new flavone with antifungal activity isolated from hops. Agric Biol Chem 1984;48: 2771-5. 5 Mizobuchi S, Sato Y. Antifungal activities of hop bitter resins and related compounds. Agric Biol Chem 1985;49:399-403. 6 Mizobuchi S, Sato Y. Antifungal activities of 2,4,6trihydroxyacylophenones and related compounds. Agric Biol Chem 1985;49:719-24. 7 Mizobuchi S, Sato Y. Antifungal activity of 2,4dihydroxyacylophenones and related compounds. Agric Biol Chem 1985;49:1327-33. 8 European Pharmacopoeia. Vol. 3. Sainte-Ruffine: Maisonneuve SA, 1975:68-71. 9 Van den Dries JMA, Baerheim Svendsen A. A simple method for detection of glycosidicbound monoterpenes and other volatile compounds occurring in fresh plant material. Flavour Fragr J 1989;4:59-61. 10 Katsiotis ST, Langezaal CR, Scheffer JJC, Verpoorte R. Comparative study of the essential oils from hops of various Humulus lupulus L. cultivars. Flavour Fragr J 1989;4:187-91. 11 Katsiotis ST, Langezaal CR, Scheffer JJC. Composition of the essential oils from leaves of various Humulus lupulus L. cultivars. Flavour Fragr J 1990;5: 97-100. 12 Janssen AM. Antimicrobial activities of essential oils - a pharmacognostical study [dissertation]. Leiden: Univ of Leiden, 1989. 13 Teuber M, Schmalreck AF. Membrane leakage in Bacillus subtilis 168 induced by the hop constituents lupulone, humulone, isohumulone and humulinic acid. Arch Mikrobiol 1973;94:159-71. 14 Schmalreck AF, Teuber M, Reininger W, Hartl A. Structural features determining the antibiotic potencies of natural and synthetic hop bitter resins, their precursors and derivatives. Can J Microbiol 1975;21: 205-12. 15 Van Sumere CF, Vande Casteele K, Hutsebaut W, Everaert E, De Cooman L, Meulemans W. RP-HPLC analysis of flavonoids and the biochemical identification of hop cultivars. EBC Monograph XIII. Niirnberg: Carl (Brauwelt-Verlag), 1987:146-75. 16 Janssen AM, Chin NLJ, Scheffer JJC, Baerheim Svendsen A. Screening for antimicrobial activity of some essential oils by the agar overlay technique. Pharm Weekbl [Sci] 1986;8:289-92.
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Methods Materials Eleven H. lupulus L. cultivars, viz. 'Brewers Gold', 'Eroica', 'Galena', 'Hallertau mittelfrfih', 'Hersbrficker sp~it', ' N o r t h e r n Brewer', 'Nugget', 'Olympic', 'Wye Challenger', 'Wye Northdown', 'Wye Target', the variety aureus and a wild type of hop were grown in the pharmacognostical garden near the Gor]aeus Laboratories, Leiden University. The cultivars were supplied by the Landen Tuinbouwschool, Vrij Technisch Instituut, Poperinge, Belgium. The hop cones were col-
Keywords Agar overlay technique Antibiotics Drug screening Essential oil
Humulus lupulus L. C.R. Langezaal, A. Chandra and J. J. C. Scheffer (correspondence): Division of Pharmacognosy, Center for Bio-Pharmaceutical Sciences, Leiden University, Gorlaeus Laboratories, P.O. Box 9502, 2300 RA Leiden, the Netherlands.
14(6) 1992
lected in the first week of September 1990, and dried at room temperature without air circulation, until crisp. Voucher specimens of the plants grown in Leiden have been deposited in the Herbarium Vadense, Agricultural University of Wageningen, the Netherlands. The micro-organisms used for the screening were: Gram-negative bacterium: Escherichia coli (ATCC 8739); Gram-positive bacteria: Bacillus subtilis (ATCC 6633) and Staphylococcus aureus (ATCC 6538); yeast: Candida albicans (ATCC 10231); fungus: Trichophyton mentagrophytes var. interdigitale (CBS 558-66).
Isolation and gas chromatographic analysis of the essential oils The dried cones of each cultivar were submitted to hydrodistillation using the Clevengertype apparatus described in the European Pharmacopoeia [8]. The distillations were performed with 40 g of cones in 2 1 of demineralized water to which approximately 5 g of calcium carbonate were added [9]. The distillation rate was 2.5 ml/min, and the distillation period 2 h. The distilled oil was t a k e n up in n-pentane. Gas chromatographic analyses were carried out on a dual channel gas chromatograph, Packard model 439 (Chrompack BV, Delft, the Netherlands), equipped with two flame ionization detectors each of which was connected with a chromatographic data processor, Chromatopac C-R3A (Shimadzu Corporation, Kyoto, Japan). Gas chromatography conditions were as follows: columns: fused silica, 50 m • 0.23 m m i.d. and 60 m • 0.25 m m i.d., coated with CP-Wax 52cb, film thickness 0.22 ~m (Chrompack BV, Middelburg, the Netherlands), and Durabond DB-1, film thickness 0.25 ttm (J & W Scientific Inc., Rancho Cordova, USA), respectively; oven temperature: programmed, 45-240~ at 3~ and subsequently isothermal at
Langezaal CR, Chandra A, Scheffer JJC. Antimicrobial screening of essential oils and extracts of some Humulus lupulus L. cultivars. Pharm Weekbl [Sci] 1992;14(6):353-6. Abstract The essential oils as well as solvent extracts of 11 hop cultivars, 1 hop variety and a wild type of hop were screened for their antimicrobial activities using the agar overlay technique. The oils were isolated from the cones of the various hop plants by hydrodistillation, the extracts were obtained by soaking the hop cones in chloroform. The oils and the extracts showed activity against the Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) and the fungus (Trichophyton mentagrophytes var. interdigitale), but almost no activity against the Gram-negative bacterium (Escherichia coli) and the yeast (Candida albicans) used in the screening. The peak area percentages of the main volatile components and the contents of the bitter acids of the extracts were determined for all cultivars using chromatographic methods. Accepted August 1992.
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Table 1
Composition of culture media used in the antimicrobial screening Medium*
Composition
CASO broth: CASO agar: ME agar: SAB broth: SAB agar: Sterilization:
15 g CASO in 500 ml demineralized water 15 g CASO + 7.5 g agar in 500 ml demineralized water 15 g malt extract + 7.5 g agar in 500 ml demineralized water 15 g Sabouraud dextrose broth in 500 ml demineralized water 15 g Sabouraud dextrose broth + 7.5 g agar in 500 ml demineralized water 120~ for 20 min at 2 k P a
*CASO: casein-peptone soymeal-peptone; ME: malt extract; SAB: Sabouraud. 240~ for 15 min; t e m p e r a t u r e of injector and detectors: 220~ and 240~ respectively; carrier gas: nitrogen; split sampling technique: ratio 1:100; sample size: 2 t~l of oil dilutions in n-pentane. The percentage composition of the oil samples was computed from the gas chromatographic peak areas without using correction factors. The identity of the eight main components of the oils was assigned by comparison of their retention times on the two columns of different polarities. In earlier studies, the identity of the components of various hop oils was also assigned using gas chromatography-mass spectrometry [10 11].
centrations of the bitter compounds in the extracts were determined as described by Verhagen [1].
Culture and test conditions of antimicrobial screening The medium composition and the culture and test conditions are summarized in the Tables 1 and 2. The bacteria, yeast and fungus were brought into the exponential phase by incubation in liquid medium. After the incubation period, test plates were inoculated with 50 ~1 of the bacterial or 100 ~1 of the fungal suspensions. The antimicrobial and antifungal activities were determined using the agar overlay technique [12]. A filter paper disc (r 6 mm) was Isolation and high pressure liquid chromatographic analysis of the extracts placed in Petri dishes (~ 90 mm) filled with 25 ml Of each cultivar or variety, a 5 g sample of of the appropriate medium and inoculated with coarsely ground cones was soaked in 100 ml of the test organism; per Petri dish one disc was used. chloroform at room t e m p e r a t u r e for 1 h. The ex- The disc was impregnated either with 2.5 ~1 of tract obtained was subsequently filtered and the one of the essential oils or of a solution of one of filtrate concentrated using a rotatory evaporator t h e extracts (20 mg/ml, in ethanol) to be tested, at room t e m p e r a t u r e until evaporation of the sol- or with an antibiotic used in order to check the vent was completed. Of each residue, 50 mg were sensitivity of the micro-organisms. The following dissolved in 9.0 ml methanol to which 1.0 ml of aqueous solutions of antibiotics were used: strepa methanolic solution of the p-nitroanilide of tomycin sulfate (0.5 mg/ml) and chloramphenicol myristic acid (internal standard; 1.32 mg/ml) was (3 mg/ml) for the bacteria, nystatin (50 mg/ml) for added. The resulting solution was analysed by the yeast; griseofulvin (50 mg/ml, in acetone) was high pressure liquid chromatography (HPLC). used for the fungus. After the incubation period The analyses were carried out using an LKB (see test conditions given in Table 2), the di2150 pump, and a UV monitor LKB 2151 ameter of the inhibition zone was measured to (Pharmacia BV, Woerden, the Netherlands) con- the nearest ram, all experiments were performed nected with a chromatographic data processor, in duplicate. Chromatopac C-R3A; for injection, a Rheodyne 7125 injector (Pharmacia) was used. The HPLC R e s u l t s a n d d i s c u s s i o n conditions were as follows; column: Chromsep, Hop extracts are described to possess anti10 cm x 4.6 mm i.d., microsphere C18, particle microbial and antifungal activities, but studies size 3 ~m (Chrompack, Middelburg); eluent: on such activities of hop essential oils were not methanol+water+phosphoric acid (85%) reported until now. Therefore, we tested the es(85+17+0.25), flow rate: 1.5 ml/min; detection: sential oils, obtained by hydrodistillation, of UV light at 314 nm; sample size: 10 ~1. The con- 11 hop cultivars, i variety and a wild type of hop Table 2
Culture methods for the micro-organisms used in the antimicrobial screening Micro-organism
Culture conditions*
Test conditions*
Bacteria Yeast Fungus
18 h at 37~ in CASO broth 18 h at 37~ in CASO broth 7 days at 27~ in SAB broth
24 h at 37~ on CASO agar 24 h at 37~ on ME agar 7 days at 27~ on SAB agar
*CASO: casein-peptone soymeal-peptone; ME: malt extract; SAB: Sabouraud. 354
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for their activities against some micro-organisms. The activities were determined u s i n g the a g a r overlay technique, and the inhibition dia m e t e r s observed for the oils are presented in Table 3. Solvent extracts obtained from cones of the same plants were also screened, using the same procedure. The p e a k a r e a percentages of some i m p o r t a n t components of the oils, and the bitter acids contents of the extracts are shown in Table 4. Although the inhibition diameters observed for the essential oils were in all cases but one smaller t h a n those observed for the antibiotics, it
is not justified to conclude t h a t the activities of the oils are thus less. The essential oils showed activity against the Gram-positive bacteria (B. subtilis and S. aureus) and the fungus (T. m e n t a g r o p h y t e s var. interdigitale), but almost no activity against the Gram-negative b a c t e r i u m (E. coli) and the yeast (C. albicans). The chloroform extracts of the hop cones caused large inhibition diameters when tested against the Gram-positive bacteria; some activity against the fungus Was observed, but almost no activities against the Gram-negative b a c t e r i u m and the yeast. Comparable results
Table 3 A v e r a g e i n h i b i t i o n d i a m e t e r s * given in m m ( i n c l u d i n g disc, r 6 mm) observed for hop essential oils and extracts when
using the organisms listed (n = 2) Cultivar/variety/ antibiotic~
Au
BG
Er
Ga
Ha
He
Hw
NB
Nu
•
O1 WC
WN
WT
Ch
St
7
16 24 20
12 20 14
NyGr
Essential oil E. B. S. C. T.
coli subtilis aureus albicans mentagrophytes var. interdigitale
.
-
7
-
-
+ 8
.
_+ 10
10 8
12 21
7
9 10
7 8
7 12
8 9
8 8
_+ 9
7 7
7 10
7
-
+
7
+
+
8
-
+
-
7
7
-
9
8
-
10
17
9
10
15
13
11
18
10
.
.
.
.
.
18
7
22
Solvent extract E. B. S. C. T.
coli subtilis aureus albicans mentagrophytes var. interdigitale
8
8
7
7
7
31 29
24 27
23 22
26 28
31 31
-
-
7
7
7
10
12
7
8
9
-
-
7
8
8
8
+
27 28
24 23
22 24
22 25
20 26
24 27
-
22 25
24 28
-
7
+
7
7
+
7
•
12
7
12
10
11
10
9
8
16 24 20
12 20 14 18 22
* - : no inhibition +: negligible inhibition. Au: var. aureus; BG: 'Brewers Gold'; Er: 'Eroica'; Ga: 'Galena'; Ha: ' H a l l e r t a u mittelfrfih'; He: 'Hersbriicker sp&t'; Hw: hop wild type; NB: ' N o r t h e r n Brewer'; Nu: 'Nugget'; 01: 'Olympic'; WC: 'Wye Challenger'; WN: 'Wye Northdown'; WT: 'Wye Target'; Ch: chloramphenicol; St: streptomycin sulfate; Ny: nystatin; Gr: griseofulvin.
Table 4 S o m e i m p o r t a n t c o m p o n e n t s o f hop essential oils a n d extracts screened for a n t i m i c r o b i a l activities
Cultivar variety*
Au
BG
Er
Ga
Ha
He
Hw
NB
Nu
O1
WC
WN
WT
10.4 12.4 35.0 8.5 1.0 1.8 1.1 1.1
44.0 11.7 26.5 1.1 0.7 1.2 0.2 0.4
65.6 11.3 1.6 2.0 t t t t
31.5 10.2 21.3 1.4 0.9 1.4 0.4 1.0
1.0 10.8 41.3 1.1 2.7 3.0 2.8 5.9
8.2 9.2 28.5 5.4 1.2 1.6 2.0 2.3
5.1 17.6 53.8 0.8 1.4 2.2 0.6 0.7
24.6 13.2 39.1 0.6 0.9 1.5 1.1 1.1
4.4 18.6 38.9 2.6 1.9 3.1 1.2 1.8
25.9 18.3 28.4 2.2 1.2 1.9 1.0 0.8
27.1 9.4 29.9 6.1 0.8 1.3 0.5 0.4
25.9 17.4 34.9 2.0 1.1 1.8 1.0 1.0
42.1 9.5 20.3 1.2 1.4 2.3 0.6 0.4
11.5 23.8
28.4 18.9
30.7 18.6
29.1 26.6
15.8 32.3
13.3 38.0
14.9 20.6
37.2 17.3
25.3 19.8
29.7 15.0
25.8 18.3
25.4 14.2
31.5 17.5
E s s e n t i a l oil~
~-Myrcene /LCaryophyllene ~-Humulene ~-Selinene ~-Cadinene 6-Cadinene /LCaryophyllene oxide a-Humulene oxide S o l v e n t extractt
a-Acids ~-Acids
*Au: var. aureus; BG: 'Brewers Gold'; Er: 'Eroica'; Ga: 'Galena'; Ha: ' H a l l e r t a u mittelfrfih'; He: 'Hersbrficker sp~it'; Hw: hop wild type; NB: ' N o r t h e r n Brewer'; Nu: 'Nugget'; Oh 'Olympic'; WC: 'Wye Challenger'; WN: 'Wye Northdown;' WT: 'Wye Target'. ~Essential oil components are represented by their gas chromatographic p e a k a r e a percentages, bitter acids by their concentrations (% wt/wt) in the extract; t: trace ( < 0.05%). 14(6) 1992
P h a r m a c e u t i s c h Weekblad Scientific edition
355
have been obtained for isolated bitter acids and related compounds or derivatives [5-7]. The extracts of the cones of the cultivar 'Hallertau mittelfriih' and the hop variety a u r e u s showed the highest activity against B. subtilis and S. aureus. However, a clear correlation between the essential oil composition and/or the content of the bitter acids, and the inhibition diameters could not be detected. Table 3 shows t h a t the inhibition diameters observed for the extracts are much larger t h a n those measured for the essential oils; this is especially true for the Gram-positive bacteria. Teuber and Schmalreck [13], using a B. subtilis strain, concluded t h a t the bactericidal activity of hop constituents against Gram-positive bacteria was a result of primary membrane leakage. In a later study, it was stated t h a t the antimicrobial properties are mainly dependent on the hydrophobic parts of the molecules [14]. Although the essential oils tested in our study are rich in hydrophobic compounds, the inhibition diameters observed for the oils were smaller t h a n those of the extracts. This is possibly due to the presence of the bitter acids and flavones [15] in the extracts, which will show a better diffusion in the medium of the Petri dishes. The in most cases r a t h e r small inhibition diameters observed for the oils and extracts when tested against the fungus, m a y be explained by evaporation of essential oil components, and degradation of the hop extract during the long period of incubation (7 days at 27 ~C). Although results obtained with the agar overlay technique should be interpreted with care [16], it can be concluded t h a t hop extracts as well as hop essential oils show antimicrobial activities, especially against the Gram-positive bacteria used in this screening (B. subtilis and S. aureus). Negligible activities against the Gramnegative bacterium (E. coli) and the yeast (C. albicans) were found for both the extracts and the essential oils.
Acknowledgements The authors express their t h a n k s to Mrs. A. Looman for technical assistance, and to the Commission of the European Communities (Brussels) and the Department of Science and Technology (New Delhi, India) for providing a fellowship (to A.C.).
356
References 1 Verhagen LC. Hop analysis. In: Linskens HF, Jackson JF, eds. Modern methods of plant analysis. New series. Vol. 7. Beer analysis. Heidelberg: Springer-Verlag, 1988:67-87. 2 Vanhoucke LC. De folklore van de hop in VlaamsBelgi~ vroeger en nu [The folklore of hop in Flemish Belgium in the past and at present]. Oudenaarde: Drukkerij Sanderus, 1964. 3 Boatwright J. Antimicrobial activity of hop oil emulsion. J Inst Brew 1976;82:334-5. 4 Mizobuchi S, Sato Y. A new flavone with antifungal activity isolated from hops. Agric Biol Chem 1984;48: 2771-5. 5 Mizobuchi S, Sato Y. Antifungal activities of hop bitter resins and related compounds. Agric Biol Chem 1985;49:399-403. 6 Mizobuchi S, Sato Y. Antifungal activities of 2,4,6trihydroxyacylophenones and related compounds. Agric Biol Chem 1985;49:719-24. 7 Mizobuchi S, Sato Y. Antifungal activity of 2,4dihydroxyacylophenones and related compounds. Agric Biol Chem 1985;49:1327-33. 8 European Pharmacopoeia. Vol. 3. Sainte-Ruffine: Maisonneuve SA, 1975:68-71. 9 Van den Dries JMA, Baerheim Svendsen A. A simple method for detection of glycosidicbound monoterpenes and other volatile compounds occurring in fresh plant material. Flavour Fragr J 1989;4:59-61. 10 Katsiotis ST, Langezaal CR, Scheffer JJC, Verpoorte R. Comparative study of the essential oils from hops of various Humulus lupulus L. cultivars. Flavour Fragr J 1989;4:187-91. 11 Katsiotis ST, Langezaal CR, Scheffer JJC. Composition of the essential oils from leaves of various Humulus lupulus L. cultivars. Flavour Fragr J 1990;5: 97-100. 12 Janssen AM. Antimicrobial activities of essential oils - a pharmacognostical study [dissertation]. Leiden: Univ of Leiden, 1989. 13 Teuber M, Schmalreck AF. Membrane leakage in Bacillus subtilis 168 induced by the hop constituents lupulone, humulone, isohumulone and humulinic acid. Arch Mikrobiol 1973;94:159-71. 14 Schmalreck AF, Teuber M, Reininger W, Hartl A. Structural features determining the antibiotic potencies of natural and synthetic hop bitter resins, their precursors and derivatives. Can J Microbiol 1975;21: 205-12. 15 Van Sumere CF, Vande Casteele K, Hutsebaut W, Everaert E, De Cooman L, Meulemans W. RP-HPLC analysis of flavonoids and the biochemical identification of hop cultivars. EBC Monograph XIII. Niirnberg: Carl (Brauwelt-Verlag), 1987:146-75. 16 Janssen AM, Chin NLJ, Scheffer JJC, Baerheim Svendsen A. Screening for antimicrobial activity of some essential oils by the agar overlay technique. Pharm Weekbl [Sci] 1986;8:289-92.
Pharmaceutisch Weekblad Scientific edition
14(6) 1992
