Antibacterial Effects of Guava Tannins and Related Polyphenols on Vibrio and Aeromonas Species
Fumi Yamanakaa, Tsutomu Hatanoa, *, Hideyuki Itoa, Shoko Taniguchib, Eizo Takahashic, and Keinosuke Okamotoc
aDepartment of Pharmacognosy, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Tsushima, Okayama 700-8530, Japan
bMedicinal Botanical Garden, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Tsushima, Okayama 700-8530, Japan
cDepartment of Pharmacogenetics, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Tsushima, Okayama 700-8530, Japan
Received: November 23rd, 2007; Accepted: February 11st, 2008
An extract obtained from a bottled tea drink of Psidium guajava L. (Myrtaceae) showed antibacterial effects on Vibrio vulnificus, V. mimicus, V. parahaemolyticus, and Aeromonas sobria. HPLC-diode array detector (DAD) analysis of an effective fraction obtained from the extract revealed the presence of several tannins and related polyphenols. To verify these results and to estimate the antibacterial effects of the polyphenols, we isolated the polyphenols from the leaves of P. guajava.
Among the polyphenols isolated, pedunculagin, castalagin, casuarinin, and stenophyllanin A were effective against Vibrio and Aeromonas species. Studies of structurally related compounds revealed that penta-O-galloyl-β-D-glucose (PGG), (-)-epigallocatechin gallate (EGCG), and alkyl gallates such as isoamyl gallate (IG) and n-octyl gallate exhibited potent antibacterial activities. The minimum bactericidal concentrations (MBCs) of three polyphenols (i.e., PGG, EGCG, and IG) that exhibited low minimum inhibitory concentrations (MICs) were then determined. Comparisons of the MBCs and MICs indicated that PGG was not bactericidal at the MIC, whereas EGCG and IG were. The effect of combinations of the three polyphenols with several antibiotics was also examined. The combination of IG and kanamycin (KM) effectively reduced the MIC of KM against V. vulnificus and V. mimicus; the combination of EGCG and tetracycline (TC) also reduced the MIC of TC against V. parahaemolyticus. Thus, polyphenols may be useful in the development of antibacterial agents against Vibrio bacteria.
Keywords: polyphenol, tannin, Vibrio, Aeromonas, antibiotic, antibacterial, Psidium guajava, Myrtaceae.
In Japan, more than 2000 people per year are affected by food poisoning caused by species of Vibrio and Aeromonas; however, only a limited number of antibiotics are available for use against these bacteria.
Still, it has been revealed that guava polyphenols are useful as antibacterial materials against Vibrio and related Aeromonas species [1]. The leaf, bark, and fruit of guava, Psidium guajava L. (Myrtaceae), are used in traditional medicine [2]. A clinical study suggested usefulness of a phytodrug derived from this plant for the treatment of diarrhea [3].
Antibacterial effects of extracts from this plant on some bacteria have also been reported [4-7]. Guava leaves are rich in tannins and polyphenols [8-10].
Moreover, several tannins and related polyphenols from various plants have antibacterial effects, and combinations of polyphenols and antibiotics are effective against methicillin-resistant Staphylococcus aureus (MRSA) and other bacteria [11-18]. A recent report indicated that intestinal chloride secretion caused by cholera toxin was inhibited by a plant extract containing proanthocyanidins (condensed tannin), and this was attributed to the interaction between the tannin and toxin [19].
We report here the antibacterial properties of the polyphenols in guava tea using Vibrio and related Aeromonas species. We prepared a guava leaf extract
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Table 1: Minimum inhibitory concentrations (MICs, μg/mL) of guava tea drink extract and its fractions a.
a A guava tea drink was passed through a Sep-pak ODS cartridge. The adsorbed compounds were then eluted with H2O, 20, 40, and 100% MeOH, and several fractions were collected.
Table 2: Minimum inhibitory concentrations (MICs, μg/mL) of 70% acetone extract from fresh leaves of guava and its fractions, obtained by solvent extraction a.
a A concentrate prepared from a 70% acetone extract made from fresh leaves was extracted with Et2O, EtOAc, and n-BuOH, respectively, and several fractions were collected.
Stenophyllanin A
Ellagic acid
Quercetin-3-O-β-D-glucuronide
Casuarinin
Gallic acid
(+)-Catechin
Wavelength
Retention time Stenophyllanin A
Ellagic acid
Quercetin-3-O-β-D-glucuronide
Casuarinin
Gallic acid
(+)-Catechin
Wavelength
Retention time
Figure 1: HPLC-DAD profile of the 20% MeOH eluate of the guava tea drink. Column, YMC ODS-A A-302; gradient elution with (A) 10 mM H3PO4/10 mM KH2PO4/CH3CN (45:45:10) and (B) 10 mM H3PO4/10 mM KH2PO4/CH3CN (35:35:30); elution profile, 0–10 min, solvent A; 10–30 min, 0–100% solvent B in
solvent A (linear gradient); flow rate, 1.0 mL/min; detection, UV at 220–400 nm.
Gallic acid
Stenophyllanin A
(+)-Catechin
Casuarinin Pedunculagin
Procyanidin B1 Castalagin
Ellagic acid
Wavelength
Retention time Hyperin
Isoquercitrin
Gallic acid
Stenophyllanin A
(+)-Catechin
Casuarinin Pedunculagin
Procyanidin B1 Castalagin
Ellagic acid
Wavelength
Retention time Hyperin
Isoquercitrin
Figure 2: HPLC-DAD profiles of the n-BuOH-soluble portion of fresh guava leaves.
Vibrio vulnificus V. mimicus V. parahaemolyticus Aeromonas sobria
Guava tea drink 256 2048 1024 1024
H2O eluate 512 >2048 1024 2048
20% MeOH eluate 64 512 512 256
40% MeOH eluate 128 1024 512 512
100% MeOH eluate 256 2048 1024 512
V. vulnificus V. mimicus V. parahaemolyticus A. sobria
Guava extract 128 1024 512 512
Et2O extract 128 2048 1024 1024
EtOAc extract 64 512 512 512
n-BuOH extract 64 512 256 256
H2O soluble residue 128 2048 2048 1024
O
O O
CH2
CO OC
HO
HO OH HO OH OH OH
HO HO HO HO
OH
COO H
CO OH
HO OH
O
HO OH
OH OH
OH COO
Stenophyllanin A
O
O O
CH2
CO OC HO
HO OH HO OH OH OH
HO HO HO HO
OH COO
COO OH
H
Casuarinin O
O O CH2
CO OC HO
HO OH HO OH OH OH
HO HO HO HO
OH COO COO
OH
Pedunculagin
O
OH HO
OH
OH OH
(+)-Catechin
O
O O
O HO
HO
OH OH
Ellagic acid HO
CO CO OH HO OH HO OH
H2C
O O
CO
O O
OC CO
O
HO OH
OH OH
OH OH
OH OH H
Castalagin O
HO
OH
OH OH O
HO
OH
OH OH
OH OH
Procyanidin B1
CO
OH OH HO
COOH
Gallic acid
HO OH
OH
HO OH
O HO
OH
OH OH
O
Hyperin O HOCH2
OH OH HO
O
O HO
OH
OH OH
O
Isoquercitrin O HOCH2
OH OH HO
O
O HO
OH
OH OH
O
Quercetin-3-O-β-D-glucuronide O
COOH
OH OH HO
O
Figure 3a: Structures of the polyphenols found in guava leaves.
O O
O CH2
OC CO HO
OH OH
HO OH
OH HO
HO HO HO
HO OH
COO
O
OH OH OH
COO CO
O
O HO
OH
OH OH OH CO
Penta-O-galloyl-β-D-glucose (PGG)
Geraniin
(-)-Epigallocatechin gallate (EGCG) Corilagin
O
O O
CO CO
O HO OH
O OH HO OH
O CO OC
OH HO OH HO OH HO
H H2C
O
O OH
OH
OH CO
O
HO OH
O OC CO
OH HO OH OH HO HO
H2C O
O OH
OH
OH
CO OH
OH OH
Figure 3b: Structures of the related compounds from other sources.
by the evaporation of a commercial guava tea drink.
The extract had antibacterial effects on three Vibrio strains and an Aeromonas strain. The minimum inhibitory concentrations (MICs) ranged between 256 and 2048 μg/mL (Table 1); V. vulnificus was the most sensitive to the extract (MIC 256 μg/mL). Since the yield of the extract indicated that the concentration of the extract in the drink was 2500 μg/mL, the concentration is high enough for the antibacterial effects. Fractionation of the extract on a Sep-pak ODS cartridge with aqueous MeOH produced a 20% MeOH eluate that had a much stronger antibacterial effect (MIC 64 μg/mL
for V. vulnificus) than the original extract. HPLC analysis has been used successfully to characterize a mixture of plant tannins [20]. HPLC-diode array detector (DAD) analysis of the active fraction (Figure 1), based on the modification of this HPLC condition, indicated that the major constituents were hydrolyzable tannins (stenophyllanin A and casuarinin), catechin, gallic acid, ellagic acid and quercetin-3-O-β-D-glucuronide.
To purify the active components, fresh guava leaves were extracted with 70% acetone (see Experimental).
The n-BuOH-soluble portion of the extract had a
Table 3: Contents (w/w %) of the polyphenols in guava leaves and guava tea drink.
Guava tea drink extract 70% Acetone extract from fresh guava leaves
Procyanidin B1 0.58 0.92
Pedunculagin - a 0.85
Castalagin 0.30 1.45
Casuarinin 0.42 0.39
Stenophyllanin A 2.06 0.87
Hyperin 0.52 0.61
Isoquercitrin - a 0.32
Quercetin-3-O-β-D-glucuronide 0.51 - a
(+)-Catechin 1.01 2.20
Ellagic acid 0.62 0.51
Gallic acid 0.88 0.50
a Not observed in the HPLC profiles.
Table 4: MICs (μg/mL) of the polyphenols found in guava leaves and related compounds.
V. vulnificus V. mimicus V. parahaemolyticus A. sobria
Procyanidin B1 a 256 >1024 1024 512
Pedunculagin a 32 512 256 128
Castalagin a 32 256 128 64
Casuarinin a 16 256 128 128
Stenophyllanin A a 32 256 128 256
Hyperin a 256 >1024 >1024 >1024
Isoquercitrin a 1024 >1024 >1024 >1024
Quercetin-3-O-β-D-glucuronide a 512 >1024 >1024 >1024
(+)-Catechin b 512 >1024 >1024 >1024
Ellagic acid b 16 512 1024 1024
Gallic acid b 16 512 128 128
Geraniin 16 256 256 256
Corilagin 16 512 256 128
PGG c 16 32 32 128
EGCG c 8 64 64 64
a Isolated from guava leaves or guava tea drink.
b Detected as the constituents of guava leaves.
c Abbreviations used: EGCG, (-)-epigallocathechin gallate; PGG, penta-O-galloyl-β-D-glucose.
greater antibacterial effect than did the fractions prepared from the guava tea drink (Table 2). The n-BuOH extract was analyzed using HPLC-DAD (Figure 2). In addition to the polyphenols found in guava tea drink, the presence of castalagin, pedunculagin, procyanidin B1, hyperin, and isoquercitrin were also indicated. The heating process used to prepare the bottled product resulted in the discrepant profiles obtained for fractions derived from fresh leaves and those derived from the guava tea drink. Such a change in profile after processing during the preparation of bottled products was shown for a tea drink made from Thea sinensis leaves [21].
We then purified the polyphenols by chromatography using Toyopearl and MCI gels, Sep-pak cartridges, and preparative HPLC. Seven polyphenols were identified based on their 1H NMR data and by co-chromatography with the authentic compounds:
procyanidin B1 [10], pedunculagin, casuarinin [8], castalagin, stenophyllanin A [22], hyperin, isoquercitrin [23]. Quercetin-3-O-β-D-glucuronide [24] was also isolated from the 20% MeOH eluate from the guava tea drink (Figure 3).
The antibacterial effects of the polyphenols found in guava leaves on Vibrio and Aeromonas species were then examined. V. vulnificus was the most sensitive to the polyphenols; the MICs of the polyphenols for V. vulnificus ranged from 8 to 512 μg/mL (Table 4). In comparison, the MICs for V. mimicus, V. parahaemolyticus, and A. sobria were somewhat higher (MIC: 32–1024 μg/mL; Table 2).
Among the polyphenols, the catechins [(+)-catechin and procyanidin B1] and flavonoid glycosides had low antibacterial effects, whereas the hydrolyzable tannins (pedunculagin, castalagin, casuarinin, and stenophyllanin A) had greater effects. Constituent polyphenolic acid (gallic acid) and a hydrolysate from tannins (ellagic acid) also had antibacterial effects. These results indicated that tannins of guava leaves were effective. The contents of the tannins and related polyphenols in the guava tea drink extract and of the fresh leaves were as shown in Table 3, based on their HPLC profiles. The content of stenophyllanin A, the representative tannin in the guava tea drink extract, corresponds to the concentration 51.5 μg/mL in the drink, and it was somewhat higher than the MIC of this tannin against V. vulnificus.
Table 5: MICs (μg/mL) of alkyl gallates.
V. vulnificus V. mimicus V. parahaemolyticus A. sobria
Methyl gallate 16 64 64 64
Ethyl gallate 16 64 64 64
n-Propyl gallate 16 64 64 64
n-Butyl gallate 16 64 64 128
Isoamyl gallate 16 32 64 128
n-Octyl gallte 4 16 16 32
n-Dodecyl gallate 64 128 128 >1024
Stearyl gallate >1024 >1024 >1024 >1024
OH HO OH
COOR
Methyl gallate: R=CH3 Ethyl gallate: R=CH2CH3 n-Propyl gallate: R=CH2CH2CH3
n-Butyl gallate: R=CH2(CH2)2CH3 Isoamyl gallate: R=CH2CH2CH(CH3)2
n-Octyl gallate: R=CH2(CH2)6CH3 n-Dodecyl gallate: R=CH2(CH2)10CH3
Stearyl gallate: R=CH2(CH2)16CH3 Figure 4: Structures of alkyl gallates.
Tannins and polyphenols with related structures (Figure 3) from other sources were then examined. Penta-O-galloyl-β-D-glucose (PGG) and (-)-epigallocatechin gallate (EGCG) had greater effects than the others. The antibacterial effects of alkyl gallates with variable alkyl chain lengths (Figure 4) were also examined. The length of the alkyl chain influenced the antibacterial effect of the compound (Table 5). Alkyl gallates with medium- length (n-octyl) chains were the most effective against Vibrio bacteria. Gallates with long alkyl chains had lesser antibacterial effects [n-dodecyl gallate (MIC 64 μg/mL), stearyl gallate (MIC > 1024 μg/mL)]. n-Octyl gallate also had potent antibacterial effects on V. mimicus, V. parahaemolyticus, and A. sobria.
Because decreases in the MICs of antibiotics have been reported in the presence of some polyphenols [11-16, 25, 26], the effects of the addition of guava polyphenols, alkyl gallates, and related compounds on the MIC of kanamycin (KM) were then examined.
The concentrations of polyphenols were set at 1/4 of their MICs, to examine the synergic effects between the antibiotics and polyphenols [11]. The results are shown in Table 6. The addition of isoamyl gallate (IG) and several polyphenols decreased the MIC of KM against V. mimicus. Therefore, we further examined the effects of IG, and also representative polyphenols EGCG and PGG, on the MICs of antibiotics. As shown in Table 7, in the presence of IG, KM showed greater antibacterial effects on
Table 6: Effect of polyphenols on the MICs (μg/mL) of kanamycin against V. mimicus.
KM a
(alone) 4
Isoamyl gallate (8 μg/mL) b 1
n-Octyl gallate (4 μg/mL) b 2
n-Dodecyl gallate (32 μg/mL) b 2
Casuarinin (64 μg/mL) b 4
Stenophyllanin A (64 μg/mL) b 4
Hyperin (256 μg/mL) b 2
(+)-catechin (256 μg/mL) b 4
EGCG (16 μg/mL) b 2
PGG (8 μg/mL) b 4
a Abbreviations used: KM, kanamycin.
b The concentrations of polyphenols were set at 1/4 of their MICs.
Table 7: Effect of polyphenols on the MICs (μg/mL) of antibiotics against Vibrio and Aeromonas species.
(A) V. vulnificus
(B) V. mimicus.
VCM ABPC TC CP KM NFLX
(alone) 512 2 1 0.5 4 0.02 IG (8 μg/mL) b 512 2 0.5 0.5 1 0.02 EGCG (16 μg/mL) b 512 4 0.5 1 2 0.02 PGG (8 μg/mL) b 512 4 0.5 1 4 0.02
(C) V. parahaemolyticus
VCM ABPC TC CP KM NFLX
(alone) >1024 1 4 1 16 0.13 IG (16 μg/mL) b >1024 4 4 1 16 0.13 EGCG (16 μg/mL) b 1024 2 1 1 16 0.25 PGG (8 μg/mL) b 1024 1 2 1 16 0.25
(D) A. sobria
VCM ABPC TC CP KM NFLX
(alone) 256 256 64 1 8 0.03
IG (32 μg/mL) b 256 512 32 0.5 16 0.06 EGCG (16 μg/mL) b 128 256 64 1 8 0.03 PGG (32 μg/mL) b 128 256 64 0.5 8 0.03
a Abbreviations used: IG, isoamyl gallate; VCM: vancomycin; ABPC, ampicillin; TC: tetracycline; CP, chloramphenicol; NFLX, norfloxacin.
b The concentrations of polyphenols were set at 1/4 of their MICs.
Table 8: MICs (μg/mL) of aminoglycosides in the presence and absence of IG against V. mimicus.
SM a TOB a GM a AMK a
(alone) 4 0.5 0.25 1
IG (8 μg/mL) 1 0.13 0.03 0.13
a Abbreviations used: SM, streptomycin; TOB, tobramycin; GM, gentamicin; AMK, amikacin.
VCM a ABPC a TC a CP a KM a NFLX a (alone) >1024 0.5 0.25 0.5 64 0.06 IG a (4 μg/ mL) b >1024 0.5 0.25 0.5 16 0.06 EGCG (2 μg/mL) b >1024 0.5 0.25 0.5 32 0.06 PGG (4 μg/mL) b >1024 0.5 0.13 0.5 32 0.06
(A)
0.0 0.2 0.4 0.6 0.8 1.0
0 6 12 18 24
Time (h)
O.D. at 655 nm
(B)
0.0 0.2 0.4 0.6 0.8 1.0
0 6 12 18 24
Time (h)
O.D. at 655 nm
Figure 5: Effect of kanamycin (KM) and isoamyl gallate (IG) on growing of bacterial cultures. (A) V. parahaemolyticus: control (○), 8 μg/mL KM (●), 32 μg/mL IG (▲), 8 μg/mL KM + 32 μg/mL IG ( ).
(B) V. mimicus: control (○), 1 μg/mL KM (●), 8 μg/mL IG (▲), 1 μg/mL KM + 8 μg/mL IG ( ).
Table 9: Minimum bactericidal concentrations (MBCs, μg/mL) of three polyphenols.
PGG EGCG IG
V. mimicus 512 (32) a 64 (64) a 32 (32) a V. parahaemolyticus 1024 (32) a 64 (32) a 64 (64) a
a The values shown in parentheses are the MICs (μg/mL).
V. vulnificus and V. mimicus relative to those in the of polyphenols. The antibacterial effect of EGCG and tetracycline (TC) on V. parahaemolyticus was greater than that observed for TC alone (Table 7). The effect of IG on the MICs of aminoglycosides was further examined using V. mimicus; all of the tested combinations were effective (Table 8). In contrast, no potentiation of the effects of the antibiotics was observed for IG in the case of V. parahaemolyticus (Table 7). The magnitude of the effect on the MIC may be affected by difference in the growth rate of the bacteria. The effect of polyphenol treatment on the growth phase of the bacteria was then examined using V. parahaemolyticus, since its growth is fastest among the four strains. IG caused a noticeable delay in the growth of V. parahaemolyticus [Figure 5(A)], although the bacterial growth was not suppressed after 24-hour-incubation. To the contrary, the growth of V. mimicus was suppressed completely by the combination of IG and KM after the incubation [Figure 5(B)].
To clarify whether the antibacterial effects were bactericidal at the MICs, the minimum bactericidal concentrations (MBCs) [26] were evaluated for the three polyphenols PGG, EGCG, and IG for V. mimicus and V. parahaemolyticus (Table 9). If the MBC was not more than twice the MIC, the effect of the compound was deemed bactericidal at the MIC.
A comparison of the MBC and MIC indicated that the effects of EGCG and IG were bactericidal, whereas those of PGG were not.
The findings described above can be summarized as follows. The polyphenols contained in guava tea extract have antibacterial effects against Vibrio and Aeromonas bacteria. HPLC-DAD analysis of the effective fraction from the guava tea drink extract indicated that the major polyphenols were stenophyllanin A and casuarinin. The presence of these polyphenols was confirmed by isolation from fresh guava leaves. The tannins pedunculagin, castalagin, casuarinin, and stenophyllanin A, which were also isolated from the leaves, had significant antibacterial effects on V. vulnificus. We subsequently examined the antibacterial effects of other tannins and related alkyl gallates, including PGG, EGCG, and n-octyl gallate, which showed potent antibacterial activity. The effects of polyphenols on the MICs of several antibiotics were also examined; IG decreased the MICs of KM on V. vulnificus and V. mimicus. In the case of V.
mimicus, IG was also effective in combination with other aminoglycoside antibiotics. EGCG decreased the MIC of TC on V. parahaemolyticus.
Since the effect of this combination on the efflux pump induced in Staphylococci was reported [27], the effect on V. parahaemolyticus can be related to the effect on the efflux system in Vibrio. Previously, the restorative effects of polyphenols on the MICs of β-lactams were ascribed to their effect on the bacterial membrane [28]. Because the antibacterial effects of aminoglycosides are due to inhibition of protein synthesis occurring inside the membrane, their effects on Vibrio species were also attributed to membrane effects, which may facilitate the penetration of aminoglycosides into bacterial cells.
The effects of tannins and related polyphenols on various bacteria have been shown to be largely dependent on their structural differences. However, non-specific binding to the bacterial biomolecules may be attributed to the antibacterial effects.
The effects of tannins and related polyphenols were generally not so potent for clinical usage. However, the polyphenols that guava leaves contain are expected to be minimally toxic, because they are used in traditional medicine and as a drink.
Experimental
General: 1H NMR spectra were recorded using acetone-d6 containing 10% D2O as the solvent on a Varian INOVA AS600 (600 MHz). The chemical shifts are given as δ (parts per million) values relative to that of the solvent signal (acetone-d6, δH
2.04) on a tetramethylsilane scale. Normal-phase HPLC was conducted on a 250 × 4.6 mm i.d. YMC- Pack SIL A-003 column (YMC, Kyoto, Japan) developed with n-hexane/MeOH/tetrahydrofuran/
HCOOH (55:33:11:1) containing oxalic acid (450 mg/L) at ambient temperature. The flow rate was set at 1.5 mL/min, and UV detection was effected at 280 nm. Reverse-phase HPLC was performed on a 150 × 4.6 mm i.d. YMC-Pack ODS-A A-302 column (YMC) developed with 10 mM H3PO4/10 mM KH2PO4/CH3CN (45:45:10) at 40˚C. The flow rate was set at 1.0 mL/min, and UV detection was effected at 280 nm. Gradient elution was also performed on the ODS column with (A) 10 mM H3PO4/10 mM KH2PO4/CH3CN (45:45:10) and (B) 10 mM H3PO4/10 mM KH2PO4/CH3CN (35:35:30).
The elution profile used solvent A for 0–10 min, followed by a linear gradient of 0–100% solvent B in solvent A for 10–30 min. The flow rate was set at 1.0 mL/min, and detection was effected at 220–400 nm on a HITACHI L-7455 DAD detector.
Preparative reverse-phase HPLC was performed on a 300 × 10 mm i.d. YMC-Pack ODS-A A-324 column (YMC) developed with 10 mM H3PO4/10 mM KH2PO4/ CH3CN (42.5:42.5:15). Column chromatography was conducted using Toyopearl HW-40 (coarse grade; Tosoh, Tokyo, Japan) and MCI-gel CHP-20P (Mitsubishi Kasei, Tokyo, Japan). Sep-pak ODS Vac cartridges (Waters, Milford, MA) were also used for fractionation of the extracts and polyphenol purification. A BioRad Model 680 microplate reader (Tokyo, Japan) was used to monitor the turbidity caused by bacterial growth.
Materials: EGCG was isolated from Camellia sinensis leaves [29], whereas PGG was prepared by treating tannic acid (Dainippon Seiyaku, Osaka, Japan) with MeOH containing an acetate buffer at pH 6 [30]. Geraniin and corilagin were isolated from
Geranium thunbergii leaves [31]. Ellagic acid, (+)-catechin and antibiotics [i.e., KM, vancomycin (VCM), ampicillin (ABPC), norfloxacin (NFLX), streptomycin (SM), tobramycin (TOB), gentamicin (GM), and amikacin (AMK)] were purchased from Sigma (St. Louis, MO). TC and chloramphenicol (CP) were purchased from Wako (Osaka, Japan), and n-butyl, isoamyl, n-octyl, n-dodecyl, and stearyl gallates and gallic acid were from Tokyo Kasei (Tokyo, Japan). Methyl, ethyl, and n-propyl gallate were purchased from Fuji Chemicals (Wakayama, Japan). Guava tea drink used in this study is a product of Yakult (Tokyo, Japan). Fresh Psidium guajava leaves were collected from a greenhouse- grown plant at the Okayama University Medicinal Botanical Garden, and the voucher specimen (MY-PG002) was kept in the Botanical Garden.
Bacterial strain: The four bacterial strains (V. vulnificus CDCA6614, V. mimicus CS14, V. parahaemolyticus RIMD2210633, and A. sobria 288) are maintained in the Department of Pharmacogenetics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences.
Fractionation of the guava tea drink: The guava tea drink (500 mL) was freeze-dried to give an extract (1250 mg). A portion (502 mg) of the extract was then subjected to a Sep-pak ODS cartridge, and the adsorbed materials were eluted with increasing concentrations of MeOH in water to give four fractions (H2O eluate, 395 mg; 20% MeOH eluate, 62 mg; 40% MeOH, 39 mg; MeOH eluate, 9 mg).
The 20% MeOH eluate was subjected to column chromatography on MCI-gel with aq. MeOH (30%→40%→50%→60%→100% MeOH) and the 50% MeOH eluate (16.9 mg) was further purified on a Sep-pak ODS cartridge to give quercetin-3-O-β-D- glucuronide (2.7 mg).
Purification of guava polyphenols: P. guajava leaves (1.7 kg) were homogenized in 70%
acetone (20.5 L in total). The filtrate was then concentrated to 4.5 L and extracted with Et2O (3 times, 6.6 L in total), EtOAc (6.6 L in total), and n-BuOH (6.6 L in total), successively, to produce Et2O (2.9 g), EtOAc (15.0 g), and n-BuOH (39.3 g) extracts and a H2O-soluble residue (97.9 g). A portion (2.0 g) of the n-BuOH extract was subjected to column chromatography using Toyopearl HW- 40C (2.2 cm i.d. x 20 cm) (70% EtOH) to produce six fractions (Fr. I–VI). Fr. II (18.9 mg) was
subjected to a Sep-pak ODS cartridge with increasing concentrations of MeOH in water (H2O→5%→10%→20%→30%→ 100% MeOH).
Evaporation of the 10% MeOH eluate gave Procyanidin B1 (5.5 mg). Fr. IV (37.9 mg) was subjected to a Sep-pak ODS cartridge, and the 10% MeOH eluate was further purified by preparative reverse-phase HPLC to yield pedunculagin (3.0 mg). Fr. V (99.9 mg) was subjected to column chromatography using an MCI gel CHP-20P (1.1 cm i.d. x 12 cm) with increasing concentrations of MeOH in water to give castalagin (1.7 mg, from the 10% MeOH eluate). A portion of the 30% MeOH eluate from the MCI column was further purified by preparative HPLC to give casuarinin (2.2 mg). Fr. VI (44.5 mg) was subjected to column chromatography using an MCI gel CHP20P (1.1 cm i.d. x 12 cm), and the 30% MeOH eluate was further purified by preparative HPLC to give stenophyllanin A (1.1 mg). Hyperin (2.5 mg) and isoquercitrin (1.3 mg) were isolated from the n-BuOH extract (500 mg) by column chromatography on MCI gel in a similar way. All of the isolated polyphenols were identified based on comparisons of their 1H NMR data with those of authentic samples and by direct comparisons of the normal- and reverse-phase HPLC data.
Procyanidin B1
1H NMR (40˚C): 2.55 (1H, dd, J= 7, 16 Hz, H-4L), 2.74 (1H, dd, J = 5, 16 Hz, H-4L), 3.93 (1H, d, J = 2 Hz, H-3U), 4.05 (1H, m, H-3L), 4.60 (1H, br s, H-4U), 4.75 (IH very broad, H-2L), 5.05 (1H, br s, H-2U), 5.90, 5.95, 5.98 (1H each, br s, H-6U, H-8U, H-6L), 6.69 (1H, dd, J = 2, 8 Hz, H-6’U), 6.71 (1H, d, J = 8 Hz, H-5’L), 6.73 (1H, d, J = 8 Hz, H-5’U), 6.77 (1H, m, H-6’L), 6.87 (1H, br s, H-2’L), 6.95 (1H, d, J = 2 Hz, H-2’U). U and L denote the upper and lower units, respectively.
Pedunculagin
1H NMR: 3.75 [dd, J = 2, 13 Hz, Glucose (Glc) H-6, α-anomer (α)], 3.82 [d, J = 13 Hz, Glc H-6, β-anomer (β)], 4.16 (dd, J = 7, 9 Hz, Glc H-5, β), 4.56 (dd, J = 7, 10 Hz, Glc H-5, α), 4.81 (t, J = 9 Hz, Glc H-2, β), 5.00 (d, J = 9 Hz, Glc H-1, β), 5.01 (dd, J = 3, 10 Hz, Glc H-2, α), 5.02 (dd, J = 7, 10 Hz, Glc H-4, α), 5.03 (t, J = 9 Hz, Glc H-4, β), 5.18 (t, J = 9 Hz, Glc H-3, β), 5.20 (dd, J = 7, 13 Hz, Glc H-6, α), 5.23 (dd, J = 7, 13 Hz, Glc H-6, β), 5.40 (d, J = 3 Hz, Glc H-1, α), 5.42 (t, J = 10 Hz,
Glc H-3, α), 6.32, 6.54, 6.59, 6.62 [each, s, hexahydroxydiphenoyl (HHDP)-H, α], 6.31, 6.50, 6.58, 6.64 (each, s, HHDP-H, β) (α:β, 8:5).
Castalagin
1H NMR: 3.98 (1H, d, J = 13 Hz, Glc H-6), 4.97 (1H, dd, J = 1, 7 Hz, Glc H-3), 5.00 (1H, dd, J = 1, 5 Hz, Glc H-2), 5.06 (1H, dd, J = 2, 13 Hz, Glc H-6), 5.21 (1H, t, J = 7 Hz, Glc H-4), 5.56 (1H, dd, J = 2, 7 Hz, Glc H-5), 5.66 (1H, d, J = 5 Hz, Glc H-1), 6.60, 6.75, 6.79 (1H each, s, aromatic-H).
Casuarinin
1H NMR: 4.04 (1H, d, J = 13 Hz, Glc H-6), 4.62 (1H, dd, J = 2, 5 Hz, Glc H-2), 4.85 (1H, dd, J = 3, 13 Hz, Glc H-6), 5.29 (1H, dd, J = 3, 8.5 Hz, Glc H-5), 5.42 (1H, t, J = 2 Hz, Glc H-3), 5.44 (1H, dd, J = 2, 8.5 Hz, Glc H-4), 5.55 (1H, d, J = 5 Hz, Glc H-1), 6.46, 6.51, 6.82 (1H each, s, HHDP-H), 7.07 (2H, s, galloyl-H).
Stenophyllanin A
1H NMR: 2.46 [1H, m, catechin (CA) H-4], 2.88 (1H, m, CA H-4), 3.94-4.04 (1H in total, m, Glc H-6), 4.11 (1H, br s, CA H-3), 4.37 (1H, s, Glc H-1), 4.65 (1H, br s, CA H-2), 4.73, 4.81, 5.26, 5.31 (each 1H br s, Glc H-2, Glc H-3, Glc H-4, Glc H-5), 4.89 (1H, dd, J = 4, 14 Hz, Glc H-6), 5.86 (1H, br s, CA H-6), 6.87 (2H, s, galloyl-H), 6.45, 6.57, 6.73, 6.81, 6.97, 7.05 (6H in total, HHDP-H).
MIC and MBC determination: A portion of the plated bacterial strain was cultured in liquid medium prior to being incubated in cation-supplemented Mueller-Hinton broth (CSMHB; Difco Laboratories, Detroit, MI) containing Ca2+ (50 μg/mL) and Mg2+
(25 μg/mL). For the cultures of V. vulnificus and V. parahaemolyticus, 3% w/v NaCl was added. The MICs were determined using a liquid microdilution method [7]. Each sample solution in 50% DMSO (20 μL) was diluted in 180 μL of CSMHB, and the solution was serially diluted on the assay plates (96-well microplates; Falcon). The final concentration of DMSO did not exceed 5%; thus, it did not affect the growth of bacterial cultures.
Bacterial cultures (5 μL/well), which were diluted with 0.85% or 3% NaCl (ca. 106 CFU/mL), were added to the wells of the plates containing the sample solutions. The plates were then incubated for 24 h at 37˚C. The MIC was defined as the lowest concentration of sample that produced no visible
turbidity. The MBC was determined as follows.
After the determination of the MIC, 20 μL each of the bacterial suspensions was plated on a Petri dish containing CSMHB-agar medium. The MBC was defined as the lowest concentration of sample that exhibited no visible growth on the agar plate. The
time course of the bacterial growth phase was monitored by the change in turbidity at 655 nm.
Acknowledgments - The NMR instruments used in this study are the property of Okayama University.
The authors thank Ms. M. Mano for the preparation of extracts from fresh leaves.
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