Efficacy of leaf extract fractions of Glycyrrhiza glabra L. against downy mildew of cucumber (Pudoperonospora cubensis)
Andrea Scherf&Jonas Treutwein&
Hubertus Kleeberg&Annegret Schmitt
Accepted:26July2012/Published online:16August2012
#KNPV2012
Abstract An ethanolic leaf extract of Glycyrrhiza glabra L.(licorice)was highly effective in former bioassays and mi-commercial trials in controlling cucumber downy mildew(efficacy up to99.0%in bioassays and83.0%in mi-commercial trials).In order to elucidate the active ingredients and the mode of action,licorice leaf extract was fractionated into six fractions of defined substance class,of which the fraction containing acidic substances(F6)showed highest efficacy(97.6%).The calculated EC50values after a probit analysis of concentration ries of crude extract and fraction F6were concentrations of1.0% (crude extract)and0.6%(fraction F6).Interestingly, the slopes of the resulting graphs were significantly different,pointing to different modes of action for the two treatments.Three flav
onoid compounds could be detected.The substances were glabranin,licoflavanon and pinocembrin.All three are known for their anti-microbial and antifungal capacity against plant and human pathogens.Besides the flavonoids,results, such as the different EC50values,indicated that other compounds may be involved in the activity of fraction F6against P.cubensis on cucumber.
Keywords Botanical.plant extract.glabranin. pinocembrin.licoflavanon.induced resistance Introduction
The roots of the Mediterranean Fabaceae Glycyrrhiza glabra(licorice)are ud in medicine since hundreds of years,uth ulcers(Kraus et al. 2004).Also,its strong antiviral capacity is ud against different dias caud by herpes virus (Curreli et al.2005).Furthermore,there is evidence that preparations of G.glabra roots also have effects on HIV(Manfredi et al.2001).Becau of the medical u and the utilization of G.glabra for production of candy or as sweetener there are many studies about substances which can be found in the roots of licorice. The substance class which were studied were mainly flavonoids and isoflavonoides(Hayashi et al.,1996a, b,2003),and some of them have long been known for their antimicrobial(Lester et al.1983;Fukui et al. 1988)and antifungal(Shain and Miller1982)activity. Fukui et al.(1988)also discovered antimicrobial and antifungal activity against human as well as plant pathogens in the upper parts of the plant.
In former bioassays and in greenhous an extract from leaves of G.glabra had a high potential for the control of the oomycete Pudoperonospora cubensis,
Eur J Plant Pathol(2012)134:755–762
DOI10.1007/s10658-012-0051-0
A.Scherf
:A.Schmitt(*)
Julius Kühn-Institut,Institute for Biological Control, Heinrichstraße243,
64287Darmstadt,Germany
e-mail:annegret.schmitt@jki.bund.de
J.Treutwein连发dnf
:H.Kleeberg
Trifolio-M GmbH,
Dr.-Hans-Wilhelmi-Weg1,
35633Lahnau,Germany
the causal agent of downy mildew in cucumber (Scherf et al.2010).This dia is a major problem in organic cucumber production and may lead to total harvest loss dependent on temperature and weather (Haggag2002;Urban and Lebeda2007).In mi-commercial trials in greenhou grown cucumbers, the efficacy of a3%G.glabra extract against downy mildew reached70to83%(Scherf et al.2010). However,information on active ingredients responsible for the efficacy against P.cubensis is lacking.
The aim of this study was therefore to identify fractions and single compounds of the crude G.glabra leaf extract involved in the activity against downy mildew of cucumbers.
Material and methods
Plant extracts
Crude plant extract was prepared from dried and finely ground leaves of Glycyrrhiza glabra L.(licorice)har-vested at NAGREF,Plant Protection Institute of Patras, Patras,Greece.The extraction wa
公共安全管理s done with a Soxhlet extractor in96%EtOH.The ud amount of EtOH was always10times the ud amount of plant powder(v/w). Extraction time varied between2h and10h depending on the amount of plant powder.The extract was concen-trated to50%concentration(w/v),i.e.the extract of50g plant powder was adjusted to a final volume of100ml.
Fractionation by shake-out procedure
For fractionation,EtOH was removed from the crude licorice extract(50%extract concentration)with a rotary evaporator.The dry residue was diluted in equal volumes of CH2Cl2and5%HCl.Concutively, emerging phas were parted and washed at least three times with equivalent portions of the respective other solvent in a paratory funnel.
The pooled CH2Cl2pha was labelled fraction F1. The aqueous pha was alkalinized with ammonia 33%to pH12and then washed again with CH2Cl2 at least three times.The resulting pooled CH2Cl2 pha was labelled fraction F2(alkaloids).The new aqueous pha was evaporated and the dry residues were washed with EtOH96%,precipitating salts were discarded.The supernatant was labelled fraction F3 (quarternary alkaloids and sugars).The CH2Cl2pha (F1)of the first fractionation step was washed with at least three equivalent portions of caustic soda5%. The remaini
ng CH2Cl2pha was evaporated and the pellet diluted in equal volumes of MeOH90%and n-hexane.The emerging phas were labelled fraction F4 (terpenes and sterols)and fraction F5(lipids and waxes). The caustic soda pha from the step before was acidi-fied with HCl to pH2and afterwards washed with at least three portions of fresh CH2Cl2.This CH2Cl2pha was labelled fraction F6(acidic substances).The aque-ous pha was discarded.From all fractions solvents were evaporated in a rotary evaporator and the dry residues were afterwards dissolved in EtOH96%.Final volumes were the same as tho of the inrted crude licorice extract.All fractions were tested in bioassays on cucumber against Pudoperonospora cubensis. Chromatography
TLC
TLC was done on plates(20cm×20cm)consisting of aluminum foil covered with silica gel60F254(Merck). Aliquots(2μl or5μl)of fractions and sub-fractions were applied with2μl or5μl capillaries.TLC plates were dried on air before they were developed in a solvent chamber with toluene-dioxane-AcOH(20:5:1). TLCs were taken out when the solvent front reached approximately11cm height,and dried on air.The spraying reagent(vanillin10mg in100ml32%phos-phoric acid)was applied with a TLC sprayer(Desaga GmbH).The TLC plate was heated at110°C until coloration of the sub-fractions developed.
Column chromatography
For column chromatography the solvent of fraction F6 (50%extract concentration)was evaporated com-pletely in a rotary evaporator.To the residue,the10-fold amount of silica gel60(0.063-0.200mm,Merck KGaA)(w/w)was added,and then CH2Cl2was added until the silica gel was covered.The mixture was stirred and finally the solvent was evaporated again. As a result,the fraction´s compounds were fixed to the silica gel.A glass column(Ø4.5cm)was filled with a thin layer of quartz sand and then solvent/silica gel60 (0.063-0.200mm,Merck KGaA)(w/w)mixture was added.The amount of silica gel was100-fold the amount of the prepared silica/fraction mixture(w/w).
Another layer of quartz sand was filled on top and then the silica/fraction mixture was carefully put on top of the prepared column.As eluent pentane-EtOAc(6:1) was ud.Eluted compounds were collected in at least 200test tubes(10ml)before the column was washed with96%MeOH to recover remaining substances which were not soluble in the first eluent.With all test tube samples a TLC was run as described above. Samples which contained the same substances were pooled to sub-fractions and the solvent was evaporated. For the bioassays dry residues were dissolved in EtOH 96%resulting in same volume and concentration as that of the inrted fraction F6.For each bioassay a fresh column chromatography was performed.
HPLC and1H-NMR-Spectroscopy
属书HPLC was carried out on a Hewlett Packard HP1090 with a photodiode array detector at292nm.For sample preparation30μl of the50%extracts were diluted with MeOH to10ml and filtered through a syringe filter (Nylon,0.2nm).The injection volume of each sample was5μl.For HPLC a Phenomenex00G-4337-E0 synergi4u MAX-RP80A250x4.6mm4micron column was ud.The chromatography was run with a MeCN/H2O gradient of70.0%for2min and afterwards from70.0%MeCN to100.0%MeCN in2min.The flow rate was0.95ml per min and the column temper-ature was40.0°C.Identification of the compounds was done via1H-NMR-spectroscopy at400MHz(Avance II 400MHz WB(A V400)Bruker BioSpin GmbH) Bioassays on potted plants
For the bioassays cucumbers of the cultivar`Chinesi-sche Schlange´were cultivated in a mixture of sand with K15pot ground(Klasmann-Deilmann GmbH)in a proportion of1:3.The plants were grown under a16h light(333μmol Quantaμm-2s-1)and8h dark regime at an average temperature of23.5±2.7°C and an average RH of49.5±15.0%until two leaves were well devel-oped,the other leaves were pricked out.Both leaves were treated by spraying the agents on the lower surface until run-off.The crude G.glabra extract(50%con-centration w/v)as well as the fractions and sub-fractions (50%concentration w/v)were diluted in de-ionized water to the final concentration of0.3%to5%(v/v).李员外
Inoculation of the lower leaf surface was done a day after treatment with an aqueous sporangia suspension of P.cubensis(5×103sporangia/ml).Downy mildew spo-rangia were obtained by washing off the leaves of highly infected cucumber plants that were kept overnight at 100%relative humidity.The resulting suspension was counted in a Fuchs-Ronthal counting chamber.After inoculation,the plants were incubated overnight at100% relative humidity in the dark.Afterwards the plants were kept at an average temperature of18.3±2.0°C and an average relative humidity of47.6±6.7%.
There were two different designs of bioassays,either: (a)the assay was replicated,independently at least four times with varying number of plants per treatment;or (b)the assay consisted of four replications with two or three plants per treatment in a randomized design.
Statistical analysis
米粉汤For statistical analysis of bioassays the median dia verity per replication from the median per plant was calculated for each bioassay.Tho data were ud for the descriptive statistics and ANOVA(WinStat for Excel2009.1).Medians as well as25and75percen-tiles and minima and maxima are pictured in box-plots of bioassays.
ANOVA with Tukey test
The data of assays with four randomized replications with two or three plants per replication and treatment underwent an ANOV A with Tukey test p<0.05.Statis-tical analysis was done with WinStat for Excel2009.1. Effective concentration(EC)values
To calculate the effective concentration values of licorice leaf extract and fraction F6resulting in a50%dia reduction(EC50),probit analysis(software MLP Version 3.08)was ud.Efficacies were calculated from the medians of dia verity per replication.Tho data were transformed in respon data with the formula respon¼nÂefficacy100n¼sample size
ðÞ:
=
荷花淀主要内容The calculated respon data were ud for further processing by probit analysis.
Chi square(χ2)
Data of the do–respon curves resulting from the probit analysis underwent a Chi square analysis
(software MLP Version 3.08)for position,parallelism and total heterogeneity p <0.05.
Results
Bioassays of extract fractions
Bioassays with cucumber plants and with P .cubensis were done with six fractions resulting from a fraction-ation of crude licorice (G.glabra )extract.The main activity was found in the fraction containing acidic substances and neutrals (Fig.1;F1).
Dia verity reached an average of 1.7%,while water treated plants showed an infection of 76.2%with P.cubensis .The efficacy of fraction F1was 97.7%and comparable to that of crude licorice ex-tract,which reached 98.8%efficacy.In contrast,the fractions which contained alkaloids and water soluble substances (Fig.1;F2and F3)were less effective against P .cubensis (Fig.1;F2and F3).For both,mean level of dia verity reached 62.5%.
Fractions F4(terpenoids and sterols)and F5(waxes and lipids),which originated from F1(Fig.1),reached efficacies of up to 41.8%and 31.1%respectively.They were more effective than fraction F2and fraction F3(efficacy 18.0%,both).However,the highest activity of all fractions in controll
ing downy mildew on cucumber was achieved by fraction F6(efficacy 97.6%)which also originated from fraction F1(Fig.1).This fraction which contained acidic substan-ces reached nearly the same efficacy as fraction F1but the variation in infection levels between single plants was slightly higher (Fig.1).
Identification of compounds from sub-fractions After column chromatography of fraction F6,a total of five sub-fractions could be parated (B1-B4and remains)with the given solvent system.With HPLC three dominant compounds of the active fraction F6could be detected (Fig.2).Tho compounds belong to the group of polyphenols and were identified via 1
H-NMR-spectroscopy as glabranin (sub-fraction
B1),
Fig.1Influence of the extract of licorice (G.glabra;lic.)and its fractions (F1-F6)on the dia verity (Infection [%])of P .cubensis on potted cucumber plants (cv.`Chinesische Schlange´)in a climate room (n 04–6per treatment in 10inde-pendent assays).Box plot:Bar 0median,cross 0min.and max.;boxes 025th percentile and 75th percentile (within 50%of the data);whisker 05th percentile and 95th
percentile
Fig.2Chromatogram from HPLC of crude licorice (G.glabra )extract,
fraction F6and parated sub-fractions (B1-B4and remains)of fraction F6.(Due to the low print quality of the original,lines and labels were reworked electronically)
pinocembrin (sub-fraction B2)and licoflavanon (sub-fraction B3).Compounds from sub-fraction B4and remains were not detectable.
Also,the chromatogram illustrated that after fraction-ation of the crude extract the amount of compounds (rembled by the peak height)detectable in fraction F6and the sub-fractions was notably lower (Fig.2).Bioassays of extract sub-fractions
Sub-fractions were tested in 3%concentration on cucumbers against P .cubensis (Fig.3a and b ).
For each of the two bioassays parate column chromatographies were done.
车位租赁合同简单版In both bioassays,a significant effect on the infection with downy mildew was shown for all sub-fractions compared to water treated plants.
志南和尚
Sub-fraction B1inhibited the development of downy mildew on cucumber in both trials to a high degree (efficacy assay a:91.7%and assay b:96.6%).Sub-fraction B2was also highly effective against
the dia (efficacy 81.7%and 75.7%)(Fig.3a and b ).Although dia levels after treatment with B2were not significantly different to tho after treat-ment with B1,sub-fraction B2tended to be less effective.Sub-fraction B3,which contains licoflava-non,was the one with the lowest efficacy against P.cubensis from all (assay a:65.8%and assay b:56.8
%).
Fig.3Influence of fraction F6(F6)from licorice (G.glabra;lic .)extract and its sub-fractions (B1-B4and remains)on dis-ea verity of P.cubensis on potted cucumber plants (cv.`Chinesische Schlange´)in two bioassays,a)and b),in a climate room (n 08–12per treatment in 4randomized replications per assay).Different letters indicate significant differences accord-ing to Tukey test (p <0.05)Box plot:Bar 0median,cross 0min.and max.;boxes 025th percentile and 75th percentile (with-in 50%of the data);whisker 05th percentile and 95th percentile.a )Mean dia verity per treatment out of median per repli-cation:water 77.5%,F618.8;B16.1%,B214.1%,B327.2%,remains 21.3.b )Mean dia verity per treatment out of median per replication:water 93.8%,F64%;lic.4.9%,B12.1%,B218.6%,B346.3%,B49.1%,remains 24.4
%
Fig.4Influence of extract of licorice (G.glabra;a)and frac-tion F6b)on the dia verity of P .cubensis on potted cucumber plants (cv.`Chinesische Schlange´)in a climate room (n 06–16per treatment in 4assays).Box plot:Bar 0median,cross 0min.and max.;boxes 025th percentile and 75th per-centile (within 50%of the data);whisker 05th percentile and 95th percentile
