A new antifungal and cytotoxic C-methylated flavone glycoside from Picea neoveitchii

Article abstract of DOI:10.1016/j.bmcl.2012.07.089

A new C-methylated flavone glycoside, 5,7-dihydroxy-3-methoxy-6-C- methylflavone 8,4′-di-O-β-d-glucopyranoside (1), was isolated from the twigs and leaves of Picea neoveitchii Mast, together with eight known compounds, 5,7,8,4′-tetrahydroxy-3-methoxy-6-me

Full text of DOI:10.1016/j.bmcl.2012.07.089

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5819–5822
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Bioorganic & Medicinal Chemistry Letters
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A new antifungal and cytotoxic C-methylated flavone glycoside
from Picea neoveitchii
⇑
Wei-Quan Chen, Zhi-Jun Song, Han-Hong Xu
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Wushan Guangzhou 510642, People’s Republic of China
Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education, South China Agricultural University, Wushan Guangzhou 510642, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
new C-methylated flavone glycoside, 5,7-dihydroxy-3-methoxy-6-C-methylflavone 8,40-di-O-b-
glucopyranoside (1), was isolated from the twigs and leaves of Picea neoveitchii Mast, together with eight
known compounds, 5,7,8,40-tetrahydroxy-3-methoxy-6-methylflavone 8-O-b-
-glucopyranoside (2)
kaempferol 3,40-di-O-b-
-glucopyranoside (3), apigenin 7-O-b- -glucopyranoside (4), tiliroside (5), mas-
sonianoside B (6), umbeliferone 7-O-b- -glucopyranoside (7), dihydroconiferin (8) and gleditschiaside A
D-
Article history:
A
Received 25 April 2012
Revised 29 June 2012
Accepted 25 July 2012
Available online 2 August 2012
D
D
D
D
(9). Their structures were elucidated on the basis of analyses of spectroscopic data. Compound 1 showed
moderate antifungal activity against tested plant pathogens (Pyricularia grisea (Cooke) Sacc., Sclerotium
rocfsii Sacc. and Alternaria mali Roberts), however, compounds 2 and 5 had obvious inhibitory effect
against S. rocfsii and A. mali, respectively. Compounds 1, 2, 3 and 9 also exhibited potent cytotoxicity
against Spodoptera litura Fabricius cells.
Keywords:
Picea neoveitchii
Pinaceae
C-Methylated flavone glycoside
Antifungal activity
Cytotoxicity
Ó 2012 Elsevier Ltd. All rights reserved.
Food shortage is a major problem in certain remote locations or
developing countries. High food prices have increased hunger and
poverty, and produced rioting in some countries. As a result, how
to raise crop yields is the urgent problem in the world. Plant fungal
diseases reduce the yield and quality of crops, causing great losses
in agriculture annually.1 For a long period of time, fungal diseases
were controlled with chemical fungicides such as carbendazim and
triadimefon. Because of the abuse of chemical antimicrobial agents,
many new problems have emerged, such as toxic reaction, drug
tolerance and residue, and so on. Regarding the negative effects
of chemical antimicrobial agents to non-target organisms and
environment, as well as the high cost of them, recent years, much
research emphasis has been focused on the more specific and envi-
ronmentally friendly materials, which are generally of botanical
origin.2,3 Therefore, it is important to find antimicrobial agents
from plants and further to exploit new plant-derived fungicides.
The genus Picea belonging to the Pinaceae family includes about
40 species around the world, which are distributed throughout the
north temperate zone, 16 species and 9 varieties occur in China.4
Previous chemical studies revealed that the plants of genus Picea
contain the constituents of diterpenes,5,6 triterpenoids,7–11 flavo-
noids,12–14 lignans,15 phenolic compounds and alkaloids.16,17 Picea
neoveitchii, an endemic species native to China with needle leaves,
and is mainly distributed at high altitudes of 1300–2000 m in Hu-
bei, Gansu and Shanxi provinces, and scattered in ravines and
mountain slopes.18,19 So far its wood has been used for building
materials, and its pine cones have been used in the folkloric med-
icine for the relief of coughs and the treatment of diabetes. Our
previous study suggested that the methanol extract of the twigs
and leaves of P. neoveitchii exhibited strong antifungal activities
against some plant pathogens. To the best of our knowledge, the
chemical constituent of P. neoveitchii has not been investigated,
this situation prompted us to investigate the bioactive constituents
of this plant. Our previous study led to the isolation of four new
flavonoids and 15 known compounds from the EtOAc fraction of
the twigs and leaves of P. neoveitchii, some of the compounds
showed antifungal activities against Fusarium oxysporum and Rhi-
zoctonia solani.20 However, many constituents in other fractions
were still not fully investigated. Therefore, further investigation
was undertaken on the water soluble fraction, which resulted in
the isolation and structure elucidation of a new C-methylated fla-
vone glycoside (1), together with eight known compounds (2–9)
(Fig. 1).21 The present paper reports the isolation, structure charac-
terization, antifungal activities and cytotoxicities of the isolated
compounds.
The air-dried and pulverized twigs and leaves of P. neoveitchii
(2.0 kg) were extracted three times with 90% aqueous EtOH at
room temperature, and the EtOH extract of P. neoveitchii was ex-
tracted with petroleum ether and EtOAc to give the petroleum
ether extract, EtOAc extract and water phase. The filtrated water
phase (87.6 g) was subjected to D101 macroporous adsorption re-
sin column and eluted with MeOH–H2O (0%, 85%, MeOH) to give Fr.
A–D. The Fr. B (35.6 g) was chromatographed over silica gel column
⇑
Corresponding author. Tel.: +86 20 85285127; fax: +86 20 38604926.
E-mail address: hhxu@scau.edu.cn (H.-H. Xu).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.089

5820
W.-Q. Chen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5819–5822
3'
OH
OR1
OR1
2'
OR1
OR1
4'
5'
8
1'
HO
O
O
HO
O
O
HO
O
O
7
9
2
6'
10
6
3
OCH3
OR1
OCH3
5
4
OH
OH
OH
2
1
3
OH
OH
HO
O
O
R1O
O
O
OH
OH
HO
O
O
OH
O
OH
O
4
5
HO
OH
O
O
OR1
OH
R2O
H3CO
O
OH
OH
6
7
O
HO
R1=
R2=
HO
OH
H3CO
R1O
O
OR2
O
O
OH
H3C
HO
HO
HO
OH
HO
8
9
OH
Figure 1. Structures of compounds 1–9.
1
eluting with CHCl3–MeOH–H2O (10:10:1, 10:10:2) and MeOH to
afford Fr. B1–B5 according to TLC analysis. The Fr. B1 (12.8 g) was
subjected to CC on polyamide and eluted with the mixture of
CHCl3–MeOH with increasing polarity (CHCl3, 30:1, 20:1, 10:1,
7:1, 4:1, 2:1, 1:1, MeOH, v/v) to divide into subfr. I–VII. Fr. I
(8.1 g) was fully purified by middle pressure liquid chromatogra-
phy (MPLC) on ODS and eluted with MeOH–H2O (10%, 20%, 30%,
40%, 50%, 60%, MeOH) to obtain Fr. I-1 to Fr. I-10 on the basis of
TLC analysis. Compound 8 (23.4 mg) was isolated from Fr. I-1 by
subjected silica gel column with a successive gradient mixture of
J = 9.0 Hz, H-30,50) in H NMR spectrum, typical of an AA0BB0 cou-
pling system, indicated the presence of a p-disubstituted B ring.
The 13C NMR spectrum (Table 1) indicated a conjugated ketone
carbonyl (dC, 178.0), characteristic of a flavone. In addition, two
anomeric proton signals at dH 4.61 (d, J = 7.8 Hz, H-100) and 4.99
(d, J = 7.2 Hz, H-1000) in the 1H NMR spectrum, as well as two ano-
meric carbon signals at dC 107.3 and 100.0 in 13C NMR spectrum
suggested the existence of two sugar moieties. Except one of the
sugar moieties, the 1H and 13C NMR spectra of 1 were similar to
those of 5,7,8,40-tetrahydroxy-3-methoxy-6-metylflavone 8-O-b-
CHCl3–MeOH (10:1, 8:1, 6:1, 5:1, 4:1, v/v). Compound
7
D-glucopyranoside. Two sugar moieties were determined to be
(43.1 mg) was obtained from Fr. I-2, and compound 9 (14.4 mg)
was isolated from Fr. I-6 through repeated crystallisation
in MeOH–CHCl3. Fr. I-9 was separated by CC on silica gel eluted
with CHCl3–MeOH (10:1, 8:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, v/v),
followed by Sephadex LH-20 CC, with acetone as eluent, to yield
compound 4 (14.5 mg). Fr. II (1.7 g) was subjected to CC on poly-
amide and eluted with MeOH–H2O (20%, 30%, 40%, 50%, 60%, 70%,
MeOH) to obtained eight subfractions, and after recrystallization,
compound 5 (8.8 mg) was isolated from Fr. II-3. The fourth Fr. IV
(1.8 g) was also fully purified by MPLC on ODS and eluted with
MeOH–H2O (10%, 20%, 30%, 40%, 50%, 60%, 70%, MeOH) to afford
Fr. IV-1 to IV-10. Compound 3 was precipitated from Fr. IV-2. Fr.
IV-5 was subjected to Sephadex LH-20 CC with MeOH as eluent,
gave a mixture, which were rechromatographed by PTLC (CHCl3–
MeOH 5:1, two times) to afford compound 6 (10.2 mg). Compound
2 (14.5 mg) was obtained from Fr. IV-9 by CC on Sephadex LH-20
eluting with acetone. After recrystallization in MeOH–CHCl3, com-
pound 1 (34.4 mg) was obtained from Fr. V.
glucopyranose by 13C NMR spectrum,23 which were confirmed
by acid hydrolysis and Co-TLC comparing with authentic samples.
Their configurations were determined as b-orientation by cou-
pling constants of two anomeric proton signals in the 1H NMR
spectrum, and the two glucopyranose moieties were determined
as
D
-form by optical rotation (½a D20
ꢀ
+54.3°, c 0.094, H2O).24 The ab-
sence of any hydrogen signals of A ring indicated two hydroxys at
A ring. These findings suggested 1 was a flavonoid with two
hydroxyls, a methoxyl, a C-methyl and two b-D-glucopyranose
moieties.
The absence of the singlet of H-3 signal at dH 6.70–6.90 and
HMBC correlation of the methoxy hydrogens with C-3 (dC 138.0)
suggested the methoxy group was at C-3 (Fig. 2). A downfield
shifted signal at dH 12.77 indicated the existence of a 5-OH group,
which was supported by long range correlations of the C-6 (dC
106.8), C-5 (dC 154.3), and C-10 (dC 103.4). In addition, the location
of C-methyl group at C-6 was confirmed by HMBC correlations of
6-methyl hydrogens with C-5, C-6, and C-7 (dC 155.3). Moreover,
the anomeric proton at dH 4.61 (d, J = 7.8 Hz, H-100) was correlated
with C-8 at dC 125.3, and another anomeric proton at dH 4.99 (d,
J = 7.2 Hz, H-1000) was correlated with C-40 at dC 159.5 in HMBC
Compound 1, obtained as yellow amorphous powder.22 It gave
pink colour with vanillin–sulfuric acid and positive reaction to the
magnesium hydrochloric acid and Molish tests suggested that 1
was a flavonoid. A molecular formula, C29H34O17 was indicated
by HR-ESI-MS at m/z 655.1861 [M+H]+, (calculated 655.1869).
The 1H NMR spectrum (Table 1) indicated the presence of an aro-
matic methoxyl dH 3.82 (3H, s) and one C-methyl dH 2.03 (3H, s).
Two doublets at dH 8.35 (2H, d, J = 9.0 Hz, H-20,60) and 7.24 (2H, d,
spectra, suggesting that two of the b-D-glucopyranose moieties
were located at C-8 and C-40, respectively. Thus, the remaining hy-
droxyl group was located at C-7. Consequently, the structure of
compound 1 was elucidated as 5,7-dihydroxy-3-methoxy-6-C-
methylflavone 8,40-di-O-b-
D-glucopyranoside.

W.-Q. Chen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5819–5822
5821
Table 1
1H and 13C NMR data of compound 1 (DMSO-d6, d in ppm, J in Hz)
Position
dH
dC
Position
dH
dC
2
3
4
5
6
7
8
9
154.6 s
138.0 s
178.0 s
154.3 s
106.8 s
155.3 s
125.3 s
146.2 s
103.4 s
123.3 s
130.5 d
116.2 d
159.5 s
7.6 q
Glc
100
4.61 (d, 1H, J = 7.2)
3.43 (t, 1H, J = 8.4)
3.29 (m, 1H)
3.20 (m, 1H)
3.28 (m, 1H)
107.3 d
74.1 d
76.6 d
69.6 d
77.4 d
60.6 t
200
300
400
500
600
3.74 (m, 1H), 3.68 (m, 1H)
Glc0
1000
2000
3000
4000
5000
6000
10
10
20, 60
30, 50
40
6-Me
3-OMe
5-OH
4.99 (d, 1H, J = 7.2)
3.27 (m, 1H)
3.32 (m, 1H)
3.39 (m, 1H)
3.40 (m, 1H)
100.0 d
73.1 d
75.8 d
68.9 d
77.1 d
60.3 t
8.35 (d, 2H, J = 9.0)
7.24 (d, 2H, J = 9.0)
2.03 (s, 3H)
3.82 (s, 3H)
12.77 (s, 1H)
3.72 (m, 1H), 3.50 (m, 1H)
59.6 q
itory percentages of 30.6%, 41.1% and 43.4%, respectively (Table 2).
Compound 2 demonstrated strong activity against S. rocfsii with a
relative inhibitory percentage of 81.0%, while compounds 3 and 4
showed moderate activity. Compound 6 displayed obvious inhibi-
tory effect against A. mali (with 88.9% relative inhibitory percent-
OH
OH
HOH2C
HO
O
HO
CH2OH
O
HO
OH
O
O
HO
O
O
age), and compounds 2, 3, 4,
5 and 8 exhibited moderate
antifungal activity. Meanwhile, compounds 2, 6 and 8 showed
moderate activity against P. grisea, and compounds 2, 5 and 6 dis-
played weak antifungal activity against Ceratocystis paradoxa.
However, all the tested compounds showed lower activities than
carbendazim against the tested plant pathogens.
O
OH
Figure 2. Key HMBC correlations of compound 1.
The cytotoxicities of all compounds against Spodoptera litura
Fabricius cell (SL) were evaluated by MTT assay.34 Compounds 1,
2, 5 and 9 exhibited potent cytotoxicity against SL cells with inhi-
bition rates of 64.4%, 62.5%, 65.0% and 62.2%, respectively, at a con-
centration of 20 mg/L (Table 3). The other compounds showed
weak cytotoxicity, while all of these compounds demonstrated
lower cytotoxicity than that of the positive control, rotenone, at
the same concentration.
In conclusion, a new C-methylated flavone glycoside, together
with eight known compounds, were isolated from the twigs and
leaves of P. neoveitchii. Their structures were established by spec-
troscopic analysis. All the isolated compounds were qualitatively
evaluated for their antifungal activities by a modified disc diffusion
The eight known compounds were identified as 5,7,8,40-tetra-
hydroxy-3-methoxy-6-methylflavone 8-O-b- -glucopyranoside
(2),13 kaempferol 3,40-di-O-b- -glucopyranoside (3),25 apigenin 7-
O-b-
-glucopyranoside (4),26 tiliroside (5),27 massonianoside B
(6),28 umbeliferone 7-O-b- -glucopyranoside (7),29 dihydroconifer-
D
D
D
D
in (8)30 and gleditschiaside A (9),31 respectively, by spectral analy-
sis comparison with those already reported in the literature.
The isolated compounds were qualitatively evaluated for their
antifungal potential using a modified disc diffusion method.32
Compound 1 showed moderate antifungal activity against Pyricu-
laria grisea, Sclerotium rocfsii and Alternaria mali with relative inhib-
Table 2
In vitro antifungal activity of compounds 1–9 against tested plant pathogens
Compd
Mycelial growtha (mm)
Ae
Growth inhibitionb (%)
Pc
Sd
Cf
Pc
Sd
Ae
Cf
1
2
3
4
5
6
7
8
9
Cg
5.7 1.5
7.6 1.0
0.0
3.3 0.5
0.0
7.0 1.8
0.0
8.6 1.0
0.0
4.8 0.6
9.4 0.5
3.7 0.6
4.8 0.9
2.6 0.7
0.0
2.8 0.8
0.0
0.0
4.3 0.6
7.2 0.9
2.4 0.8
5.8 1.0
5.5 0.5
8.8 1.0
0.0
3.1 0.6
0.0
9.9 0.6
0.0
6.2 1.0
0.0
30.6 8.0
40.9 5.4
0.0
17.7 2.7
0.0
37.6 9.7
0.0
46.2 5.4
0.0
41.4 5.2
81.0 4.3
31.9 5.2
41.4 7.8
22.4 6.0
0.0
24.1 6.9
0.0
0.0
43.4 6.1
72.7 9.1
24.2 8.0
58.6 10.1
55.6 5.1
88.9 10.1
0.0
31.3 6.1
0.0
100.0
0.0
24.1 3.9
0.0
0.0
0.0
7.1 0.7
5.6 0.6
1.5 0.5
0.0
0.0
25.7 0.4
27.6 2.7
21.8 2.3
5.8 1.9
0.0
0.0
100.0
18.6 1.4
11.6 1.0
100.0
100.0
Data represent the mean S.D. of three separate experiments. Each filter paper disc was preloaded with 100
control.
lg of the target compounds in MeOH, the same to the positive
a
Mycelial growth inhibitory zone diameter (mm).
Relative mycelial growth inhibitory percentage (%).
P = Pyricularia grisea.
S = Sclerotium rocfsii Sacc.
A = Alternaria mali.
C = Ceratocystis paradoxa.
b
c
d
e
f
g
C = Carbendazim, positive control.

5822
W.-Q. Chen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5819–5822
13. Jung, M. J.; Choi, J. H.; Chung, H. Y.; Jung, J. H.; Choi, J. S. Fitoterapia 2001, 72,
Table 3
Cytotoxicity of compounds 1–9a against Spodop-
tera litura cells
943.
14. Jung, M. J.; Jung, H. A.; Kang, S. S.; Hwang, G. S.; Choi, J. S. Arch. Pharm. Res.
2009, 32, 1699.
Compound
Inhibitory rates (%)
15. Kawamura, F.; Kawai, S.; Ohashi, H. Phytochemistry 1997, 44, 1351.
16. Schneider, M. J.; Brendze, S.; Montali, J. A. Phytochemistry 1995, 39, 1387.
17. Tzong, H. L.; Ming, H. Y.; Chi, I. C.; Ching, K. L.; Yi, Y. S.; Yueh, H. K. Chem. Pharm.
Bull. 2006, 54, 693.
18. Wang, M. L.; Zhang, D. S.; Ma, Q. X.; Qi, Y. F. J. Anhui Agri. Sci. 2010, 22, 12040.
19. Dai, Z. L.; Li, H. X.; Cheng, C. H.; Yang, X. F.; Ding, G. L.; Wang, B.; Wang, M.;
Zhou, J. P. J. Beijing Forestry Univ. 2010, 32, 247.
1
2
3
4
5
6
7
8
64.4 1.7
62.5 3.8
23.1 1.0
7.8 2.4
65.0 5.6
17.9 1.2
15.3 4.3
4.0 0.9
20. Song, Z. J.; Chen, W. Q.; Du, X. Y.; Zhang, H.; Lin, L. J.; Xu, H. H. Phytochemistry
2011, 72, 490.
21. Plant material: The twigs and leaves of P. neoveitchii were collected from the
Three Gorges, Hubei Province, PR China, in June 2008, and were identified by
Professor Li PT (College Of Forestry, South China Agricultural University, PR
China). A voucher specimen (No. 11614) was deposited in the Wuhan Botanical
Garden, Chinese Academy of Sciences.
9
62.2 1.8
74.4 2.4
Rotenoneb
Data represent the mean S.D. of three separate
experiments.
Test concentration of each compound is
20 mg/L.
22. Physical and spectroscopic data of compound 1: 5,7-Dihydroxy-3-methoxy-6-C-
methyl-flavone 8,40-di-O-b-
D-glucopyranoside, yellow amorphous powder,
a
molecular formula: C29H34O17; mp 180–182 °C; ½a D20
ꢁ42.3° (c 0.086 MeOH);
ꢀ
1H NMR (600 MHz, DMSO-d6) and 13C NMR (150MHz, DMSO-d6) data see Table
1; HR-ESI-MS m/z: 655.1861 [M+H]+ (calculated for C29H35O17, 655.1869).
23. Yao, C. S.; Zhang, X. H.; Zhang, W.; Shen, Y. H.; Xu, Y. L. Nat. Prod. Dev. Res. 2004,
16, 102.
b
Positive control (20 mg/L).
24. Acid hydrolysis and sugar analysis: Compound 1 (2 mg) was dissolved in 2 N
HCl (H2O–MeOH 1:1 2 mL) and refluxed with magnetic stirring in a water bath
at 80 °C for 4 h. After cooling, the reaction mixture was diluted with H2O (3 mL)
and extracted three times with EtOAc. The aqueous layer were neutralized
with 2 N NaOH, then concentrated and dried to furnish a monosaccharide
residue. The residue was purified through Sephadex LH-20 column eluting
with CHCl3–MeOH (1:1) to afford sugar, which was identified as glucose by Co-
method, and their cytotoxicities all evaluated against S. litura cell
by MTT assay. Compound 2 demonstrated strong activity against
S. rocfsii, while compound 6 displayed obvious inhibitory effect
against A. mali. The compounds 1, 2, and 6 are considered to be po-
tential as antimicrobial agents, and would provide more potent
derivative with a suitable modification. Moreover, compounds 1,
2, 5 and 9 exhibited potent cytotoxicity against SL cells at a con-
centration of 20 mg/L. These findings indicated that the twigs
and leaves of P. neoveitchii is a promising source of valuable bioac-
tive compounds and is worthy of further investigation. Ours is the
first phytochemical analysis on the chemical constituents of P. neo-
veitchii, and so adds to the chemotaxonomic data of the Pinaceae
family.
TLC with authentic sample. The glucose was determined as D-form by optical
rotation (½a 2D0
+54.3°, c 0.094, H2O).
ꢀ
25. Rikke, N.; Tadao, K. Phytochemistry 1999, 51, 1113.
26. Vanhoenacker, G.; Van Rompaey, P.; De Keukeleire, D.; Sandra, P. Nat. Prod. Lett.
2002, 16, 57.
27. Budzianowski, J.; Skrzypczak, L. Phytochemistry 1995, 38, 997.
28. Shen, Y. B.; Kojima, Y.; Terazawa, M. J. Wood Sci. 1999, 45, 332.
29. Reisch, J.; Achenbach, S. H. Phytochemistry 1992, 31, 4376.
30. Mazur, M.; Hope-Ross, K.; Kadla, F. J.; Sederoff, R.; Chang, H. M. J. Wood Chem.
Technol. 2007, 27, 1.
31. Phan, V. K.; Nguyen, H. V.; Chau, V. M.; Nguyen, T. D. Tap Chi Hoa Hoc 2008, 46,
636.
Acknowledgments
32. The agricultural pathogenic fungi including Pyricularia grisea (Cooke) Sacc.,
Sclerotium rocfsii Sacc., Alternaria mali Roberts and Ceratocystis paradoxa (Dade)
Moreau were obtained from the Key Laboratory of Natural Pesticides and
Chemical Biology, South China Agricultural University. Antifungal activity
The work was supported by the grant from the National Depart-
ment Public Benefit Research Foundation of China (No. 200903052)
and China Postdoctoral Science Foundation (No. 20110490900).
assay was evaluated using previously reported method with
a minor
modification.20,33 Briefly, sterile potato dextrose agar (PDA) medium was
prepared and distributed into Petri plates of 90 mm diameter (each plate
contained 15 mL of PDA medium). Sterilized filter paper discs (5 mm in
diameter) were preloaded each with 10 lL of a solution of the tested
Supplementary data
compound containing 10 mg/mL in MeOH, and the discs were allowed to air
dry, then the discs were placed at the centre of the PDA plates. Four plugs of
fungal inoculums, 5 mm in diameter, were placed upside down at the quarter
circle points 20 mm in radius around the drug-loaded disc in the Petri dishes.
Blank control discs were treated with MeOH, and carbendazim was used as a
positive control. The inoculated plates were incubated at 29 °C for 2–4 days,
when the growth of fungi in the blank control would have reached the centre
of the plates, the inhibitory zone was measured in mm. The growth inhibition
of the tested compounds were calculated against carbendazim using the
following formula: [inhibitory zone of treatment (mm)/inhibitory zone of
positive control (mm)] ꢂ 100% (Table 2). Each treatment was run in triplicate.
33. Vivek, K. B.; Seung, Y. S.; Hak, R. K.; Sun, C. K. Ind. Crops Prod. 2008, 27, 136.
34. The Spodoptera litura Fabricius cell (SL) was obtained from Key Laboratory of
Natural Pesticide and Chemical Biology, Ministry of Education, South China
Agricultural University and cultured in Grace’s insect culture medium (Gibco-
BRL, America) containing 9% new born calf serum at 27.5 °C. Cells in the
logarithmic phase of growth were used in all experiments. The cytotoxicity of
the compounds on SL cell was evaluated by MTT assay, as previously
reported.35 The test compounds were dissolved in DMSO, and diluted with
culture medium to the test concentrations. The contrast group was cultured
with medium containing 0.5% DMSO. The cell was seeded in each well of 96-
well plates with 0.1 mL culture medium for 24 h, and then the cell was treated
with tested compounds. After 24 h, the cell was incubated with MTT solution
(0.5 mg/mL) for 4 h and subsequently dissolved in 0.1 mL DMSO. The
absorbance was measured on a Bio-Rad ELISA reader at 570 nm.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
07.089.
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