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Analysis of Antifungal Components in the Galls of Melaphis chinensis and Their Effects on Control of Anthracnose Disease of Chinese Cabbage Caused by Colletotrichum higginsianum

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Fungal pathogens caused various diseases which resulted in heavy yield and quality losses on plants of commercial interests such as fruits, vegetables, and flowers. In our preliminary experimental results, the methanol extracts of four species of medicinal plants Melaphis chinensis, Eugenia caryophyllata, Polygonumcuspidatum, andRheumofficinalepossessedantifungal activity to causal agent of cabbage anthracnose, Colletotrichum higginsianum.Thus it was conducted to identify and quantify the chemical constituents in these herbs and to assess the antifungal effects of these compounds. Among the tested principles, the indicator compound methyl gallate from M. chinensis was the most effective one against the conidial germination. In addition, it exhibited significant effects of controlling anthracnose disease of Chinese cabbage caused by C. higginsianum PA-01 in growth chamber.These results indicate that M. chinensis may be potential for further development of plant-derived pesticides for control of anthracnose of cabbage and other cruciferous crops.
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Research Article
Analysis of Antifungal Components in the Galls of
Melaphis chinensis and Their Effects on Control of
Anthracnose Disease of Chinese Cabbage Caused by
Colletotrichum higginsianum
Ping-Chung Kuo,1Ting-Fang Hsieh,2Mei-Chi Lin,1Bow-Shin Huang,1
Jenn-Wen Huang,3and Hung-Chang Huang4
1Department of Biotechnology, National Formosa University, Yunlin 632, Taiwan
2Floriculture Research Center, Taiwan Agricultural Research Institute, Council of Agriculture, Executive Yuan, Yunlin 646, Taiwan
3Department of Plant Pathology, National Chung Hsing University, Taichung 402, Taiwan
4Plant Pathology Division, Taiwan Agricultural Research Institute, Council of Agriculture, Executive Yuan, Wufeng,
Tai c h u ng 413, Tai w a n
Correspondence should be addressed to Ping-Chung Kuo; pcckuoo@nfu.edu.tw and Ting-Fang Hsieh; tsieh@tari.gov.tw
Received  July ; Revised  September ; Accepted  September 
Academic Editor: Hasan Uslu
Copyright ©  Ping-Chung Kuo et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Fungal pathogens caused various diseases which resulted in heavy yield and quality losses on plants of commercial interests such
as fruits, vegetables, and owers. In our preliminary experimental results, the methanol extracts of four species of medicinal plants
Melaphis chinensis, Eugenia cary ophyllata, Polygonum cuspidatum,andRheum ocinale possessedantifungalactivitytocausalagent
of cabbage anthracnose, Colletotrichum higginsianum. us it was conducted to identify and quantify the chemical constituents in
these herbs and to assess the antifungal eects of these compounds. Among the tested principles, the indicator compound methyl
gallate from M. chinensis was the most eective one against the conidial germination. In addition, it exhibited signicant eects
of controlling anthracnose disease of Chinese cabbage caused by C. higginsianum PA- in growth chamber. ese results indicate
that M. chinensis may be potential for further development of plant-derived pesticides for control of anthracnose of cabbage and
other cruciferous crops.
1. Introduction
ere are numerous reports indicating that tissues of some
plant species contain antifungal substances, including rhi-
zomes of Curcuma longa [], seeds of Cassia tora [],
stem/leaves and owers of Lavandula stoechas [], and others.
For example, seeds of mustard (Brassica juncea cv. Bau Sin)
arerichinglucosinolateandenzymatichydrolysisofthis
compound resulted in the release of allyl isothiocyanate that
is highly toxic to Rhizoctonia solani K¨
uhn AG-, causal agent
of root rot of cabbage []. Some plant species with antifungal
properties are also used as medicinal plants. For example,
galls of Melaphis chinensis [,]andleavesofAloe vera []
contained toxic substances against plant pathogenic fungi.
e n-hexane fraction of a cinnamon (Cinnamomum cassia)
extract exhibited signicant inhibition on mycelia growth of
R. solani []. Various essential oils also displayed signicant
antifungal activity, such as those from Hypericum linarioides
[], Pistacia lentiscus [], Metasequoia glyptostroboides [],
and Silene armeria []. e essential oils of cinnamon
leaves (Cinnamomum zeylanicum)andclovebuds(Eugenia
caryophyllata) also showed highly antifungal activity against
Botrytis cinerea []. Chu et al. reported that the aqueous
extracts of Coptis chinensis (goldthread), Polygonum cuspida-
tum (Japanese knotweed), Cinnamomum cassia (cinnamon),
Rheum ocinale (Chinese rhubarb), Polygonum multio-
rum,andEugenia caryophyllata (clove) showed inhibitory
eects to conidial germination of Oidium murrayae [].
Hindawi Publishing Corporation
Journal of Chemistry
Volume 2015, Article ID 850103, 12 pages
http://dx.doi.org/10.1155/2015/850103
Journal of Chemistry
Water-soluble extracts of clove completely inhibited the
conidial germination and mycelial growth of C. higginsianum
at the concentration of % (w/v). In addition, clove oil and
eugenol were equally eective in reducing disease severity
of anthracnose caused by this pathogen in greenhouse [].
Although the antifungal activities of various plants were
extensively reported, there were relatively few studies regard-
ing the antifungal principles in the plant extracts.
Colletotrichum species are important fungal pathogens
causing anthracnose disease of numerous economically
important crops, including legumes, ornamentals, vegetables,
and fruit trees [] and thus are responsible for severe
yield losses of cabbage crops in commercial elds in Taiwan
[]. Although these diseases could be successfully con-
trolled by the synthetic chemical fungicides, the utilization
of synthetic fungicides led to the development of resistance
and environment pollution. e biological control of plant
diseases which is recognized as use of metabolites from
the natural source is an eco-friendly resolution []. In
our preliminary experimental results (Tab l e  ), forty herbal
extracts were examined for their antifungal activity against
C. higginsianum PA-. Most of them displayed inhibition
of the fungus and among the tested methanol extracts,
four species of medicinal plants, Melaphis chinensis, Eugenia
caryophyllata,Polygonum cuspidatum,andRheum ocinale,
exhibited the inhibitory percentages between 80.64 ± 3.16
and 91.92±3.00%atthetestedconcentration(g/mL).
e experimental data indicated that these extracts possessed
antifungal activity to causal agent of cabbage anthracnose
C. higginsianum. However, the chemical nature of the anti-
fungal substances in these Chinese herbs remains unknown.
erefore, the objectives of this study were to identify the
compounds and their antifungal activity in four species of
medicinal plants. In addition, the indicator compounds were
used as standards to quantitatively analyze these medici-
nal plants with the aid of high performance liquid chro-
matography (HPLC) and the validation examinations were
performed to conrm that these methods were precise and
reliable for quality evaluation. us they could be utilized
to control the quality of herbal preparations to ensure their
antifungal activities. Moreover, their eects of controlling
anthracnose disease of Chinese cabbage caused by Col-
letotrichum higginsianum PA- in growth chamber were also
examined.
2. Materials and Methods
2.1. General Procedure. All the solvents including the HPLC-
grade methanol were purchased from Merck KGaA (Darm-
stadt, Germany). e chemical structures of the indicator
compounds were identied by comparison of their spectro-
scopicandphysicaldatawiththosereportedintheliterature.
eir purities were better than .% as determined by
HPLC. Plant materials were extracted using a Major Sci-
ence LM-R shaking incubator. High performance liquid
chromatography (HPLC) was performed on a Shimadzu
LC-ATseries pumping system equipped with a Shi-
madzu SPD-AUV-Vis detector, a Gemini u C column
(. mm × mm, m), and a SIL-AF autosampling
system.
2.2. Fungal Pathogen and Plant Materials. Two i s olates (PA-
 and PA-) of C. higginsianum were used in this study. ey
were isolated from diseased leaves of cabbage (Brassica rapa
L. Chinese group) grown in Yunlin, Taiwan. e cultures were
maintained on potato dextrose agar (PDA, Difco, USA). Dry
powders of all the examined medicinal herbs were purchased
from herbal stores in Yunlin, Taiwan. All the purchased
materials for the experiments were authenticated by Dr. T. F.
Hsieh and the voucher specimens (PCKuo TFHsieh -
) were deposited in the herbarium of Department of
Biotechnology, National Formosa University, Yunlin, Taiwan.
Seeds of Chinese cabbage (B. rapa L.) were put on a number
lterpaper(cmindiameter,ToyoRoshiCo.,Japan)
moistened in water and kept in a Petri dish at room temper-
ature (–C) for  day. e germinated seeds were sown
in peat moss in plastic pots,  cells/tray, and  seed/cell.
Aer one week, individual seedlings were transplanted to
plastic pots ( cm in diameter) lled with Stender peat
substrates (Stender AG, Germany),  plants/pot, and kept in
agreenhouseforfourweekswithdailywatering.
2.3. Eect of the Methanol Extracts on Conidial Germination
of C. higginsianum. e methanol extracts were tested for
inhibition of conidial germination of C. higginsianum PA-
 according to the method of Lee and Dean []. e
isolatewasgrownonoatmealagar(g/L)at
C under
continuous uorescent light. Conidia were harvested from -
to -day-old cultures and the solution was ltered to collect
conidial suspension. Ten microliters of conidial suspension
(5conidia/mL) was mixed with ten microliters of dierent
extracts. e cultures were placed in a moistened plastic box,
incubated at C for  h, and examined for germination
of conidia under a compound microscope. ere were six
replicates for each sample ( conidia/replicate). Sterile
distilled water was used as negative control and azoxystrobin
was used as positive control. Inhibition rate of conidia for
each treatment was calculated by
Inhibition (%)
=1−conidial germinated with tested compound
conidial germinated in control
×100%.()
2.4. Extraction and Fractionation of Medicinal Plants. e
galls of M. chinensis (. g) were extracted with methanol
under reux (. L ×× h), and the crude extracts were
concentrated in vacuo to give a brown syrup (MCR, . g).
e crude extract was partitioned between ethyl acetate
and water to aord ethyl acetate solubles (MCRE, . g)
and water extracts (MCRW, . g), respectively. e buds
of E. caryophyllata (. g) were extracted with methanol
under reux (. L ×× h), and the crude extracts were
concentrated to give a brown syrup (EC, . g). e crude
Journal of Chemistry
T : e preliminary antifungal screening of the dierent herbal extracts on conidial germination of Colletotrichum higginsianum PA-.
Sample Inhibition percentage (%)aSample Inhibition percentage (%)a
Anemarrhena asphodeloides . ±. Lithospermum erythrorhizon . ±.
Arctium lappa . ±. Lycium barbarum . ±.
Cassia angustifolia bMelaphis chinensis 89.86 ±2.00
Cassia tora . ±. Morus alba
Carthamus tinctorius Paeonia lactiora .±.
Cinnamomum cassia . ±. Polygala tenuifolia
Crataegus pinnatida . ±. Polygonum cuspidatum 80.64 ±3.16
Cuscuta chinensis .±. Prunella vulgaris . ±.
Epimedium brevicornum . ±. Prunus armeniaca . ±.
Equisetum hyemale . ±. Pueraria lobata . ±.
Eucommia ulmoides . ±. Rheum ocinale 91.92 ±3.00
Eugenia caryophyllata 87.37 ±4.74 Salvia miltiorrhiza . ±.
Forsythia suspensa . ±. Scrophularia ningpoensis . ±.
Gardenia jasminoides . ±. Scutellaria barbata .   ±.
Gentiana scabra . ±. Smilax glabra . ±.
Hedyotis diusa . ±. Sophora avescens . ±.
Houttuynia cordata Sophora tonkinensis . ±.
Isatis indigotica . ±. Taraxacum mongolicum . ±.
Leonurus japonicus . ±. Zingiber ocinale . ±.
Ligusticum chuanxiong . ±. Ziziphus jujuba . ±.
aPercentage of inhibition at  𝜇g/mL (X dilution) concentration. (𝑛=6). bNo inhibition was found.
extract was partitioned between ethyl acetate and water to
aord ethyl acetate solubles (ECE, . g) and water extracts
(ECW, . g), respectively. e roots of P. c u s p i d a t u m (. g)
were extracted with methanol under reux (.L ××
h), and the crude extracts were concentrated to give a
brown syrup (PC, . g). e crude extract was partitioned
between chloroform and water to aord chloroform solubles
(PCC, . g) and water extracts (PCW, . g), respectively. e
roots of R. ocinale (. g) were extracted with methanol
under reux (. L ×× h), and the crude extracts were
concentrated to give a brown syrup (RO, . g). e crude
extract was partitioned between chloroform and water to
aord chloroform solubles (ROC, . g) and water extracts
(ROW, . g), respectively.
2.5. Purication and Identication of Indicator Compounds.
e methods for purication and identication of indicator
compounds in the four medicinal plants were described as
follows.
(I) M. chinensis. e ethyl acetate soluble fraction (MCRE,
. g) of the crude extract was applied to a silica gel column
and then eluted with chloroform and step gradient of ethyl
acetate ( :  to : , v/v) to yield  fractions. Fraction  was
subjected to silica gel column chromatography eluted with
n-hexane and acetone ( : , v/v) to yield methyl gallate (2)
(. g). Fraction  was further resolved on a silica gel column
eluted with chloroform and acetone ( : ) to give gallic acid
(1) (. g).
(II) E. caryophyllata. e ethyl acetate soluble fraction (ECE,
. g) of the crude extract was puried with silica gel column
chromatography and eluted with n-hexane and step gradient
of ethyl acetate ( : to  : , v/v) to yield  fractions. Fraction
 was further puried on a silica gel column eluted with
chloroformandethylacetate(:,v/v)togiveeugenol(3)
(. mg).
(III) P. cuspidatum and R. ocinale.ForP. c u s p i d a t u m ,the
chloroform soluble fraction (PCC, . g) of the crude extract
was puried with silica gel column chromatography and
eluted with chloroform and step gradient of ethyl acetate
( :  to  : , v/v) to yield fractions. Fraction  was further
recrystallized with chloroform and ethyl acetate to aord
physcion (6) (. mg). Fraction was further resolved
on a silica gel column eluted with chloroform and step
gradient of methanol ( :  to : , v/v) to yield emodin (4)
(. mg). For R. ocinale, the chloroform soluble fraction
(ROC, . g) of the crude extract was puried with silica
gel column chromatography and eluted with chloroform and
step gradient of methanol ( :  to  : , v/v) to aord 
fractions. Fraction  was further puried with the assistance
of silica gel column chromatography eluted with n-hexane
andacetone(:,v/v)togivechrysophanol(5) (. mg).
2.6. Chromatography. e six indicator compounds used in
chromatographic analysis were gallic acid (1) and methyl
gallate (2)fromM. chinensis,eugenol(3)fromE. caryophyl-
lata,emodin(4)andphyscion(6)fromP. c u s p i d a t u m ,and
chrysophanol (5)fromR. ocinale. e analytic conditions
Journal of Chemistry
for these chemical constituents were determined by HPLC
according to the reported methods in the literature [].
2.7. Preparation of Standard Solutions, Calibration Cur ves, and
Validation of the Analytical Methods. e standard solutions
and calibration curves for the six indicator compounds were
preparedaccordingtothemethodsreportedintheliterature
[]. e reproducibility and precision of detection were
measured by repeatedly injecting a ready-made sample pool
andexpressedastherelativestandarddeviationoftheresults.
To determine the variance of samples within a day, the same
samples were tested at dierent times within the day. e
variance between days was determined by assaying the spiked
samples over three consecutive days at the same time each
day. e limit of detection (LOD) was determined as the
lowest detectable concentration with acceptable accuracy and
precision and three times above the noise level (S/N ).
e recovery of the indicator compounds was evaluated using
three dierent concentrations covering the linear range of the
standardcurveandthepeakheightswerecomparedtothe
standard compounds to calculate the recovery data.
2.8. Eect of the Methanol Extracts, Partially Puried Frac-
tions, and Indicator Compounds on Conidial Germination
of C. higginsianum. Each of the four methanol extracts,
partially puried fractions, and the six indicator compounds
from the plant extracts were tested for inhibition of conidial
germination of C. higginsianum, isolates PA- and PA-, as
described previously [].
2.9.EectofGallicAcidandMethylGallateonControl
of Anthracnose Disease of Chinese Cabbage Caused by C.
higginsianum PA-01 in Growth Chamber. To determine the
eect of indicator compounds, gallic acid, and methyl gallate,
on control of anthracnose disease of Chinese cabbage, each
dilution of gallic acid and methyl gallate derived from galls
of M. chinensis with the concentration of , , , and
 g/mL was sprayed on -week-old Chinese cabbage
plants until running water one day prior to the inoculation
of C. higginsianum PA-. Plants sprayed with sterile distilled
water were used as controls. ere were three replicates (pots)
for each treatment. Conidial suspensions of C. higginsianum
wereinoculatedoneachplantatmL/plantand
5coni-
dia/mL, using a compressed air-sprayer (SIL-AIR, Werther
International, Italy). All pots were placed in moist plastic bags
and kept in a growth chamber at Cunderhdiurnal
illumination. e plastic bags were removed aer one-day
incubation and the plants were examined for lesion number
and infection area in  cm diameter of leaf spot at , , and 
days aer inoculation.
2.10. Statistical Analysis. Data collected from all the experi-
ments in this study were analyzed for statistical signicance
using analysis of variance (ANOVA). Means of treatments
in each experiment were separated using Duncan’s multiple
range tests. e analytical results are expressed as mean ±
standard deviation (SD). Relative standard deviations (RSDs)
were calculated from those values. In addition, the mean
values of lesion number and lesion area on infection leaves
were analyzed by the least signicant dierence (LSD) test.
3. Results and Discussion
3.1. Antifungal Activities of the Crude Extracts. e antifungal
eects of the forty herbal extracts on conidial germination
of C. higginsianum PA-  a re display e d i n Table .Among
the examined samples, four species of medicinal plants,
including Melaphis chinensis, Eugenia caryophyllata,Poly-
gonum cuspidatum,andRheum ocinale,displayedsigni-
cant antifungal activity against C. higginsianum PA- . us
these four extracts were selected as the targets of developing
new botanical pesticides. Although the synthetic fungicides
successfully controlled the plant diseases sometimes, they
also contributed to increasing the population of fungicide-
resistant pathogens []. Natural plant metabolites are gener-
ally considered as safe to the humans and environment since
these chemical compounds are easily decomposed in the soil
and would not exhibit long-term eects to the environment
[]. us more and more reports were focused on the
development of new plant-derived pesticide preparations
recently [,,,,], but comparatively few studies related
to the antifungal principles in the bioactive extracts were
completed. ese new preparations would be hopeful to
reduce the damage caused by traditional synthetic fungicides
andinthemeanwhiletosuppressthediseasedevelopment
eectively. Detailed chemical analysis of the constituents in
the plant extracts is helpful to conrm the active compounds
and control the quality of the plant-derived pesticide prepa-
rations.
3.2. Identication of Indicator Compounds in the Medicinal
Plant Extracts. e indicator compounds (Figure ), includ-
ing gallic acid (1)[] and methyl gallate (2)[]from
the galls of M. chinensis,eugenol(3)[]frombudsofE.
caryophyllata,emodin(4)[]andphyscion(6)[]from
the roots of P. c u s p i d a t u m ,andchrysophanol(5)[]from
the roots of R. ocinale, were puried and characterized by
comparison of their spectral and physical data with those
reported in the literature. e purity of all the indicator
compounds except physcion (6) as determined by HPLC was
better than .%.
3.3. Optimization of the HPLC Condition and Method Val-
idation. e optimized HPLC analytical conditions for the
medicinal plant extracts were designed as displayed in the
experimental section. e calibration curve parameters and
limits of detection (LOD) for the indicator compounds were
displayed in Tab l e  . e precision of the HPLC method
developed was evaluated through the intraday and interday
experiments. Among the linear ranges, the RSDs for all the
indicator compounds of the intraday and interday precisions
were found to be less than . and .%, respectively
(Table ). e recovery of the indicator compounds was
determined by the addition of a sample with known con-
centration to the standard solution, and the mean recovery
rate was found to be in the ranges from . to .%
Journal of Chemistry
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
HO HO OCH3
OCH3
Gallic acid (1)Methyl gallate (2)
O
OO
O
O
O
Emodin (4) Chrysophanol (5) Physcion (6)
Eugenol (3)
CO2CH3
CO2H
H3CH3CH3C
F : Chemical structures of the indicator compounds 16.
with satisfactory RSDs in the ranges between . and .%
(Table ).
In the present study, the indicator compounds in M.
chinensis,E. caryophyllata,C. cassia,P. c u s p i d a t u m ,and
R. ocinale had been extracted and puried. e indicator
compounds were further used as standards to quantitatively
analyze these traditional Chinese medicines with the aid of
HPLC and the validation examinations were carried out to
conrm that these methods were precise and reliable for
quality evaluation. In the development of the HPLC method
for the quantitative determination of indicator compounds,
several solvent systems and separation columns were evalu-
ated and compared. Detection wavelength was also optimized
in this work. e maximum number and the heights of the
peaks of the constituents were obtained and the baseline
of chromatogram was stable. e reproducibility of the
analytical method was performed and the results showed
that it was satisfactory with the RSDs below .% for any
of the indicator compounds (data not shown). e precision
and recovery tests all displayed that the established HPLC
chromatographic methods were valid for the quantitative
determination of the indicator compounds and also con-
venient and feasible as tools for species authentication and
quality assessment of the herbal raw materials.
3.4. Quantitative Determination of Indicator Compounds in
the Medicinal Plant Extracts. e developed HPLC chro-
matographic analytical methods were applied to assess the
contents of the indicator compounds in the extracts of
corresponding plant materials and the data were displayed
in Table . e contents of methyl gallate (2) and eugenol
(3) in the ethyl acetate soluble fractions of methanol extracts
of M. chinensis and E. caryophyllata, respectively, were more
than % and they indicated that these constituents were the
major component in the plant extracts. In contrast, emodin
(4), chrysophanol (5), and physcion (6) were less than %
in the methanol extracts of P. c u s p i d a t u m and R. ocinale.
e reproducibility of the analytical results was satisfactory
with the RSDs below .% for all the examined indicator
compounds.
3.5. Antifungal Activities of the Methanol Extracts, Partially
Puried Fractions, and Indicator Compounds. e antifungal
eects of the extracts and fractions on conidial germi-
nation of C. higginsianum PA- and PA- are d i s played
in Table . Most of the crude extracts and low polarity
fractions displayed inhibitions of the conidial germination
of the fungal pathogen. Among the tested samples, the ethyl
acetate fraction of the methanol extracts of M. chinensis
exhibited the most signicant antifungal activities towards
C. higginsianum PA-  a n d PA-  w i t h t h e IC50 values of
. and . g/mL, respectively. e antifungal eects of
the indicator compounds 16and the reference compound
azoxystrobin were displayed in Table .Compounds15
all exhibited the inhibitory eects against C. higginsianum
PA-withtheIC
50 values less than . g/mL, and
comparatively compounds 14and 6showed the signicant
inhibition of the conidial germination of C. higginsianum PA-
 with the IC50 values ranging from . to . g/mL,
respectively. e major component methyl gallate (2)in
the most active fraction (MCRE) displayed the most sig-
nicant antifungal eects with the IC50 values of .
Journal of Chemistry
T : Calibration curve parameter, limits of detection (LOD), precision, and recovery for the indicator compounds.
Compound Calibration curve Correlation coecients
(2)
Linear range
(g/mL)
LOD
(g/mL)
Concentration
(g/mL)
Intraday precision Interday precision Spiked concentration
(g/mL)
Recovery
(%)
RSD
(%)
Mean ±SD (RSD %)
1=12112+33065 . .–. .
.. ±. (.) . ±. (.) . . ±. .
. . ±. (.) . ±. (.) . . ±. .
. . ±. (.) . ±. (.) . . ±. .
2=11797+53348 . .–. .
.. ±. (.) . ±. (.) . . ±. .
. . ±. (.) . ±. (.) . . ±. .
. . ±. (.) . ±. (.) . . ±. .
3=12313+376 . .–. .
. . ±. (.) . ±. (.) . . ±. .
. . ±. (.) . ±. (.) . . ±. .
. . ±. (.) . ±. (.) . . ±. .
4=83352359078 . .–. .
. . ±. (.) . ±. (.) . . ±. .
. . ±. (.) . ±. (.) . . ±. .
. . ±. (.) . ±. (.) . . ±. .
5=92840376375 . .–. .
. . ±. (.) . ±. (.) . .  ±. .
. . ±. (.) . ±. (.) . . ±. .
. . ±. (.) . ±. (.) . . ±. .
6=54687123241 . .–. .
. . ±. (.) . ±. (.) . . ±. .
. . ±. (.) . ±. (.) . . ±. .
. . ±. (.) . ±. (.) . . ±. .
Journal of Chemistry
T : Contents of each indicator compound in the crudes and fractions of the examined herb extracts.
Compound Samples
MCR MCRE MCRW EC ECE ECW PC PCC PCW RO ROC ROW
1. ±. (2.90)a. ±. (.) .±. (.) ——
2. ±. (.) . ±. (.) . ±. (.) ——
3—
b——. ±. (.) . ±. (.) N.D.c——— —
4—. ±. (.) . ±. (.) N.D. . ±. (.) . ±. (.) N.D.
5—N.D. N.D. N.D. . ±. (. ) .±. (.) N.D.
6—. ±. (.) . ±. (.) N.D. . ±. (.) . ±. (.) N.D.
ae contents of each compound were presented as mean ±S.D. (RSD %) (%, g/g sample). bNot determined. cNot detectable.
Journal of Chemistry
T : Antifungal activity of crude extracts and fractions of Chinese medicinal herbsa.
Sample
C. higginsianum PA- C. higginsianum PA-
Concentration
(g/mL)
Inhibition of conidial germination
(%)b
IC
(g/mL)
Concentration
(g/mL)
Inhibition of conidial germination
(%)
IC50
(g/mL)
MCR
. . ±.∗∗∗
.
. . ±.∗∗∗
.
. . ±.∗∗∗ . . ±.∗∗∗
. . ±.∗∗∗ . . ±.∗∗∗
MCRE
. . ±.∗∗∗
.
. . ±.∗∗∗
.
. . ±.∗∗∗ . . ±.∗∗∗
. . ±.∗∗∗ . . ±.∗∗∗
MCRW
. . ±.∗∗∗
.
. . ±.∗∗∗
.
. . ±.∗∗∗ . . ±.∗∗∗
. . ±.∗∗∗ . . ±.
EC
. . ±.∗∗∗
.
. . ±.∗∗∗
.
. . ±.∗∗∗ . . ±.∗∗∗
. . ±.∗∗∗ . . ±.∗∗∗
ECE
. . ±.∗∗∗
.
. . ±.∗∗∗
.
. . ±.∗∗∗ . . ±.∗∗∗
. . ±.∗∗∗ . . ±.∗∗∗
ECW
. . ±.∗∗∗
.
. . ±.∗∗
.
. . ±.∗∗∗ . . ±.∗∗∗
. . ±.∗∗∗ . . ±.∗∗∗
PC
. . ±.∗∗∗
.
. . ±.
.
. . ±.∗∗∗ . . ±.∗∗∗
. . ±.∗∗∗ . . ±.∗∗∗
PCC
. . ±.∗∗∗
.
. . ±.
.
. . ±.∗∗∗ . . ±.∗∗∗
. . ±.∗∗∗ . . ±.∗∗∗
PCW
. .±.∗∗∗
c
. . ±.
c
. . ±.∗∗∗ . . ±.
. . ±.∗∗∗ . . ±.
RO
. . ±.∗∗∗
.
. . ±.
.
. . ±.∗∗∗ . . ±.∗∗∗
. . ±.∗∗∗ . . ±.∗∗∗
ROC
. . ±.∗∗∗
.
. . ±.
.
. . ±.∗∗∗ . . ±.∗∗∗
. . ±.∗∗∗ . . ±.∗∗∗
ROW
. . ±.∗∗∗
.
. . ±.
.
. . ±.∗∗∗ . . ±.∗∗∗
. . ±.∗∗∗ . . ±.∗∗∗
a𝑛=6;bmean ±S.D.; ∗∗𝑃 < 0.01;∗∗∗𝑃 < 0.001;cIC >. 𝜇g/mL and not determined.
and . g/mL towards C. higginsianum PA- and PA-
(Figure ), respectively, compared to the reference synthetic
pesticide azoxystrobin (IC50 . and . g/mL against C.
higginsianum PA- and PA-, resp.). Among the examined
samples, most of them displayed signicant inhibition of
the conidial germination of the pathogen and this indi-
cated that these plant-derived pesticide preparations were
promising.
3.6.EectofGallicAcidandMethylGallateonControl
of Anthracnose Disease of Chinese Cabbage Caused by C.
higginsianumPA-01inGrowthChamber.Suppression of
Chinese cabbage anthracnose by indicator compounds, gallic
acid, and methyl gallate was dependent on the concentration,
where lesion area (%) and lesion number per  cm in diameter
of infected leaf were decreased by increasing concentrations
of each indicator compound (Table ). In general, disease
Journal of Chemistry
T : Antifungal activity of indicator compoundsa.
Compound
C. higginsianum PA- C. higginsianum PA-
Concentration
(g/mL)
Inhibition of conidial germination
(%)b
IC
(g/mL)
Concentration
(g/mL)
Inhibition of conidial germination
(%)
IC
(g/mL)
1
. . ±.∗∗∗
.
. . ±.∗∗∗
.
. . ±.∗∗∗ . . ±.∗∗∗
. . ±.∗∗∗ . . ±.∗∗∗
2
. . ±.∗∗∗
.
. . ±.∗∗∗
.
. . ±.∗∗∗ . . ±.∗∗∗
. . ±.∗∗∗ . . ±.∗∗∗
3
. . ±.∗∗∗
.
. . ±.∗∗∗
.
. . ±.∗∗∗ . . ±.∗∗∗
. . ±.∗∗∗ . . ±.∗∗∗
4
. . ±.∗∗∗
.
. . ±.
.
. . ±.∗∗∗ . . ±.∗∗∗
. . ±.∗∗∗ . . ±.∗∗∗
5
. . ±.∗∗∗
.
. . ±.
d
. . ±.∗∗∗ . . ±.
. . ±.∗∗∗ . . ±.
6
. . ±.∗∗∗
c
. . ±.
.
. . ±.∗∗∗ . . ±.∗∗∗
. . ±.∗∗∗ . . ±.∗∗∗
Azoxystrobin
. . ±.∗∗∗
.
. . ±.∗∗∗
.
. . ±.   ∗∗∗ . . ±.∗∗∗
. .±.∗∗∗ . . ±.∗∗∗
a𝑛=6;bmean ±S.D.; ∗∗∗𝑃 < 0.001;cIC >. 𝜇g/mL and not determined; dIC >. 𝜇g/mL and not determined.
T : Eect of gallic acid and methyl gallate on control of anthracnose disease of Chinese cabbage caused by Colletotrichum higginsianum
PA-  in gro wth cha m ber.
Treatment Conc. (g/mL) days
days days
LNLA (%) LN LA (%) LN LA (%)
CK  .a .a.a.a.a.a
Gallic acid
 .b.b.b.b.b.b
 .cd .cd .bc .bc .cd .c
 .cd .cd .bcd .bc .de .c
 .cd .cd .cde .cd .f.d
Methyl gallate
 .c.c.bc .bcd .bc .c
 .c.cd .  bcd .cd .cd .c
 .d.  d.de.d.ef .d
 .e.e.e.e.  g.e
LSD. . . . . . .
1Days aer inoculation. LN: lesion number per cm in diameter of infected leaves and LA (%): percentage of lesion area per  cm in diameter of infected
leaves. Data in each column with the same letter are signicantly dierent according to LSD test in 𝑃 = 0.05.
suppression by methyl gallate was better than by gallic acid
in the same concentration (Figure ). For example, the lesion
number was not a signicant dierence in treatment of
methyl gallate with . at  g/mL and in treatment of
gallic acid with . at  g/mL  days aer inoculation.
Similarity, lesion area (%) was also not a signicant dierence
in treatment of methyl gallate with . at g/mL and
in treatment of gallic acid with . at g/mL  days
aer inoculation (Tabl e  ). It means that lower concentration
of methyl gallate displayed better eects for disease control
than higher concentration of gallic acid. e results would
be valuable for the discovery of new plant-derived pesticide
preparations.
4. Conclusion
e present investigation results indicate that the methanol
extracts of M. chinensis, E. caryophyllata,P. c u s p i d a t u m ,
 Journal of Chemistry
AP
(a)
C
(b)
F : Eect of methyl gallate on inhibition of conidial germination of Colletotrichum higginsianum PA-. (a) An irregular brown
appressorium (AP) formed aer conidial germination in distilled water (check); and (b) conidium (C) failed to germinate in the solution
with . g/mL of methyl gallate (b ar scale =  m).
CK (A) (B)
F : Eect of gallic acid ( g/mL) (A) and methyl gallate
( g/mL) (B) on control of anthracnose disease of Chinese
cabbage caused by Colletotrichum higginsianum PA-.
and R. ocinale may be potential for further development
of plant-derived pesticides for control of anthracnose of
cabbage and other cruciferous crops. e developed HPLC
analytical methods are convenient and feasible tools for
species authentication and quality assessment of the herbal
raw materials. ey are helpful to monitor the contents of
active principles in the herbs for developing new botanical
pesticides.
According to the experimental data in the present study,
methylgallateshowedonly/activityofthepesticide
azoxystrobin; however, the herbal extracts would be safer and
less dangerous to the ecosystem. ese traditional Chinese
medicines could be studied further for their cytotoxicity and
synergistic eects of dierent combinations. It would be also
potential to study the antifungal mechanism in the future.
Abbreviation List
HPLC: High performance liquid chromatography
PDA: Potato dextrose agar
LOD: Limit of detection
SD: Standard deviation
RSD: Relative standard deviation.
Conflict of Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.
Acknowledgments
e authors are grateful for the nancial support from the
Council of Agriculture, Executive Yuan, Taiwan, awarded
toDr.P.C.Kuo.isstudyissupportedinpartbygrants
awardedtoDr.T.F.HsiehandDr.P.C.KuofromtheMinistry
of Science and Technology, Taiwan.
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... Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License (Iqbal and Javaid, 2012;Jabeen et al., 2014;Anil and Raj, 2015;Abd El-Ghany et al., 2015). Researchers have also isolated many potential antifungal compounds from plants such as β-amyrin from Melia azedarach L. (Jabeen et al., 2011), tow flavonoids 7-O-glucoside and (-)-epi catechin from Azadirachta indica (Kanwal et al., 2011), and methyl gallate from Melaphis chinensis (Kuo et al., 2015). Gond et al. (2015) reported that lipopeptides secreted by bacterial endophytes naturally occurring in many maize varieties inhibit pathogens and for Martins et al. (2015) fungicidal effects proved to be dependent on the occurrence of phenolic compounds in tested extracts. ...
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