Anti-Salmonella Activity of Alkyl Gallates
J. Agric. Food Chem., Vol. 50, No. 23, 2002 6693
Hexyl gallate (hexyl 3,4,5-trihydroxybenzoate) (8) was obtained in
58% yield as a colorless solid: 1H NMR (270 MHz, CDCl3) δ 0.88 (t,
J ) 6.5 Hz, 3H, -CH3), 4.18-4.28 (br, 2H, -OCH2), 6.4-5.8 (br,
3H, exchangeable with D2O, -ArOH), 7.20 (s, 2H, ArH); IR (KBr)
3389, 2932, 1688, 1615, 1449, 1314, 1258, 1026 cm-1
.
Geranyl gallate (3,7-dimethylocta-2,6-dienyl 3,4,5-trihydroxyben-
zoate) (9) was obtained in 57% yield as colorless powder: mp 67.0-
1
68.5 °C; H NMR (300 MHz, CDCl3) δ 1.60 (s, 3H, -CH3), 1.67 (d,
J ) 0.6 Hz, 3H, -CH3), 1.75 (d, J ) 1.2 Hz, 3H, -CH3), 2.08 (m,
4H, -CH2), 4.79 (d, J ) 7.2 Hz, 2H, -OCH2), 5.09 (m, 1H, -CH),
5.43 (qt, J ) 7.2, 1.2 Hz, 1H, -CH), 6.12 (br s, 3H, -OH), 7.31 (s,
2H, ArH); IR (KBr) 3549, 3406, 3288, 1686, 1614, 1537, 1312, 1244,
1229, 1198, 1022 cm-1; EI-MS (m/z) 306 (M+), 237, 170, 153 (base
peak), 136, 121, 93, 69, 41; HRMS-FAB (m/z) [M + H]+ calcd for
C17H23O5 307.1546, found 307.1561.
Test Strains. The test strains, Salmonella choleraesuis ATCC 35640,
Escherichia coli ATCC 9637, Pseudomonas aeruginosa ATCC 10145,
Enterobacter aerogenes ATCC 13048, and Proteus Vulgaris ATCC
13315, used for this study were purchased from American Type Culture
Collection (Manassas, VA). The strain of Ps. aeruginosa IFO 3080
was available from our previous work.
Medium. NYG broth (0.8% nutrient broth, 0.5% yeast extract, 0.1%
glucose) was used for the antibacterial assay. Nutrient broth was
obtained from BBL Microbiology System (Cockeysville, MD). Yeast
extract was purchased from Difco Lab (Detroit, MI).
Precultivation. The cells of S. choleraesuis ATCC 35640 were
precultured in 3 mL of NYG broth without shaking at 37 °C for 16 h.
The preculture was used for the following antibacterial assay and time
kill study.
Figure 1. Chemical structures of alkyl gallates and related compounds.
dilute aqueous citric acid solution, saturated aqueous NaHCO3 solu-
tion, and water, dried over MgSO4, and evaporated. The crude products
were purified by chromatography (SiO2; elution with CHCl3-MeOH,
98:2, v/v). Structures of the synthesized esters were established
by spectroscopic methods (IR, MS, and NMR) (Figure 1).
Their analogues, described in this paper, were synthesized in the same
manner.
Antibacterial Assay. Broth macrodilution methods were followed
as previously described (9) with slight modifications. Briefly, serial
2-fold dilutions of the test compounds were prepared in DMF, and 30
µL of each dilution was added to 3 mL of NYG broth. These were
inoculated with 30 µL of preculture of S. choleraesuis. After incubation
of the cultures at 37 °C for 24 h, the minimum inhibitory concentration
(MIC) was determined as the lowest concentration of the test compound
that demonstrated no visible growth. The minimum bactericidal
concentration (MBC) was determined as follows. After the determi-
nation of the MIC, 100-fold dilutions with drug-free NYG broth from
each tube showing no turbidity were incubated at 37 °C for 48 h. The
MBC was the lowest concentration of the test compound that was not
visible in the drug-free cultivation. The concentration of DMF in each
medium was always 1%. The highest concentration tested was 3200
µg/mL unless otherwise noted.
Measurement of Oxygen Uptake. Exponentially growing Ps.
aeruginosa IFO 3080 cells were harvested and washed with saline by
centrifugation. The cells were suspended in 50 mM phosphate buffer
(pH 7.0) to give approximately 1 mg of dry cells per milliliter. The
test compound dissolved in DMSO was added to the reaction mixture,
and the O2 consumption was measured polarographically at 30 °C with
an OBH 100 oxygen electrode (10).
Preparation of Bacterial Cell Membrane. Exponentially growing
Ps. aeruginosa IFO 3080 cells were harvested by centrifugation and
then washed twice with distilled water. The cell paste was suspended
in 50 mM Tris-HCl buffer (pH 7.4) containing 0.5 M sucrose and 20
mM MgCl2, and then it was disrupted by ultrasound using a Sonifier
450 at 10 kc for 2 min at 4 °C. After centrifugation of the cell
suspension at 15000g for 20 min, the supernatant was centrifuged at
105000g for 90 min. The resultant precipitate was washed by
centrifugation at 105000g for 60 min with 10 mM Tris-HCl buffer (pH
7.4) containing 0.5 M sucrose and 10 mM MgCl2. The precipitate was
resuspended in the same buffer (11).
Tridecyl gallate (tridecyl 3,4,5-trihydroxybenzoate) (3) was obtained
in 77% yield as a colorless solid: 1H NMR (270 MHz, CDCl3) δ 0.88
(t, J ) 6.5 Hz, 3H, -CH3), 4.24 (t, J ) 6.5 Hz, 2H, -OCH2), 7.16 (s,
2H, ArH); IR (KBr) 3520, 3495, 3010, 2960, 1686, 1615, 1480, 1420,
1395, 1270, 1140 cm-1
.
Dodecyl gallate (dodecyl 3,4,5-trihydroxybenzoate) (2) was obtained
in 65% yield as a colorless solid: 1H NMR (270 MHz, CDCl3) δ 0.88
(t, J ) 6.5 Hz, 3H, -CH3), 4.24 (t, J ) 6.5 Hz, 2H, -OCH2), 7.16 (s,
2H, ArH); IR (KBr) 3515, 3490, 3000, 2960, 1680, 1610, 1480, 1395,
1270, 1145 cm-1. Dodecyl gallate was also purchased from Sigma
Chemical Co. but was recrystallized prior to use.
Undecyl gallate (undecyl 3,4,5-trihydroxybenzoate) (4) was obtained
in 63% yield as a colorless solid: 1H NMR (270 MHz, CDCl3) δ 0.88
(t, J ) 6.5 Hz, 3H, -CH3), 4.24 (t, J ) 6.5 Hz, 2H, -OCH2), 7.16 (s,
2H, ArH); IR (KBr) 3500, 3450, 2960, 2890, 1685, 1620, 1480, 1395,
1280, 1160 cm-1
.
Decyl gallate (decyl 3,4,5-trihydroxybenzoate) (5) was obtained in
74% yield as a colorless solid: 1H NMR (270 MHz, CDCl3) δ 0.88 (t,
J ) 6.5 Hz, 3H, -CH3), 4.24 (t, J ) 6.5 Hz, 2H, -OCH2), 7.16 (s,
2H, ArH); IR (KBr) 3391, 2924, 1686, 1615, 1448, 1312, 1258, 1028
cm-1; HRMS-EI (m/z) [M]+ calcd for C17H26O5 310.1778, found
310.1788.
Nonyl gallate (nonyl 3,4,5-trihydroxybenzoate) (6) was obtained in
86% yield as colorless needles from benzene-n-pentane: mp 96.7-
97.3 °C; 1H NMR (300 MHz, CDCl3-CD3OD) δ 0.88 (t, J ) 6.6 Hz,
3H, -CH3), 4.25 (t, J ) 6.6 Hz, 2H, -OCH2), 7.21 (s, 2H, ArH); IR
(KBr) 3489, 3439, 3333, 1670, 1625, 1604, 1531, 1300, 1256, 1024
cm-1; HRMS-EI (m/z) [M]+ calcd for C16H24O5 296.1630, found
296.1624.
Octyl gallate (octyl 3,4,5-trihydroxybenzoate) (1) was obtained in
89% yield as a colorless solid: 1H NMR (270 MHz, CDCl3) δ 0.88 (t,
J ) 6.5 Hz, 3H, -CH3), 4.24 (t, J ) 6.5 Hz, 2H, -OCH2), 7.15 (s,
2H, ArH); IR (KBr) 3325, 2926, 1686, 1614, 1468, 1314, 1246, 1028
cm-1; HRMS-EI (m/z) [M]+ calcd for C15H22O5 282.1469, found
282.1473.
Enzyme Assay. NADH oxidase activity was assayed by measuring
the decrease in the absorbance at 340 nm. The reaction mixture
contained 0.1 M Tris-HCl buffer (pH 7.5), 200 µM NADH, and
membrane fraction (equivalent to 2 mg of protein) (12).
Heptyl gallate (heptyl 3,4,5-trihydroxybenzoate) (7) was obtained
in 71% yield as a colorless solid: 1H NMR (270 MHz, CDCl3) δ 0.88
(t, J ) 6.5 Hz, 3H, -CH3), 4.24 (t, J ) 6.5 Hz, 2H, -OCH2), 7.16 (s,
2H, ArH); IR (KBr) 3389, 2920, 1685, 1620, 1448, 1320, 1258, 1140
RESULTS AND DISCUSSION
In our previous report, propyl (C3), octyl (C8), and dodecyl
(C12) gallates were tested for their antimicrobial activity against
cm-1
.