Antimicrobial Activity of Alk(en)yl Sulfides Found in Essential by ipm13571


									Food Sci. Biotechnol. Vol. 13, No. 2, pp. 235 ~ 239 (2004)


                                                                                                        + The Korean Society of Food Science and Technology

Antimicrobial Activity of Alk(en)yl Sulfides Found in Essential Oils of
Garlic and Onion
Jay Wook Kim, Jung Eun Huh1, Suk Hun Kyung1 and Kyu Hang Kyung
Department of Food Science, Sejong University, Seoul 143-747, Korea
 Applied Biology and Chemistry, Konkuk University, Seoul 143-701, Korea

    Abstract Alk(en)yl sulfides derived from S-alk(en)yl-L-cysteine sulfoxides in garlic and onion were chemically synthesized,
    and their antimicrobial activities were determined. Both garlic oil (GO) and onion oil (OO) showed equal antimicrobial
    activities, with minimum inhibitory concentrations of 25 and 100 ppm against Candida utilis ATCC42416 and Staphylococcus
    aureus B31, respectively. Number of carbons in alk(en)yl group and sulfur atoms in molecules influenced antimicrobial
    activity of sulfides. Sulfides with single sulfur atom were not antimicrobial, and sulfides with three or four sulfur atoms were
    highly inhibitory against growths of C. utilis ATCC42416 and S. aureus B31. Dimethyl trisulfide was inhibitory against yeast
    growth, but not to bacterium growth, whereas opposite was observed for dipropyl trisulfide and dipropyl tetrasulfide.
    Antimicrobial activities of GO and OO appeared to be determined by the concentrations of individual constituent sulfides.
    Keywords: garlic, onion, garlic oil, alk(en)yl sulfides, antimicrobial activity

Introduction                                                          stronger antimicrobial activity (9) than those with less sulfur
                                                                      atoms such as DADS.
The antimicrobial activity of Allium (1, 2) is due to volatile           DATS, thermally generated from alliin in the absence of
sulfur compounds derived from S-allyl-L-cysteine sulfoxide,           alliinase activity, was believed to be the primary antimicrobial
a non-protein amino acid found in vegetables, by the action           compound of heated garlic (12). Dimethyl trisulfide (DMTS),
of an enzyme, cysteine sulfoxide lyase. Alliin (S-allyl-L-            for example, had an MIC of 20 ppm for all four yeasts
cysteine sulfoxide), the major S-alkenyl-L-cysteine sulfoxide         tested, while showing MICs of 200 ppm or higher for
in garlic, is degraded into allicin (allyl 2-propenethiosulfinate).   various bacteria (13).
This thiosulfinate is the principal antimicrobial agent of garlic        Many researchers have studied the antiyeast effects of
(3). Alliin (substrate) and alliinase (enzyme) are located in         plants (14-17). The active compounds are mostly essential oils
different cells of the garlic cloves (4). Therefore, thiosulfinate    and phenolic compounds. Some sulfur compounds originating
is generated only after the vegetable tissues are injured, and        from vegetables, including DMTS and allyl isothiocyanate
the enzymes react with their substrates. Naturally, heating           (AITC), are more effective at inhibiting the growth of
at high temperatures decreases the antimicrobial activity of          yeasts (13) than that of bacteria.
garlic (4) due to the inactivation of enzyme alliinase by heat.          Our objectives were to synthesize alk(en)yl sulfides with
This is also expected to apply to onion.                              different alk(en)yl groups and with different numbers of
   Cavallito and his coworkers (1, 5, 6) found that allicin           sulfur atoms, and to determine antimicrobial activities of the
rapidly decomposed into diallyl sulfide and neither aqueous           sulfides to explain the antimicrobial activity of essential
garlic extracts lacking allicin nor those with garlic oil (GO)        oils of garlic and onion.
or diallyl sulfides were antimicrobial. Accordingly, there have
been only few investigations concerning the antimicrobial
activity of GO and its constituting sulfides, because early           Materials and Methods
pioneers of garlic antimicrobial activity concluded that GO           Materials Garlic oil (GO) and onion oil (OO), products
and its sulfide were not antimicrobial.                               of Grupo Tecnaal Co. (Zapopan, Mexico), were obtained
   GO is produced by heating the crushed garlic to boiling            from Hyangwon Spice Co. (Seoul, Korea) as gifts. Diallyl
temperature and collecting the resulting vapor as a distillate        monosulfide (DAMS), DADS, and DATS were purchased
(7). During the heating process, allicin in crushed garlic is         from Sigma Chem. Co (St. Louis, MO, USA), Fluka Chem.
converted into various kinds of sulfides (8, 10, 11), with            Co. (Munich, Germany), and LKT Laboratories Inc. (St. Paul,
diallyl disulfide (DADS) being the most abundant.                     MN, USA), respectively. Diethyl disulfide (DEDS), dipropyl
   GO and its diallyl constituents were recently found to be          disulfide (DPDS), dimethyl monosulfide (DMMS), dimethyl
antibacterial (8-10). Sulfides, e.g. diallyl trisulfide (DATS),       disulfide (DMDS), dimethyl trisulfide (DMTS), and Tween
diallyl tetrasulfide (DATTS), diallyl pentasulfide (DAPS),            80 were purchased from Aldrich Chem Co. (Milwaukee,
and diallyl hexasulfide (DAHS), with greater number of                WI, USA).
sulfur atoms were found in GO and are known to possess
                                                                      Synthesis of diallyl tetrasulfide (DATTS) A solution of
*Corresponding author: Tel: 82-2-3408-3225; Fax: 82-2-3408-3319       20 mM each allylmercaptan and triethylamine in 50 mL
 E-mail:                                         anhydrous diethyl ether was stirred under argon at −78oC.
 Received November 30, 2003; accepted March 26, 2004
                                                                      A solution of 10 mM sulfur monochloride in 50 mL
236                                                                                                                          J.W. Kim et al.

anhydrous diethyl ether was added dropwise over 0.5 hr.                 Microbial strains and culture conditions Staphylococcus
The reaction mixture was stirred for additional 1.5 hr, and             aureus B31 and Candida utilis ATCC42416 were gifts from
subsequently quenched with 25 mL H2O. The organic phase                 Dr. Henry P. Fleming (Food Fermentation Laboratories,
was washed 3×25 mL H2O or until the aqueous phase                       USDA/ARS, North Carolina State Univ., Raleigh, NC, USA).
became clear. The organic phase was dried over anhydrous                Microorgarisms cultures were stored at −64oC in basal media
MgSO4. This mixture was vacuum-filtered and evaporated.                 containing 16% glycerol. The basal media were tryptic soy
The resulting yellow residue was dissolved in dichloro-                 broth (TSB; Difco Laboratories, Detroit, MI, USA) for S.
methane and separated by silica gel columns using hexane                aureus B31 and YMPG broth (0.3% yeast extract, 0.3% malt
as the eluent. Synthesis of dimethyl-, diethyl-, and dipropyl           extract, 0.5% peptone, 1% glucose; Difco Laboratories)
tetrasulfides were carried out in the same manner used for              for C. utilis ATCC42416. For resuscitation, frozen cultures
diallyl tetrasulfide (18, 19).                                          were streaked onto agar media of the same composition
                                                                        used for growth, and an isolated colony was picked and
Synthesis of diproyl trisulfide (DPTS) A solution of 10                 cultivated at least twice in the growth medium before using
mM each propylmercaptan and triethylamine in 25 mL                      a 24-hr culture for final inoculation of microorganisms. C.
anhydrous diethyl ether was stirred under argon at −78oC.               utilis ATCC42416 was grown aerobically by shaking at
A solution of 10 mM sulfur dichloride in 50 mL anhydrous                150 rpm (KSI-200L Shaker; Korea Environmental Control
diethyl ether was added dropwise over 0.5 hr. The reaction              Co., Ltd., Kyunggi-do, Korea). Ten microliters of a 10×
mixture was stirred for 0.5 hr after the addition was complete.         diluted aliquot of bacterial seed culture were inoculated
A solution of 10 mM propanethiol and (MM) triethylamine                 into 10 mL of the appropriate broth in 16×150 mm glass
in 25 mL diethyl ether was added dropwise over 0.5 hr at                culture tubes and statically incubated. One hundred microliters
−78oC, and the reaction mixture was stirred for an additional           of a 10× diluted aliquot of yeast seed culture were inoculated
0.5 hr. The reaction mixture was transferred into a separatory          into 100 mL of YMPG broth in Erlenmeyer flasks. The
funnel, washed with 2×25 mL portions of water, 2×25 mL                  numbers of viable cells were estimated as colony-forming
portions of 1 N NaOH solution, and with 25 mL portions                  units (CFU)/mL by spiral plating (Spiral Autoplate System;
of water until neutral pH was achieved. The organic phase               Spiral Biotech Inc., Bethesda, MD, USA) onto plate count
was separated, dried with anhydrous magnesium sulfate,                  agar (Difco Laboratories) and incubating for 24 to 48 hr.
filtered, and evaporated. The resulting residue was dissolved           All growth studies were performed at 30oC.
in dichloromethane and separated by Silica gel columns
using hexane as the eluent. Synthesis of diethyl trisulfide             Determination of minimum inhibitory concentration (MIC)
was carried out in the same manner used for dipropyl                    Each material [dialk(en)yl sulfide] was individually dissolved
trisulfide (20). All Synthesized alk(en)yl sulfides were                in the appropriate growth medium with 0.05% (w/v) Tween
characterized by NMR (1H NMR and 13C NMR: Bruker                        80 as a surfactant to make a 500-ppm (w/v) stock solution
DPX-400, 9.4T/spectrometer 400MHz/ Dual 5 mm probe;                     and filter-sterilized. Stock solutions of test materials were
Karlsruhe, Germany) and GC/MS (Trace GC/Finnigan TQS                    diluted with heat-sterilized culture broth to give the desired
7000 system; Restek, PA, USA) (Table 1). The GC column                  final concentrations. Medium at the desired test concentration
(30 m capillary; J&W Scientific Inc., Folsom, CA, USA)                  was inoculated with microorganisms to give initial numbers
was coated with DB-5 (0.32 m thickness), and the oven                   between 104-105 CFU/mL and incubated at 30oC for 24
temperature was increased from 60 to 200oC at 10oC/min,                 and 48 hr for S. aureus B31 and C. utilis ATCC42416,
with holding at initial and final temperatures for 3 min                respectively. The sensitivity of the test organisms was
each. The carrier gas was He, and injection volumn was 2                expressed as the minimum inhibitory concentration (MIC
µL. Electron impact ionization voltage, ionization current,             in ppm). Experiments were performed in duplicates, and
and source temperature were −70 eV, 400 µA , and 185oC,                 the higher values were recorded as the MIC. A complete
respectively (Table 1).                                                 absence of growth based on the viable count (Spiral Autoplate

Table 1. NMR and GC/MS analysis for characterized synthesis of dialk(en)yl sulfides
     Dialk(en)yl                              NMR                                                 GC-MS                          Purity
      Sulfides1)                     (1H NMR or 13C NMR)                                  (m/z, relative intensity)               (%)
                    H NMR(400MHz, CDCl3): δ5.23(4H, m), 5.87(2H, m),
      DATTS                                                                       210(M+, 31), 146(100), 73(79)                    85
                    H NMR(400MHz, CDCl3): δ2.59(6H, s)
      DMTTS                                                                       157.8(M+,100), 78.9(60), 63.9(19)                86
                     C NMR(100MHz, CDCl3): δ23.68
                    H NMR(400MHz, CDCl3): δ1.34(6H,t,J=7.4Hz),
      DETTS                                                                       185.9((M+,100), 92.9(55.5), 64(21.1)             85
                    H NMR(400MHz, CDCl3): δ1.02(6H, t,J=7.4Hz),
      DPTTS                                                                       213.8(M+, 100), 107.8, 129.7, 171.8              82
                   1.79(m,4H), 2.91(4H,t,J=7.4Hz)
                    H NMR(400MHz, CDCl3): δ0.9(3H), 1.75(2H), 2.8(2H),            179.8(M+, 11.10), 114.9(100),
       DPTS                                                                                                                        83
                   3.5(2H), 5.15(2H), 5.8(1H)                                     72.93(81.6), 73.95(22.7)
       DETS        1
                    H NMR(400MHz, CDCl3): δ1.3(6H), 2.9(4H)                                                                        81
 DATTS; diallyl tetrasulfide, DMTTS; dimethyl tetrasulfide, DETTS; diethyl tetrasulfide, DPTTS; dipropyl tetrasulfide, DPTTS; dipropyl trisul-
 fide, DETS; diethyl trisulfide.
Antimicrobial Activity of Alk(en)yl Sulfides Found in Essential Oils of Garlic and Onion                                        237

System, Spiral Biotech Inc.) after the incubation period            Table 2. Minimum inhibitory concentrations of alk(en)yl sulfides
was regarded as non-growth.                                         against Candida utilis ATCC424161) and Staphylococcus aureus
Effect of cysteine on antiyeast activity of DATS DATS                                                             MIC (ppm)
was dissolved in the appropriate YMPG broth with 0.05%                               Alk(en)yl sulfides     C. utilis   S. aureus
(w/v) Tween 80 as a surfactant to make a 500-ppm (w/v)                                                    ATCC42416        B31
stock solution and filter-sterilized. Stock solution of DATS                        Garlic oil                 25          100
was diluted with YMPG broth containing cysteine (0 to 2             Essential oil
                                                                                    Onion oil                  25          100
mM) to give the desired final concentrations. After 3 hr at
                                                                      Alk(en)yl     Dimethyl monosulfide     >500         >500
room temperature, YMPG broth containing cysteine with                  sulfides     Diethyl monosulfide      >500         >500
DATS at the desired test concentration was inoculated with                          Diallyl monosulfide      >500         >500
C. utilis ATCC42416 to give initial numbers of 2.2-2.4×104                          Dipropyl monosulfide     >500         >500
CFU/mL and incubated at 30oC for 48 hr. The sensitivity                             Dimethyl disulfide       >500         >500
of the test organism was expressed as the MIC.                                      Diethyl disulfide        >500         >500
                                                                                    Diallyl disulfide         110         >500
                                                                                    Dipropyl disulfide        130         >500
Results and Discussion                                                              Dimethyl trisulfide        15         >500
The garlic oil (GO) used in this work was analyzed by                               Diethyl trisulfide         20           40
reverse-phase HPLC as previously described (10). It contained                       Diallyl trisulfide         7            40
                                                                                    Diproyl trisulfide       >500           50
16 different kinds of sulfides, of which alk(en)yl groups                           Dimethyl tetrasulfide      10           30
were either diallyl-, dimethyl- or methyl allyl sulfides with                       Diethyl tetrasulfide       4            50
different numbers of sulfur atoms (10). Other studies (8,                           Diallyl tetrasulfide       4            30
11) reported findings of 14 to 17 kinds of sulfides in GO.                          Dipropyl tetrasulfide    >500           60
Garlic sulfides have only allyl- and methyl- groups, because        1)
                                                                      MICafter 48 hr of incubation, basal medium was YMPGB.
garlic contains S-allyl-L-cysteine sulfoxide and S-methyl-          2)
                                                                     MIC after 24 hr of incubation, basal medium was TSB.
L-cysteine sulfoxide as precursors of sulfides (7, 11).
   GO used in this work contained highest amounts of diallyl
disulfide (DADS; 30.6%) and diallyl trisulfide (DATS;               under the experimental conditions.
30.1%) and small amounts of alk(en)yl sulfides as follows:             Among disulfides, Not much difference in antiyeast
DAMS (2.8%), DATTS (14.1%), DAPS (3.6%), DAHS                       activity was observed between diallyl and dipropyl derivatives;
(1.4%), methyl allyl monosulfide (0.4%), methyl allyl disulfide     however, DADS had a marginal advantage over dipropyl
(2.6%), methyl allyl trisulfide (4.7%), methyl allyl tetrasulfide   disulfide (DPDS; Table 2). Dimethyl and diethyl disulfides,
(2.1%), methyl allyl pentasulfide (0.9%), methyl allyl              on the other hand, did not inhibit the yeast growth. This
hexasulfide (1.8%), DMDS (0.7%), DMTS (0.2%), DMTTS                 indicates that saturation/unsaturation of alk(en)yl groups
(3.0%), and DMPS (0.5%) (10). Others also mentioned that            has less influence on the activity of disulfides than the number
DADS was highest in their GO. DADS comprised more                   of carbon atoms. All disulfides tested were not inhibitory
than half of GO as reported by O'Gara et al. (8), with              to the growth of S. aureus.
DATS being the second most highest. GO of Lawson et al.                Among trisulfides the diallyl derivative had approximately
(11) contained high amounts of mixed sulfides, methyl allyl         two- and threefold stronger antiyeast effects as the dimethyl
disulfide and trisulfide probably due to the effect of climatic     and diethyl derivatives, respectively. Disulfide with allyl
condition during cultivation of garlic. Garlic grown in cooler      group (MIC of DADS = 110 ppm) showed higher inhibition
climatic condition is known to have higher allyl to methyl          against the growth of C. utilis than the that with methyl
ratio (21).                                                         group (MIC of DMDS = >500 ppm), but much lower was
   Sulfides with three or more sulfur atoms of our GO sample        observed in trisulfides (MIC of DATS = 7 ppm; MIC of
(10) and those of Lawson et al. (11) comprised about 62             DMTS = 15 ppm). Dipropyl trisulfide (DPTS) was not
and 58% of all sulfides, whereas those of O'Gara et al. (8)         inhibitory against the growth of C. utilis. At present, actions
contained much less (29%). Thus, because sulfides with              of the sulfides in terms of their inhibitory activity against
greater number of sulfur atoms are known to have higher             the yeast are unexplainable. DMTS, significantly antiyeast
antimicrobial activity, GO of O'Gara et al. (8) is expected         (MIC 15 ppm), was not antibacterial (MIC >500 ppm).
to be least potent among the three GOs.                             Other trisulfides showed similar antibacterial activities
   The antimicrobial potencies of GO and OO were very               against S. aureus.
similar, with MICs of 25 and 100 ppm for C. utilis ATCC42416           Diallyl and diethyl tetrasulfides showed extremely potent
and S. aureus B31, respectively (Table 2), even though the          antiyeast activities (MIC = 4 ppm). The antiyeast potency of
constituent sulfur compounds of the two oils are quite              these sulfides was comparable to that of allyl isothiocyanate
different. Artificially synthesized sulfides were tested for        (AITC), a natural sulfur compound found in mustard with
antimicrobial activity against the growths of C. utilis, and        extremely potent antimicrobial activity, showing MICs of
S. aureus to elucidate the antimicrobial activities of GO           1-5 ppm against various yeasts (13, 22, 23). Dimethyl
and OO.                                                             tetrasulfide (DMTTS) also showed very potent antiyeast
                                                                    activity; MIC being 10 ppm. All tetrasulfides showed potent
Effects of different alk(en)yl groups on the antimicrobial          antibacterial activities, with MICs ranging from 30 ppm for
activity of sulfides All monosulfides showed no antimicrobial       DMTTS to 60 ppm for dipropyl tetrasulfide (DPTTS). The
activity (MIC>500 ppm) against C. utilis and S. aureus              antibacterial activity of tetrasulfides increased as the number
238                                                                                                                       J.W. Kim et al.

of carbons in the alk(en)yl group decreased (Table 2).

Effects of number of sulfur atoms on the antimicrobial
activity of sulfides The number of sulfur atoms available
is important in conferring the potency of antimicrobial activity
against dialk(en)yl sulfides (8, 10, 13). Addition of a single
sulfur atom to disulfides dramatically increased antimicrobial
activity of sulfides (trisulfides). However, there are two
exceptions to this observation, the antibacterial activity of
DMTS and the antiyeast activity of dipropyl sulfides. Among
the dipropyl sulfides, only dipropyl disulfide (DPDS) was
antiyeat. Potency of the antimicrobial activity of GO is
expected to be dictated by the concentrations of different
sulfides. Various samples of GO have shown a wide variation
in their contents of different sulfides (8, 10, 11). GO analyzed
by O'Gara et al. (8) contained 53 and 10% DADS and
DAMS, respectively, both of which were found by Kim et
al. (10) to be only very weakly antimicrobial, while the GO
samples evaluated in the current study and that of Lawson
et al. (11) contained approximately 60% sulfides with
three or more sulfur atoms, and thus would be expected to
show more potent antimicrobial activity than that of O'Gara
                                                                   Fig. 1. Change of minimum inhibitory concentration of DATS
et al. (8).                                                        against Candida utilis ATCC 42416 depending on cysteine
    Full-scale analysis on the sulfur compounds of OO is           concentration. MIC after 48 hr of incubation, initial number
not available. Presumably, sulfides with more than six sulfur      (CFU/mL) was 2.2-2.4×104, basal medium was YMPGB.
atoms exist at very small concentrations, below detection
level or resist analysis. These sulfides should have even more
potent antiyeast activities than those with fewer sulfur           unlike the SO-, -S-S-, and S- groups, could react with
atoms.                                                             sulfhydryl enzymes. Sulfides, especially those with three
    Thiosulfinates, including allicin, are known to be             or more sulfur atoms, apparently possess potent antimicrobial
antimicrobial (1, 3, 5) due to their S(O)-S- group, which is       activity.
believed to react with the SH group of cellular proteins to
generate mixed disulfides (3, 5). Therefore, the antimicrobial
activity of thiosulfinates is inactivated by cysteine (3, 5,
24). Various sulfides, including DADS, DMDS, DPDS, DATS,            1. Cavallito CJ, Bailey JH. Allicin, the antibacterial principle of
DMTS, and DATTS are antimicrobial (Table 2), with stronger             Allium sativum. I: Isolation, physical properties and antimicrobial
                                                                       action. J. Am. Chem. Soc. 66: 1950-1951 (1944)
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