全 文 ：AntifungalActivityofExtractsfromClerodendrumBungeiLeavesagainst
TwoSpeciesofPhytopathogens
YINLi-guo, LINNa, WEIQin＊ , ZHANGChao, ZHOULi-jun
KeyLabofSouthwestSpecialEconomicPlantProteitionandUtilization, YibinColege, Yibin644007
Abstract　ThetestwasundertakentorevealtheantifungalactivityofextractsfromClerodendrumbungeileavesagainstPestalotiafunerealandRhizoctonia
solani, theresultsshowedthatoptimalconditionforbestantifungalactivityofextractsagainstPestalotiafunerealandRhizoctoniasolaniareasfolows:mate-
rial-liquidratioof1∶6, 75% ethanolasextractingsolvent, refluxat90℃for1.5h.Thesubstanceswithgooddissolubilityinethanolandwatersolution
suchasorganicacid, bioflavonoidandalkaloidaremainantifungalbioactivesubstancesinClerodendrumbungei.
Keywords　Clerodendrumbungeileaves;Pestalotiafunereal;Rhizoctoniasolani;Antifungalactivity
Received:March18, 2008　　Accepted:March31, 2008
SupportedbyDoctorStartupFoundationofYibinColege(2005B02).
＊Correspondingauthor.Tel:0831-3545069;E-mail:Weiqin2001
-67@163.com
　　Roseglorybower(ClerodendrumbungeiSteud.), ade-
ciduousshrubofVerbenaceae, distributeswidelyfromNorth
China, ShaanxiProvincetotheprovincesinthesouthofthe
YangtzeRiver, withdiferentChinesenameinvariousrelat-
edreferencebooks, forexample, DahongbaoandChoubabao
in, Dahonghuain<
GuizhouCivilBarkColection>.Alitsroot, stemandleaf
possesmedicinalvalue, assumewarminpropertyandbiter
inflavour, withmanyeficaciesincludingbloodactivating
andstasisremoving, detumescenceanddetoxification, clear-
ingheatremovingdampnesandacesodyne.Additionaly,
modernmedicalresearchesshowedthatroseglorybowerhas
antitumorandimmuneenhancementefect, andcancuredis-
eases, suchasmastitis, nephritis, arthritis, eczema, tooth-
ache, hemorhoidsandexania[ 1-8] .Studiesaboutitschemi-
calcomponentspresentedroseglorybowerisrichinplant
polyphenols, alkaloid, organicacid, aminoacids, sugar, bi-
oflavonoidandvolatileoil[ 9] .Nowadaysthelargelyusedsyn-
theticorganicpesticidesplaygreatrolesincroppestmanage-
mentandstableyield, buttheirowntoxicitycausesevere
negativeinfluencetoenvironmentandhumanbeing.Soex-
ploitingnewbotanicalpesticideswithlowtoxicityandstrong
efectisakeyapproachtoaboveproblem.Herewereport
theantifungalactivityofrosegloryboweragainstPestalotia
funerealandRhizoctoniasolani.
Materialsandmethods
Materialsandreagents
Roseglorybowerleaveswerecolectedandthendried,
crushedforstorage.ThesamplingplotisQiuchangTownof
CuipingDistrict, YibinCity, SichuanProvince.Teststrains
PestalotiafunerealandRhizoctoniasolaniwerefromKeyLa-
boratoryofProtectionandUtilizationofSpecialEconomic
PlantinSouthwestChina.
Altheethanol, acetone, ethylacetate, ethyletherand
petroleumetherfortestareanalyticalreagents.
Experimentalinstruments
ConstanttemperaturebiochemicalincubatorLRH-70
(ShanghaiYihengTechnologyCo., Ltd.), constanttemper-
aturewatertrough(BeijingChanganScientificInstrument
Company)andrefluxextractionpump.
Preparationofextractsfromroseglorybowerleaves
10.0gdriedroseglorybowerleafpowderwasscaledand
placedinextractionvesel, additionof60 mlextractingsol-
vent.Theextractingsolventwasleachedanddiscardedby
vacuumconcentration, thendissolvedwithDMSOandsetthe
volumeat100 ml.Firstlyoptimalextractingsolventwas
screened, thenforoptimizationoftheconcentrationofselect-
edsolventandextractingtime.
Detectionofantifungalactivity
Filterpapermethodwasadoptedtotestofantifungalac-
tivityofpreparedextracts.Theproceduresareasfolows:the
strainswereinoculatedonPDAmediumandsubcultured3
timesforactivation;themediumslantwaswashedwith2 ml
salinesolutionbyinjectionforpreparingbacterialsuspen-
sion;understerilecondition, 0.1 mlbacterialsuspension
wasaddedintomedium( 9 cm), coatedtoevennes,
meanwhile, immersionoffilterpapers( 7 cm)inextracts;
thenthefilterpapersweredepositedonplate, andinoculated
inbiochemicalincubatorat28 ℃ lasting48 hfordetermi-
ningantifungaldiameters.Theconcentrationsofbacterial
suspensioninthistestare0.42×106 -0.61×106 cfu/mlfor
PestalotiafunerealandRhizoctoniasolani, respectively.
Resultsandanalysis
Antifungalefectofextractsofvariousextractingsol-
vents
Diferentextractingsolventsofethanol, ethylether, pe-
troleumether, acetone, waterandethylacetateweresetto
screenoptimalextractingsolvent.Refluxat90, 45, 45, 55,
100, 70 ℃ corespondingtoabovesolventsfor1h.Thepro-
cedurewasasabovedescribed.
AsshowninTable1, theextractsofethanol, acetone
andethylacetateasumedobviousantifungalefectagainst
PestalotiafunerealandRhizoctoniasolani, theextractsof
othertwoextractingsolventsperformedpoor.Theoptimalan-
AgriculturalScience＆Technology, 2008, 9(1):143-145
Copyright 2008, InformationInstituteofHAAS.Alrightsreserved. PlantProtection
tifungalefectforextractingsolventsisethanolandfortested
phytopathogensisPestalotiafunereal.
Table1　Theantifungaleffectofextractsofdiferentextractingsol-
vents
Extractingsolvent
(Temperature∥℃)
Antifungaldiameter∥mm
Pestalotiafunereal Rhizoctoniasolani
Ethanol(90) 8.5 8.5
Acetone(45) 7.9 7.8
Ethylacetate(45) 7.5 7.4
Ethylether(55) 7.3 7.2
Petroleumether(100) 7.3 7.2
Water(70) 7.1 7.2
Antifungalefectofextractsofdiferentethanolconcen-
trations
Accordingtoaboveextractspreparationprocedure, dif-
ferentconcentrationsofethanolaqueoussolutionwereregar-
dedasextractingsolvents.Refluxat90 ℃ forpreparingex-
tracts.Theefectwasexaminedbyantifungaltest.Thede-
tectionresultswereshowninTable2.
Table2　Theantifungaleffectofextractsofdiferentethanolconcen-
trations
Ethanolconcentration
%
Antifungaldiameter∥mm
Pestalotiafunereal Rhizoctoniasolani
30 7.4 7.3
45 7.6 7.6
60 8.6 8.7
75 8.8 8.8
90 8.6 8.5
100 8.5 8.5
　　Theextractsof75% ethanolpresentedbestantifungal
efectagainstPestalotiafunerealandRhizoctoniasolani, ex-
tractsofethanolwithotherconcentrationsalsomanifestedan-
tifungalefectandtheefectdecreasedobviouslywiththe
concentrationofethanolreduced.Thewatersolublematers
suchassugarandaminoacidsextractedeasilywithlower
ethanolconcentration, andresultantlyconcentrationofanti-
fungalactivematersdecreased.
Efectsofextractingtimeonantifungalefect
Basedonaboveresults, extractingtimewasoptimized
with75%ethanolaqueoussolutionasextractingsolvent.The
timeof1, 0.5, 1.5, 2, 2.5, 3 hweresetforscreeningoptimal
refluxduration.Preparingextractsaccordingtoaboveproce-
dure.Thentheefectwasexaminedbyantifungaltest.
Antifungalefectofextractsenhancedwithextracting
timeprolonged, bestantifungalefectappearedundertheex-
tractingtimeof1.5h(Table3).However, whenextracting
timewaslongerthan3 h, theantifungalefectweakened,
whichprobablybecauseofactivematersweredissolvedun-
derhightemperatureforalongtime.
Table3　Effectsofextractingtimeonantifungalefect
Extractingtime
h
Antifungaldiameter∥mm
Pestalotiafunereal Rhizoctoniasolani
0.5 7.5 7.6
1 8.4 8.3
1.5 8.8 8.8
2 8.8 8.8
2.5 8.8 8.8
3 8.7 8.6
Conclusion
AntifungalactivityofextractsfromClerodendrumbungei
leaveswerestudiedthroughfilterpapermethod.Theresults
revealedoptimalconditionforbestantifungalactivityofex-
tractsagainstPestalotiafunerealandRhizoctoniasolaniare
material-liquidratioof1∶6, 75% ethanolasextractingsol-
vent, refluxat90 ℃ for1.5 h.Therearemanyplantpoly-
phenols, alkaloid, organicacid, aminoacids, sugar, biofla-
vonoidandvolatileoilinroseglorybower, andweknowplant
polyphenols, alkaloid, organicacid, bioflavonoidandvola-
tileoilareendowedwithgooddisolubilityinethanoland
watersolution[ 10] , whichisthemainreasonforexplaining
75% ethanolisthebestextractingsolvent;meanwhile, ami-
noacidsandsugarsarefreelysolubleinwater, butavailthe
growthofmicroorganisms, consequently, antifungalactivity
ofextractsweakenedwiththeincreaseofwatercontent.
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144 AgriculturalScience＆TechnologyVol.9, No.1, 2008
臭牡丹叶提取液对 2种植物病菌的抑制作用研究
尹礼国 ,林娜 ,魏琴＊ ,张超 ,周黎军　(宜宾学院西南特色经济植物保护与利用重点实验室 , 四川宜宾 644007)
　　笔者以广泛引发植物病害的松赤枯病菌(Pestalobiafune-
rea)和玉米纹枯病菌(RhizoctoriasolaniKuha)为检测菌 ,对臭牡
丹的抗植物病菌活力进行研究 。
1　材料与方法
1.1　材料　臭牡丹叶采自四川省宜宾市翠屏区邱场乡 ,及时晒
干后粉碎 ,密封保存;松赤枯病菌和玉米纹枯病菌由宜宾学院西
南特色经济植物保护与利用重点实验室提供 。 LRH-70型电热
恒温生化培养箱 (上海一恒科技有限公司)、电热恒温水浴锅
(北京长安科学仪器厂)、回流浸提装置 。
1.2　臭牡丹叶提取液的制备　称取 10.0g干燥臭牡丹叶粉 ,倒
入浸提瓶 ,分别加 60ml溶剂回流浸提 ,抽滤得提取液 ,真空浓缩
除溶剂 ,用二甲基亚砜溶解 ,定容至 100ml。筛选出最佳溶剂 ,
然后按上述步骤对比溶剂适于提取的浓度及浸提时间进行优
化 。
1.3　提取液抑菌效果检测　采用滤纸片法 。将菌种接种于马
铃薯培养基 ,活化 3次后 ,用注射器吸取 2ml生理盐水冲洗真菌
斜面 ,制得菌悬液 ,测得菌浓度分别为松赤枯病菌 0.42×106 ～
0.61×106 cfu/ml、玉米纹枯病菌 0.51×106 ～ 0.67×106 cfu/ml。
在无菌条件下 ,吸取 0.1ml菌悬液至培养基(φ9cm)上 ,涂布均
匀 ,将灭菌后的滤纸片(φ7 mm)浸泡在各提取液中 ,取出后放到
平板上 ,在 28℃生化培养箱中培养 48h后 ,测定抑菌圈直径 。
2　结果与分析
2.1　几种溶剂的提取液抑菌作用　设置不同溶剂按“1.2”制备
提取液 ,进行抑菌试验 。由表 1可知 ,乙醇、丙酮和乙酸乙酯提
取液对松赤枯病菌和玉米纹枯病菌的抑制作用较明显 ,而水 、乙
醚 、石油醚提取液的抑菌作用较弱 。其中以乙醇提取液的抑菌效
果最佳 ,提取液对松赤枯病菌的抑制作用强于玉米纹枯病菌。
2.2　不同浓度乙醇溶液提取液的抑菌作用　按照提取液制备
程序 ,以不同浓度乙醇水溶液为溶剂 , 90 ℃下提取 ,制备提取
液 ,通过抑菌试验测得抑菌圈直径(表 2)。由表 2可知 , 75%乙
醇提取液对松赤枯病原真菌和玉米纹枯病原真菌的抑制作用最
大 ,其他浓度的乙醇提取液对 2种病原菌也有抑制作用 ,随着浓
度降低 ,其抑制作用明显下降。这是由于随着乙醇浓度降低 ,有
益于微生物生长的水溶性物质如糖类 、氨基酸浸出比率增加 ,而
抑菌活性物质浸出比率下降所致 。
2.3　浸提时间对抑菌效果的影响　在上述试验基础上 ,以 75%
乙醇水溶液为溶剂 ,设置 0.5、1.0、1.5、2.0、2.5、3.0 h6个浸提
时间 ,按操作步骤制备提取液。通过抑菌试验测得抑菌圈直径
基金项目　宜宾学院博士启动基金课题(2005B02)资助。
作者简介　尹礼国(1979-),男 ,湖北大冶人 ,硕士 ,讲师 ,从事天然产
物化学及应用研究。 ＊通讯作者。
收稿日期　 2008-03-18　　修回日期　 2008-03-31
(表 3)。随着浸提时间延长 ,浸提液的抑菌作用增强;提取时间
为 1.5h时 ,抑菌效果达到最大值;当提取时间延长至 3 h,抑菌
作用减弱 ,这是由于长时间高温作用使得活性物质分解所致 。
　　表 1 不同溶剂的提取液的抑菌效果
溶剂
(提取温度∥℃)
抑菌圈直径∥mm
松赤枯病菌 玉米纹枯病菌
乙醇(90) 8.5 8.5
丙酮(45) 7.9 7.8
乙酸乙酯(45) 7.5 7.4
乙醚(55) 7.3 7.2
石油醚(100) 7.3 7.2
水(70) 7.1 7.2
　　表 2 不同浓度乙醇提取液的抑菌效果
乙醇浓度
%
抑菌圈直径∥mm
松赤枯病菌 玉米纹枯病菌
30 7.4 7.3
45 7.6 7.6
60 8.6 8.7
75 8.8 8.8
90 8.6 8.5
100 8.5 8.5
　　表 3 浸提时间对提取液抑菌作用的影响
提取时间
h
抑菌圈直径∥mm
松赤枯病菌 玉米纹枯病菌
0.5 7.5 7.6
1 8.4 8.3
1.5 8.8 8.8
2 8.8 8.8
2.5 8.8 8.8
3 8.7 8.6
3　结论
采用滤纸片法对臭牡丹叶提取液的抑菌作用进行了研究 ,
结果表明 ,以料液比 1∶6、75%乙醇为溶剂 ,在 90 ℃下回流浸提
1.5h的提取液对松赤枯病菌和玉米纹枯病菌的抑菌作用最好 。
据报道 ,臭牡丹含有植物多酚 、生物碱、有机酸、氨基酸 、糖类 、生
物类黄酮和挥发性物质 ,其中植物多酚 、生物碱 、有机酸 、生物类
黄酮和挥发性物质在乙醇及其水溶液中有较好的溶解性 ,这是
以 75%乙醇水溶液为溶剂的提取液抑菌性最强的主要原因;而
氨基酸和糖类物质易溶于水 ,但有利于微生物的生长 ,所以当乙
醇水溶液中水的比例提高时 ,其抑菌作用减弱 。
(上接第 103页)
3　讨论
将 bel基因导入恢复系 ,用于父母本混播制种 ,通过抽穗期
喷施苯达松去除恢复系 ,可实现制种机械化 ,革新了杂交水稻
种子生产技术 ,具有广阔的应用前景 。该试验结果表明 ,以父 、
母本重量混合比 1∶5进行混播制种 ,在父本抽穗期以前 ,喷施
48%苯达松水剂 3.375 L/hm2以上 ,父本几乎被完全杀死 ,母
本结实率高达 70%以上 ,制种纯度达 98.5%,杂交稻 F1的发
芽率为 95.5%,完全可以生产出达到国家标准的杂交水稻种
子。在生产过程中 ,应注意选择父本始穗至抽穗期喷施苯达
松 ,迟于该时期会影响杂交种的纯度 。进行混播制种组合选育
时 ,存在要求父母本的播始历期相近 、对母本的纯度要求更严
格、阴雨天施药父本杀死率低等难题 ,有待于进一步研究 。
145YINLi-guoetal.AntifungalActivityofExtractsfromClerodendrumBungeiLeavesagainstTwoSpeciesofPhytopathogens