中草药提取物对几种植物病原菌的抗菌活性研究
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摘要
以苹果炭疽病菌、番茄枯萎病菌、梨黑星病菌为供试病菌，对7种中草药提取物的抗菌活性进行了研究，并且对不同提取方法进行了探讨，研究结果如下：
1．不同提取方法的提取率及提取物抗菌活性表明，不同溶剂提取效率均以索氏抽提法和超声波法为高；同种提取方法以氯仿、95％乙醇提取效率高。从提取物抗菌活性来看，同种溶剂不同提取方法提取物活性差异不大，但以索氏提取物和超声波提取物抗菌活性略高。实验室提取可以95％乙醇做溶剂采用超声波法提取较为理想。
2．7种中药材提取物对供试植物病原菌的抗菌活性研究表明，中药材CCB对苹果炭疽病菌有良好的抑制作用，氯仿、95％乙醇提取物抑菌效果在72h时，对苹果炭疽病菌的EC_(50)分别为0.5474g／L、1.4785g／L；95％乙醇提取物经过液液分配得到有机相和水相，其石油醚相和乙酸乙酯相抑制效果较好，其EC_(50)分别为2.8092g／L、0.3246g／L，说明抗菌活性物质极性中等偏低；乙酸乙酯相进一步柱层析，得到不同馏分，生测结果表明乙酸乙酯洗脱C1馏分活性最强，EC_(50)为0.058g／L，进一步说明活性成分的极性为中等；C1馏分用制备型薄层分离，在254nm紫外光下观察，R_f=0.5处有一个红色带，经过生测其对苹果炭疽病菌的EC_(50)为0.18g／L，而分离的色素进行生测活性很低，说明R_f=0.5的组分为实验跟踪的活性成分。
3．中药材CCB鲜样、干样及植株不同部位活性研究表明，植株干样、鲜样对苹果炭疽病菌的抑制作用没有差异，但其整株活性明显高于植株各个器官部分(根、茎、叶)的活性，根、茎、叶提取物中以叶的活性最差。
4．中药材CCB与化学药剂比较研究表明，中药材CCB提取物对苹果炭疽病菌的抑制作用低于化学药剂多菌灵、代森锰锌，后者对苹果炭疽病菌的EC_(50)分别为0.13mg／L、24.4728mg／L，而中药材CCB柱层析C1馏分的EC_(50)为58mg／L，制备型薄层分离得到的活性物质的EC_(50)为180mg／L。
5．中药材CCB粗提物活体实验表明，其对苹果炭疽病具有一定的保护和治疗作用，但保护作用优于治疗作用，且在浸果1d后接菌保护作用最高。以多菌灵做参比，中药材CCB粗提物在10g/L浓度下防效与多菌灵(100mg／L)防效相当，甚至略高，持效期比多菌灵短，但两种药剂总体防效都不是很高，有待进一步研究其他的作用机理。
6．中药材CCB抗菌活性成分的分离、鉴定。对其95％乙醇提取物采用
系列极性溶剂液液分配，活性成分进行柱层析，进行梯度洗脱，不同馏分结
合生测确定活性馏分为O，活性馏分口经过制备薄层分离得到本实验跟踪
的活性组分。薄层分离以苯：甲醇（28：5）为展开剂，上行展开，活性组分
的RPO．5。比较薄层分离组分和柱层析CI馏分的紫外吸收光谱，具有相同
的吸收波长，分别为Z14nm、282urn；分别以Z14nm、282urn为检测波长，
进行HPLC分析，均有一个明显的主峰，保留时间分别为2．71 6min、2．843min；
活性组分进行GC分析得到一个主峰13．363min。为了进一步明确活性组分，
进行 GC－MS分析，从总离子流图上看到 11．88min、12．83min两个离子峰，
分别对这两个峰进行质谱分析，具有相同的分子离子峰m／e 2 86，基峰分别为
120．9、133．9，对比质谱图库得到两个离子峰为黄酮类、黄酮醇类化合物，这
一结果与前面的紫外吸收光谱、化学性状相一致。
In this paper,studies were conducted to screen the antifungal activity of organic solvent extract from seven kinds herbs on the three plant pathogen (Venturia inaequalis (Cooke)Wine,Fusarium oxysporum f.sp.lycopeerisci (Sacc.) Syder et.hanson),Colletotrichum gloeosporioides (Penz) Penz and Sacc.),The results were summarized as follows :
l.The extract efficiency of Soxhlet method and Supersonic method was superior to the other methods by the same solvent .In the same method,the extract efficiency of chlorioform and 95% ethanol is higher. As far as antifungal activity was concerned,the antifungal activity of every method extract was similar by the same solvent,but Soxhlet method and supersonic method were a little better.
2.Seven kinds of herbs were screened,and the extract of herb CCB had strong antifungal activity on the Colletotrichum gloeosporioides and the ECso of which were 0.5474g/L and 1.4785g/L respectively in 72 hours by chlorioform and 95% ethanol;the extract of 95% ethanol was isolated by liquid extraction method,the fraction of petroleum ether and ethyl acetate had strong antifungal activity on the Colletotrichum gloeosporioides,the ECso of which were 2.8092 g/L and 0.3264g/L respectively;the fraction of ethyl acetate was isolated by liquid solid column chromatography,The effluent Cl showed strong antifungal activity,the ECso of which was 0.058g/L,which showed that antifungal substance was intermoderate polarity;The effluent Cl was isolated further by Preparative TLC,two band were showed,one had colour in visual light range,which had nearly no antifungal activity,the other was red band in UV light of 254 nm,which had antifungal activity on the Colletotrichum gloeosporioides,the ECso was 0.18g/Land the Rf was 0.5.
So we can conclude that the substance of Rf=0.5 was the antifungal substance traced in experiment.
3.The extracts of fresh sample and dry sample of herb CCB and its every part were tested,and the antifungal activity of fresh sample and dry sample was nearly similar on the Colletotrichum gloeosporioides,but the antifungal activity of fresh sample and dry sample was higher than that of root,stem and leaf. The antifungal activity of leaf was the lower on the Colletotrichum gloeosporioides than that of
r
root and stem.
4. Compared with chemical fungicides including carbendazim and mancozeb,antifungal activity of extact from herb CCB was lower. The ECso of two fungicides were 0.13mg/L,24.4728 mg/L respectively,but the ECso of the effluent Cl from liquid solid column chromatography of herb CCB was only 58 mg/L.
5.The control efficiency of the crude extract of herb CCB to apple anthracnose in vivo was studied,which showed that the extract of the herb CCB had protective and therapeutic action for apple anthracnose,with the protective action superior to therapeutic action.The protective ation of herb CCB was highest,when the apple was incubated after 1 d,which was conducted by extract of herb CCB. Compared with carbendazim,the control efficiency of the crude extract of herb CCB(10g/L) and carbendazim(100mg/L) was nearly similar,and the lasting period of latter was longer,but the control efficiency of both was not as great.
6.The herb CCB powder was extracted by 95% ethanol .the extract of 95% ethnol was defatted with petroleum ether,then followed by solvent partition to obtain ethyl acetate extract which was fractioned on a column of silica gel with gradient petroleum ether-ethyl acetate -methanol system .It was found the effluent Cl possessed the antifungal substance .Therefore the effluent Cl was rechromatographed on PTLC with benzen-methanol system as a mobile phase (28+5,v/v),and obtained active substance which was analyzed and identified by means of chemical reaction,UV,HPTLC,HPLC,GC,GC-MS,suggesting the active substance may be flavonoids.
1．不同提取方法的提取率及提取物抗菌活性表明，不同溶剂提取效率均以索氏抽提法和超声波法为高；同种提取方法以氯仿、95％乙醇提取效率高。从提取物抗菌活性来看，同种溶剂不同提取方法提取物活性差异不大，但以索氏提取物和超声波提取物抗菌活性略高。实验室提取可以95％乙醇做溶剂采用超声波法提取较为理想。
2．7种中药材提取物对供试植物病原菌的抗菌活性研究表明，中药材CCB对苹果炭疽病菌有良好的抑制作用，氯仿、95％乙醇提取物抑菌效果在72h时，对苹果炭疽病菌的EC_(50)分别为0.5474g／L、1.4785g／L；95％乙醇提取物经过液液分配得到有机相和水相，其石油醚相和乙酸乙酯相抑制效果较好，其EC_(50)分别为2.8092g／L、0.3246g／L，说明抗菌活性物质极性中等偏低；乙酸乙酯相进一步柱层析，得到不同馏分，生测结果表明乙酸乙酯洗脱C1馏分活性最强，EC_(50)为0.058g／L，进一步说明活性成分的极性为中等；C1馏分用制备型薄层分离，在254nm紫外光下观察，R_f=0.5处有一个红色带，经过生测其对苹果炭疽病菌的EC_(50)为0.18g／L，而分离的色素进行生测活性很低，说明R_f=0.5的组分为实验跟踪的活性成分。
3．中药材CCB鲜样、干样及植株不同部位活性研究表明，植株干样、鲜样对苹果炭疽病菌的抑制作用没有差异，但其整株活性明显高于植株各个器官部分(根、茎、叶)的活性，根、茎、叶提取物中以叶的活性最差。
4．中药材CCB与化学药剂比较研究表明，中药材CCB提取物对苹果炭疽病菌的抑制作用低于化学药剂多菌灵、代森锰锌，后者对苹果炭疽病菌的EC_(50)分别为0.13mg／L、24.4728mg／L，而中药材CCB柱层析C1馏分的EC_(50)为58mg／L，制备型薄层分离得到的活性物质的EC_(50)为180mg／L。
5．中药材CCB粗提物活体实验表明，其对苹果炭疽病具有一定的保护和治疗作用，但保护作用优于治疗作用，且在浸果1d后接菌保护作用最高。以多菌灵做参比，中药材CCB粗提物在10g/L浓度下防效与多菌灵(100mg／L)防效相当，甚至略高，持效期比多菌灵短，但两种药剂总体防效都不是很高，有待进一步研究其他的作用机理。
6．中药材CCB抗菌活性成分的分离、鉴定。对其95％乙醇提取物采用
系列极性溶剂液液分配，活性成分进行柱层析，进行梯度洗脱，不同馏分结
合生测确定活性馏分为O，活性馏分口经过制备薄层分离得到本实验跟踪
的活性组分。薄层分离以苯：甲醇（28：5）为展开剂，上行展开，活性组分
的RPO．5。比较薄层分离组分和柱层析CI馏分的紫外吸收光谱，具有相同
的吸收波长，分别为Z14nm、282urn；分别以Z14nm、282urn为检测波长，
进行HPLC分析，均有一个明显的主峰，保留时间分别为2．71 6min、2．843min；
活性组分进行GC分析得到一个主峰13．363min。为了进一步明确活性组分，
进行 GC－MS分析，从总离子流图上看到 11．88min、12．83min两个离子峰，
分别对这两个峰进行质谱分析，具有相同的分子离子峰m／e 2 86，基峰分别为
120．9、133．9，对比质谱图库得到两个离子峰为黄酮类、黄酮醇类化合物，这
一结果与前面的紫外吸收光谱、化学性状相一致。
In this paper,studies were conducted to screen the antifungal activity of organic solvent extract from seven kinds herbs on the three plant pathogen (Venturia inaequalis (Cooke)Wine,Fusarium oxysporum f.sp.lycopeerisci (Sacc.) Syder et.hanson),Colletotrichum gloeosporioides (Penz) Penz and Sacc.),The results were summarized as follows :
l.The extract efficiency of Soxhlet method and Supersonic method was superior to the other methods by the same solvent .In the same method,the extract efficiency of chlorioform and 95% ethanol is higher. As far as antifungal activity was concerned,the antifungal activity of every method extract was similar by the same solvent,but Soxhlet method and supersonic method were a little better.
2.Seven kinds of herbs were screened,and the extract of herb CCB had strong antifungal activity on the Colletotrichum gloeosporioides and the ECso of which were 0.5474g/L and 1.4785g/L respectively in 72 hours by chlorioform and 95% ethanol;the extract of 95% ethanol was isolated by liquid extraction method,the fraction of petroleum ether and ethyl acetate had strong antifungal activity on the Colletotrichum gloeosporioides,the ECso of which were 2.8092 g/L and 0.3264g/L respectively;the fraction of ethyl acetate was isolated by liquid solid column chromatography,The effluent Cl showed strong antifungal activity,the ECso of which was 0.058g/L,which showed that antifungal substance was intermoderate polarity;The effluent Cl was isolated further by Preparative TLC,two band were showed,one had colour in visual light range,which had nearly no antifungal activity,the other was red band in UV light of 254 nm,which had antifungal activity on the Colletotrichum gloeosporioides,the ECso was 0.18g/Land the Rf was 0.5.
So we can conclude that the substance of Rf=0.5 was the antifungal substance traced in experiment.
3.The extracts of fresh sample and dry sample of herb CCB and its every part were tested,and the antifungal activity of fresh sample and dry sample was nearly similar on the Colletotrichum gloeosporioides,but the antifungal activity of fresh sample and dry sample was higher than that of root,stem and leaf. The antifungal activity of leaf was the lower on the Colletotrichum gloeosporioides than that of
r
root and stem.
4. Compared with chemical fungicides including carbendazim and mancozeb,antifungal activity of extact from herb CCB was lower. The ECso of two fungicides were 0.13mg/L,24.4728 mg/L respectively,but the ECso of the effluent Cl from liquid solid column chromatography of herb CCB was only 58 mg/L.
5.The control efficiency of the crude extract of herb CCB to apple anthracnose in vivo was studied,which showed that the extract of the herb CCB had protective and therapeutic action for apple anthracnose,with the protective action superior to therapeutic action.The protective ation of herb CCB was highest,when the apple was incubated after 1 d,which was conducted by extract of herb CCB. Compared with carbendazim,the control efficiency of the crude extract of herb CCB(10g/L) and carbendazim(100mg/L) was nearly similar,and the lasting period of latter was longer,but the control efficiency of both was not as great.
6.The herb CCB powder was extracted by 95% ethanol .the extract of 95% ethnol was defatted with petroleum ether,then followed by solvent partition to obtain ethyl acetate extract which was fractioned on a column of silica gel with gradient petroleum ether-ethyl acetate -methanol system .It was found the effluent Cl possessed the antifungal substance .Therefore the effluent Cl was rechromatographed on PTLC with benzen-methanol system as a mobile phase (28+5,v/v),and obtained active substance which was analyzed and identified by means of chemical reaction,UV,HPTLC,HPLC,GC,GC-MS,suggesting the active substance may be flavonoids.
引文
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