Isolation of Phytoalexins and Phytoncides
Sophoraflavanone G (2(S)-5,7,2′,4′-tetrahydroxy-8-lavandulyflavanone) isolated
from Sophora spp. (Leguminosae) was used in the anti-MRSA activity test [21].
Calozeyloxanthone was isolated from the root bark of Callophyllum moonii as a
yellow crystalline compound (thin-layer chromatography (TLC) single spot, melt-
ing point 236–237 °C). It was identified from the plant material collected from the
Kanneliya forest in the southern province of Sri Lanka. The plant specimens were
compared with herbarium specimens (specimen no. 24994) at the Royal Botanic
Gardens, Peradeniya, Sri Lanka and a voucher specimen was deposited at the nat-
ural products laboratories of the Institute of Fundamental Studies [22].
-Mangostin was isolated as follows. Stem bark of G. mangostana L. (1 kg) was
dried, powdered, and extracted with hexane, methylene chloride, and methanol,
respectively. Silica gel column chromatography (Fluka 6074,1 particle size
7.5 Isolation of Phytoalexins and Phytoncides 141
0.063~0.2 mm with hexane, methylene chloride, and methanol as solvents) of the
hexane extract (11.9 g) and methylene chloride extract (25 g) gave two major com-
pounds: -mangostin (11.6 g, 1.16%) and â-mangostin (6.4 g, 0.64%) as yellow
needles. These structures were confirmed by direct comparison with authentic
samples and spectral data [23].
The stilbene oligomers (gnemonol B and genetin E) isolated from gnetaceous
plants were donated by Professor Dr M. Iinuma (Gifu Pharmaceutical University)
[24, 25].
Minimum Inhibitory Concentration
MIC values were determined by the agar dilution method of the Japanese Society
of Chemotherapy [26] using a micro-inoculater (Sakuma Seisakusho Co., Ltd., To-
kyo). MIC of the five strains of VRE and three strains of VSE described above were
measured as 250, 32, 200, 200, and 16 μg mL–1, while MIC values of the nine
strains of MRSA and three strains of methicillin were measured as 12.5, 400, 25,
12.5, 400, 1600, 25, 12.5, and 400 μg mL–1, respectively [27].
Synergism of Antibacterial Compounds with Commercially Available Antibiotics
Antimicrobial compounds were prepared in 50% dimethylsulfoxide solution. A so-
lution of phytoalexin (or phytoncide) in combination with respective antibiotics
was prepared by the doubling dilution method with sterilized water, and each solu-
tion was poured into sterilized plastic Petri dishes separately. Sterilized MH agar
8 mL (MH agar poured into phytoalexin (or phytoncide) alone or the antibiotic
alone) was poured into the above Petri dishes and mixed. After cooling, the MIC
values of phytoalexin or phytoncide alone, the antibiotics alone, and their combina-
tions, were examined. The fraction inhibitory concentration (FIC) indices were cal-
culated by the method of Didry et al. [28], and the interactive effects of the phytoa-
lexin or phytoncide and the commercial antibiotics were examined.
FIC index values were judged as follows: FIC index ≤0.5: synergetic effect; FIC
index 0.5–1.0: partially synergetic effect; FIC index >1.0: no synergetic effect; FIC
index ≥2.0: antagonistic effect.
142 7 Anti-MRSA and Anti-VRE Activities of Phytoalexins and Phytoncides Isolated from Tropical Plants
Antibacterial Activities
7.8.1
Sophoraflavanone G
The MIC values of sophoraflavanone G against 27 strains of MRSA are shown in
Table 7.1. The values ranged from 3.13 to 6.25 μg mL–1.
7.8 Antibacterial Activities 143
Table 7.1 Antibacterial activities of sophoraflavanone G (SFG) against 27 strains
of methicillin-resistant Staphylococcus aureus (MRSA).
Strain no. Coagulase type MIC values to methicillin MIC values to SFG
μg mL–1) (μg mL–1)
1[a] II 1600 6.25
2[a] II 100 6.25
3 II 1600 6.25
4 II 1600 6.25
5 VII 3200 6.25
6 II 1600 6.25
7 II 1600 6.25
8 II 3200 3.12
9 VII 800 3.12
10[a] II 400 3.12
11[a] II 800 3.12
12 III 12.5 6.25
13 II 400 6.25
14 III 12.5 6.25
15 VII 12.5 6.25
16 II 400 6.25
17 II 50 6.25
18 II 400 6.25
19[a] II 400 3.12
20[a] II 800 6.25
21 II 400 6.25
22 II 400 6.25
23 II 800 6.25
24 II 1600 3.12
25 II 800 3.12
26 II 800 6.25
27 II 400 6.25
Strain nos 1–9: MRSA isolates from Osaka Medical Center for Cancer and Cardiovascular Diseases.
Strain nos 11–19: MRSA isolates from Osaka National Hospital.
Strain nos 19–27: MRSA isolates from Kitano Hospital.
a These strains were used for the test of synergism.
7.8.2
Calozeyloxanthone
The MIC values of calozeyloxanthone against each of the two strains of VRE and
VSE are shown in Table 7.2. The antibacterial activity of calozeyloxanthone against
VRE and VSE was strong and MIC values observed were 6.25 μg mL–1 and
12.5 μg mL–1, respectively.
144 7 Anti-MRSA and Anti-VRE Activities of Phytoalexins and Phytoncides Isolated from Tropical Plants
Table 7.2 Antibacterial activity of calozeyloxanthone against two strains of vancomycinresistant
enterococci (VRE) and vancomycin-sensitive enterococci (VRE).
MIC (μg mL–1)
Calozeyloxanthone Gentamicin
Enterococcus faecalis ATCC 51575 (VRE) 12.5 400
Enterococcus faecium ATCC 51559 (VRE) 6.25 25
Enterococcus faecalis ATCC 12953 (VSE) 12.5 12.5
Enterococcus faecalis ATCC 8459 (VSE) 6.25 6.25
MIC, minimum inhibitory concentration.
7.8.3
-Mangostin
Table 7.3 shows the anti-VRE activity of -mangostin and â-mangostin, and Table
7.4 shows the anti-MRSA activity of -mangostin and â-mangostin, respectively.
-Mangostin was found to be active against five strains of VRE and nine strains
of MRSA, with MIC values ranging from 6.25 to 12.5 μg mL–1, respectively.
Table 7.3 MIC values of -mangostin and â-mangostin mangostin against five strains of
vancomycin-resistant enterococci (VRE) and three strains of vancomycin-sensitive enterococci
(VSE).
MIC (μg mL–1)
-Mangostin â-Mangostin Gentamicin
Enterococcus faecalis ATCC 51299 (VRE)[a] 3.13 25 >100
Enterococcus faecalis ATCC 51575 (VRE)[a] 3.13 25 >100
Enterococcus faecium ATCC 51559 (VRE)[a] 3.13 25 6.25
Enterococcus faecium KIHC-237 (VRE)[b] 3.13 25 6.25
Enterococcus gallinarum KIHC-241 (VRE)[b] 6.25 25 3.13
Enterococcus faecalis IFO 12965 (VSE)[c] 6.25 25 12.5
Enterococcus faecium IFO 3535 (VSE)[c] 3.13 25 6.25
Enterococcus faecalis ATCC 8459 (VSE)[c] 3.13 25 6.25
MIC, minimum inhibitory concentration.
a Purchased from American Type culture Collection (ATCC).
b Supplied from Kobe Institute of Public Health.
c Purchased from Institute for Fermentation of Osaka (IFO), Japan.
7.8.4
Gnemonol B and Gnetin E
Tables 7.5 and 7.6 show the MIC values of gnemonol B and gnetin E against VRE,
MRSA, VSE, and MSSA.
7.8 Antibacterial Activities 145
Table 7.4 MIC values of -mangostin and â-mangostin against nine strains of methicillinresistant
S. aureus (MRSA) and three strains of methicillin-sensitive S. aureus (MSSA).
MIC (μg mL–1)
-Mangostin â-Mangostin Gentamicin
MRSA-1[a] 6.25 >100 25
MRSA-2[a] 6.25 >100 3.13
MRSA-3[a] 6.25 >100 1.56
MRSA-4[b] 6.25 >100 3.13
MRSA-5[b] 6.25 >100 6.25
MRSA-6[b] 6.25 >100 0.2
MRSA-7[c] 6.25 >100 0.2
MRSA-8[c] 12.5 >100 6.25
MRSA-9[c] 6.25 >100 >100
MSSA 1 (S. aureus IFO 13276)[d] 6.25 >100 0.2
MSSA 2 (S. aureus IFO 12732)[d] 6.25 >100 0.2
MSSA 3 (S. aureus IFO 3080)[d] 6.25 >100 0.2
MIC, minimum inhibitory concentration.
a Donated from Osaka Medical Center for Cancer and Cardiovascular Diseases, Japan.
b Donated from Osaka National Hospital, Japan.
c Donated from Kitano Hospital, Japan.
d Purchased from Institute for Fermentation of Osaka (IFO), Japan.
Table 7.5 MIC values of gnemonol B and gnetin E against five strains of vancomycin-resistant
enterococci (VRE) and three strains of vancomycin-sensitive enterococci (VSE).
MIC (μg mL–1)
Gnemonol B Gnetin E Gentamicin
Enterococcus faecalis ATCC 51299 (VRE)[a] 12.5 12.5 >100
Enterococcus faecalis ATCC 51575 (VRE)[a] 12.5 12.5 >100
Enterococcus faecium ATCC 51559 (VRE)[a] 12.5 12.5 6.25
Enterococcus faecium KIHC-237 (VRE)[b] 12.5 25 6.25
Enterococcus gallinarum KIHC-241 (VRE)[b] 12.5 25 3.13
Enterococcus faecalis IFO 12965 (VSE)[c] 12.5 25 12.5
Enterococcus faecium IFO 3535 (VSE)[c] 12.5 25 6.25
Enterococcus faecalis ATCC 8459 (VSE)[c] 12.5 25 6.25
MIC, minimum inhibitory concentration.
a Purchased from American Type culture Collection (ATCC).
b Supplied from Kobe Institute of Public Health.
c Purchased from Institute for Fermentation of Osaka (IFO), Japan.
Gnemonol B was found to be active against five strains of VRE and nine strains
of MRSA with MIC values of 12.5 and 6.25 μg mL–1, respectively. Gnemonol B was
also active against the three strains of VSE and MSSA with MIC values of 12.5 and
6.25 μg mL–1, respectively. Gnetin E also exhibited activities against five strains of
VRE, nine strains of MRSA, and three strains of VSE and MSSA with MIC values
ranging from 12.5 to 25 μg mL–1.
7.8.5
Sophoraflavanone G (2(S)-5,7,2′,4′-tetrahydroxy-8-lavandulyflavanone) isolated
from Sophora spp. (Leguminosae) was used in the anti-MRSA activity test [21].
Calozeyloxanthone was isolated from the root bark of Callophyllum moonii as a
yellow crystalline compound (thin-layer chromatography (TLC) single spot, melt-
ing point 236–237 °C). It was identified from the plant material collected from the
Kanneliya forest in the southern province of Sri Lanka. The plant specimens were
compared with herbarium specimens (specimen no. 24994) at the Royal Botanic
Gardens, Peradeniya, Sri Lanka and a voucher specimen was deposited at the nat-
ural products laboratories of the Institute of Fundamental Studies [22].
-Mangostin was isolated as follows. Stem bark of G. mangostana L. (1 kg) was
dried, powdered, and extracted with hexane, methylene chloride, and methanol,
respectively. Silica gel column chromatography (Fluka 6074,1 particle size
7.5 Isolation of Phytoalexins and Phytoncides 141
0.063~0.2 mm with hexane, methylene chloride, and methanol as solvents) of the
hexane extract (11.9 g) and methylene chloride extract (25 g) gave two major com-
pounds: -mangostin (11.6 g, 1.16%) and â-mangostin (6.4 g, 0.64%) as yellow
needles. These structures were confirmed by direct comparison with authentic
samples and spectral data [23].
The stilbene oligomers (gnemonol B and genetin E) isolated from gnetaceous
plants were donated by Professor Dr M. Iinuma (Gifu Pharmaceutical University)
[24, 25].
Minimum Inhibitory Concentration
MIC values were determined by the agar dilution method of the Japanese Society
of Chemotherapy [26] using a micro-inoculater (Sakuma Seisakusho Co., Ltd., To-
kyo). MIC of the five strains of VRE and three strains of VSE described above were
measured as 250, 32, 200, 200, and 16 μg mL–1, while MIC values of the nine
strains of MRSA and three strains of methicillin were measured as 12.5, 400, 25,
12.5, 400, 1600, 25, 12.5, and 400 μg mL–1, respectively [27].
Synergism of Antibacterial Compounds with Commercially Available Antibiotics
Antimicrobial compounds were prepared in 50% dimethylsulfoxide solution. A so-
lution of phytoalexin (or phytoncide) in combination with respective antibiotics
was prepared by the doubling dilution method with sterilized water, and each solu-
tion was poured into sterilized plastic Petri dishes separately. Sterilized MH agar
8 mL (MH agar poured into phytoalexin (or phytoncide) alone or the antibiotic
alone) was poured into the above Petri dishes and mixed. After cooling, the MIC
values of phytoalexin or phytoncide alone, the antibiotics alone, and their combina-
tions, were examined. The fraction inhibitory concentration (FIC) indices were cal-
culated by the method of Didry et al. [28], and the interactive effects of the phytoa-
lexin or phytoncide and the commercial antibiotics were examined.
FIC index values were judged as follows: FIC index ≤0.5: synergetic effect; FIC
index 0.5–1.0: partially synergetic effect; FIC index >1.0: no synergetic effect; FIC
index ≥2.0: antagonistic effect.
142 7 Anti-MRSA and Anti-VRE Activities of Phytoalexins and Phytoncides Isolated from Tropical Plants
Antibacterial Activities
7.8.1
Sophoraflavanone G
The MIC values of sophoraflavanone G against 27 strains of MRSA are shown in
Table 7.1. The values ranged from 3.13 to 6.25 μg mL–1.
7.8 Antibacterial Activities 143
Table 7.1 Antibacterial activities of sophoraflavanone G (SFG) against 27 strains
of methicillin-resistant Staphylococcus aureus (MRSA).
Strain no. Coagulase type MIC values to methicillin MIC values to SFG
μg mL–1) (μg mL–1)
1[a] II 1600 6.25
2[a] II 100 6.25
3 II 1600 6.25
4 II 1600 6.25
5 VII 3200 6.25
6 II 1600 6.25
7 II 1600 6.25
8 II 3200 3.12
9 VII 800 3.12
10[a] II 400 3.12
11[a] II 800 3.12
12 III 12.5 6.25
13 II 400 6.25
14 III 12.5 6.25
15 VII 12.5 6.25
16 II 400 6.25
17 II 50 6.25
18 II 400 6.25
19[a] II 400 3.12
20[a] II 800 6.25
21 II 400 6.25
22 II 400 6.25
23 II 800 6.25
24 II 1600 3.12
25 II 800 3.12
26 II 800 6.25
27 II 400 6.25
Strain nos 1–9: MRSA isolates from Osaka Medical Center for Cancer and Cardiovascular Diseases.
Strain nos 11–19: MRSA isolates from Osaka National Hospital.
Strain nos 19–27: MRSA isolates from Kitano Hospital.
a These strains were used for the test of synergism.
7.8.2
Calozeyloxanthone
The MIC values of calozeyloxanthone against each of the two strains of VRE and
VSE are shown in Table 7.2. The antibacterial activity of calozeyloxanthone against
VRE and VSE was strong and MIC values observed were 6.25 μg mL–1 and
12.5 μg mL–1, respectively.
144 7 Anti-MRSA and Anti-VRE Activities of Phytoalexins and Phytoncides Isolated from Tropical Plants
Table 7.2 Antibacterial activity of calozeyloxanthone against two strains of vancomycinresistant
enterococci (VRE) and vancomycin-sensitive enterococci (VRE).
MIC (μg mL–1)
Calozeyloxanthone Gentamicin
Enterococcus faecalis ATCC 51575 (VRE) 12.5 400
Enterococcus faecium ATCC 51559 (VRE) 6.25 25
Enterococcus faecalis ATCC 12953 (VSE) 12.5 12.5
Enterococcus faecalis ATCC 8459 (VSE) 6.25 6.25
MIC, minimum inhibitory concentration.
7.8.3
-Mangostin
Table 7.3 shows the anti-VRE activity of -mangostin and â-mangostin, and Table
7.4 shows the anti-MRSA activity of -mangostin and â-mangostin, respectively.
-Mangostin was found to be active against five strains of VRE and nine strains
of MRSA, with MIC values ranging from 6.25 to 12.5 μg mL–1, respectively.
Table 7.3 MIC values of -mangostin and â-mangostin mangostin against five strains of
vancomycin-resistant enterococci (VRE) and three strains of vancomycin-sensitive enterococci
(VSE).
MIC (μg mL–1)
-Mangostin â-Mangostin Gentamicin
Enterococcus faecalis ATCC 51299 (VRE)[a] 3.13 25 >100
Enterococcus faecalis ATCC 51575 (VRE)[a] 3.13 25 >100
Enterococcus faecium ATCC 51559 (VRE)[a] 3.13 25 6.25
Enterococcus faecium KIHC-237 (VRE)[b] 3.13 25 6.25
Enterococcus gallinarum KIHC-241 (VRE)[b] 6.25 25 3.13
Enterococcus faecalis IFO 12965 (VSE)[c] 6.25 25 12.5
Enterococcus faecium IFO 3535 (VSE)[c] 3.13 25 6.25
Enterococcus faecalis ATCC 8459 (VSE)[c] 3.13 25 6.25
MIC, minimum inhibitory concentration.
a Purchased from American Type culture Collection (ATCC).
b Supplied from Kobe Institute of Public Health.
c Purchased from Institute for Fermentation of Osaka (IFO), Japan.
7.8.4
Gnemonol B and Gnetin E
Tables 7.5 and 7.6 show the MIC values of gnemonol B and gnetin E against VRE,
MRSA, VSE, and MSSA.
7.8 Antibacterial Activities 145
Table 7.4 MIC values of -mangostin and â-mangostin against nine strains of methicillinresistant
S. aureus (MRSA) and three strains of methicillin-sensitive S. aureus (MSSA).
MIC (μg mL–1)
-Mangostin â-Mangostin Gentamicin
MRSA-1[a] 6.25 >100 25
MRSA-2[a] 6.25 >100 3.13
MRSA-3[a] 6.25 >100 1.56
MRSA-4[b] 6.25 >100 3.13
MRSA-5[b] 6.25 >100 6.25
MRSA-6[b] 6.25 >100 0.2
MRSA-7[c] 6.25 >100 0.2
MRSA-8[c] 12.5 >100 6.25
MRSA-9[c] 6.25 >100 >100
MSSA 1 (S. aureus IFO 13276)[d] 6.25 >100 0.2
MSSA 2 (S. aureus IFO 12732)[d] 6.25 >100 0.2
MSSA 3 (S. aureus IFO 3080)[d] 6.25 >100 0.2
MIC, minimum inhibitory concentration.
a Donated from Osaka Medical Center for Cancer and Cardiovascular Diseases, Japan.
b Donated from Osaka National Hospital, Japan.
c Donated from Kitano Hospital, Japan.
d Purchased from Institute for Fermentation of Osaka (IFO), Japan.
Table 7.5 MIC values of gnemonol B and gnetin E against five strains of vancomycin-resistant
enterococci (VRE) and three strains of vancomycin-sensitive enterococci (VSE).
MIC (μg mL–1)
Gnemonol B Gnetin E Gentamicin
Enterococcus faecalis ATCC 51299 (VRE)[a] 12.5 12.5 >100
Enterococcus faecalis ATCC 51575 (VRE)[a] 12.5 12.5 >100
Enterococcus faecium ATCC 51559 (VRE)[a] 12.5 12.5 6.25
Enterococcus faecium KIHC-237 (VRE)[b] 12.5 25 6.25
Enterococcus gallinarum KIHC-241 (VRE)[b] 12.5 25 3.13
Enterococcus faecalis IFO 12965 (VSE)[c] 12.5 25 12.5
Enterococcus faecium IFO 3535 (VSE)[c] 12.5 25 6.25
Enterococcus faecalis ATCC 8459 (VSE)[c] 12.5 25 6.25
MIC, minimum inhibitory concentration.
a Purchased from American Type culture Collection (ATCC).
b Supplied from Kobe Institute of Public Health.
c Purchased from Institute for Fermentation of Osaka (IFO), Japan.
Gnemonol B was found to be active against five strains of VRE and nine strains
of MRSA with MIC values of 12.5 and 6.25 μg mL–1, respectively. Gnemonol B was
also active against the three strains of VSE and MSSA with MIC values of 12.5 and
6.25 μg mL–1, respectively. Gnetin E also exhibited activities against five strains of
VRE, nine strains of MRSA, and three strains of VSE and MSSA with MIC values
ranging from 12.5 to 25 μg mL–1.
7.8.5
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