天山云杉自毒作用与自毒物质的鉴定
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摘要
天山云杉(Picea schrenkiana)是中亚和亚洲中部山地特有种,天山山地最主要的地带性森林植被,主要分布在天山北坡和天山南坡,在昆仑山西部北坡也有少量的分布。天山云杉的森林干扰体制决定了其现有林分以纯林为主,林分面积约为52.84万hm2,占新疆山地天然林有林地面积的50%。天山云杉是天山乃至新疆森林生态系统的主要树种和生命之源,对水源涵养发挥着重要作用。然而随着人类活动对森林生态系统干扰强度的不断加大,以及长时间无火干扰的缓解,天山云杉林的自然更新问题日益突出。已有研究结果认为,在现有森林干扰体制下,自毒作用是影响天山云杉天然更新成败的关键因素。基于此,本研究通过人工气候箱中模拟天山云杉种子萌发和幼苗生长的条件,在对天山云杉凋落物和针叶水浸提液(原始水相)、乙醚、乙酸乙酯、正丁醇萃取相及剩余水相进行生物测定的基础上,采用GC-MS-MS初步鉴定了乙醚、乙酸乙酯、正丁醇萃取相的化学成分,并采用硅胶柱层析分离、重结晶等技术从自毒效应最强的乙醚萃取相中分离纯化出化合物3,4-二羟基苯乙酮,检测了其自毒和化感效应。研究主要结果如下：
1.凋落物原始水相、乙醚、乙酸乙酯和正丁醇萃取相都具有自毒效应。种子萌发试验中,凋落物水浸提液乙醚萃取相具有最强的抑制效应(IC50=5mg/mL),而正丁醇萃取相的抑制效应最弱(IC50>10mg/mL)；幼苗生长试验中,1.25mg/mL凋落物水浸提液乙醚萃取相对幼苗生长具有显著的抑制作用(p<0.05),1.25mg/mL正丁醇萃取相能够促进其幼苗生长,但与对照比效果不显著,大于或等于2.5mg/mL正丁醇萃取相对幼苗生长都具有显著的抑制效应(p<0.05)。从三种有机萃取相的化学组成中,初步鉴定出了包括酚酸、长链脂肪酸、单宁酸和吲哚类物质在内的17种化合物。乙醚萃取相中分离纯化出的3,4-二羟基苯乙酮为一新的化感物质。
2.针叶原始水相、乙醚、乙酸乙酯和正丁醇萃取相都具有自毒效应。种子萌发试验中,针叶水浸提液乙醚萃取相具有最强的抑制效应(IC50=5.84mg/mL),而正丁醇萃取相的抑制效应最弱(IC50>10mg/mL)；幼苗生长试验中,1.25mg/mL针叶水浸提液乙醚萃取相对幼苗生长具有显著抑制作用(p<0.05),而大于或等于2.5mg/mL正丁醇萃取相对幼苗生长具有显著抑制效应(p<0.05)。从三种有机萃取相的化学组成中,初步鉴定出了包括酚酸、长链脂肪酸、单宁酸、吲哚类和黄酮类物质在内的多种化合物。3,4-二羟基苯乙酮存在于乙醚萃取相中。
3.3,4-二羟基苯乙酮的自毒效应强于化感效应。3,4-二羟基苯乙酮处理液浓度为2.5mM,显著抑制天山云杉种子萌发(p<0.05);0.5mM即能显著抑制幼苗生长(p<0.05)。6个浓度的3,4-二羟基苯乙酮处理液对双子叶植物白萝卜、辣椒和黄瓜种子萌发无影响。处理液浓度在0.1mM、0.5mM、1mM,显著促进洋葱种子萌发(p<0.05)。处理液浓度在1mM和10mM,显著促进水稻种子萌发(p<0.05),10mM时,种子发芽势高达对照的166.69%。与对照相比,3,4-二羟基苯乙酮处理液浓度大于等于1mM,显著抑制白萝卜和黄瓜幼苗生长(p<0.05);处理液浓度大于等于10mM,显著抑制辣椒和洋葱幼苗生长(p<0.05),处理液浓度为0.5mM,显著促进洋葱幼苗生长；处理液浓度大于等于2.5mM,显著抑制水稻幼苗生长(p<0.05)。
Picea schrenkiana, the most important zonal vegetation of Tianshan Mountains in China, is an endemic species in Middle Asia and Mountains of Asia. In China, it is mainly distributed in the northern and southern slopes of Tianshan Mountains, and the northern slope of the western part of Kunlun Mountain, accounting for half of the woodland area in Xinjiang province with the area of528,400hm2. As the main tree species and the source of life of Tianshan Mountains and even the forest ecosystems of Xinjiang, P. schrenkiana plays an important role in water conservation. However, with the disturbance on forest ecosystems resulted from human activities, natural regeneration issue of P. schrenkiana is more prominent without fire interference relief. Previous results showed that autotoxicity might be the main cause of the issue for the current forest interference system. Therefore, our study tested the potential autotoxicity of aqueous extract from P. schrenkiana litters and leaves. Water extract from P. schrenkiana litters and leaves was partitioned against diethyl ether, ethyl acetate, and n-butanol sequentially. The organic fractions, original water extract and the residual water after the organic solvent extraction were tested for inhibitory activity on seed germination and seedling growth under climatic cabinate conditions. Furthermore, preliminary identification of the chemical composition of each organic fraction of P. schrenkiana litters and leaves were undertaken by GC-MS-MS analysis.3,4-dihydroxyacetophenone was also isolated and purified from diethyl ether fraction by Silica gel column chromatography and recrystallization. Autotoxicity and allelopathy effects of3,4-dihydroxyacetophenone were also bioassayed. The results were as following:
1. Among the five fractions, it showed that the original water extract, diethyl ether fraction, ethyl acetate fraction and n-butanol fraction of P. schrenkiana litters all exhibited different phytotoxic effect. In the seed germination experiments, diethyl ether fraction exhibited the strongest phytotoxicity and reduced seed germination at low concentration (IC50=5mg/mL) and n-butanol fraction had the least effect (IC50>10mg/mL). In the seedling growth experiments, diethyl ether fraction at concentration of1.25mg/mL had significant inhibitory effect on seedling growth (p<0.05). n-Butanol fraction at1.25mg/mL could slightly stimulate seedling growth; while the fraction at concentrations higher than or equal to2.5mg/mL all showed obviously inhibitory effect on seedling growth (p<0.05). Phemolics, long-chain fatty acids, tannic acids and indoles were preliminary identified in the three fractions of aqueous extracts from P. schrenkiana litters.3,4-dihydroxyacetophenone, which was identified but not reported as an allelochemical or autotoxin previously, was isolated and purified from the diethyl ether fraction.
2. Among the five fractions, it showed that the original water extract, diethyl ether fraction, ethyl acetate fraction and n-butanol fraction of P. schrenkiana leaves all exhibited different phytotoxic effect. In the seed germination experiments, diethyl ether fraction exhibited the strongest phytotoxicity and reduced seed germination at low concentration (IC50=5.84mg/mL) and n-butanol fraction had the least effect (IC50>10mg/mL). In the seedling growth experiments, diethyl ether fraction at concentration of1.25mg/mL had significant inhibitory effect on seedling growth (p<0.05); while n-butanol fraction at concentrations higher than or equal to2.5mg/mL could show obviously inhibitory effect on seedling growth (p<0.05). Phemolics, long-chain fatty acids, tannic acids, indoles and flavonoids were preliminary identified in the three fractions of aqueous extracts from P. schrenkiana leaves.3,4-dihydroxyacetophenone was also isolated and purified from the diethyl ether fraction.
3. Autotoxicity effect of3,4-dihydroxyacetophenone was stronger than its allelopathy effect.3,4-dihydroxyacetophenone treatment solution at concentration of2.5mM had significant inhibitory effect on seed germination of P. schrenkiana (p<0.05); at concentration of0.5mM could significant inhibitory effect on seedling growth of P. schrenkiana (p<0.05). Six different concentrations of3,4-dihydroxyacetophenone treatment solutions had no effect on seed germination of Raphanus sativus, Capsicum frutescens and Cucumis sativus. The treatment solutions at concentrations of0.1mM,0.5mM,1mM could significantly promote seed germination of Allium cepa.(p<0.05) The treatment solutions at concentrations of1mM,10mM could significantly promote seed germination of Oiyza sativa (p<0.05), and seed germination vigor was up to166.69%of control at lOmM. Compared with control, concentrations of3,4-dihydroxyacetophenone treatment solutions higher than or equal to1mM exhibited significant inhibitory effects on seedling growth of R. sativus and C. sativus (p<0.05). Concentrations at higher than or equal to10mM had significant inhibitory effects on seedling growth of C. frutescens and A. cepa (p<0.05), concentrations at0.5mM could significantly promote seedling growth of A. cepa. Concentrations at higher than or equal to2.5mM had significant inhibitory effects on seedling growth of C. frutescens and O. sativa (p<0.05).
1.凋落物原始水相、乙醚、乙酸乙酯和正丁醇萃取相都具有自毒效应。种子萌发试验中,凋落物水浸提液乙醚萃取相具有最强的抑制效应(IC50=5mg/mL),而正丁醇萃取相的抑制效应最弱(IC50>10mg/mL)；幼苗生长试验中,1.25mg/mL凋落物水浸提液乙醚萃取相对幼苗生长具有显著的抑制作用(p<0.05),1.25mg/mL正丁醇萃取相能够促进其幼苗生长,但与对照比效果不显著,大于或等于2.5mg/mL正丁醇萃取相对幼苗生长都具有显著的抑制效应(p<0.05)。从三种有机萃取相的化学组成中,初步鉴定出了包括酚酸、长链脂肪酸、单宁酸和吲哚类物质在内的17种化合物。乙醚萃取相中分离纯化出的3,4-二羟基苯乙酮为一新的化感物质。
2.针叶原始水相、乙醚、乙酸乙酯和正丁醇萃取相都具有自毒效应。种子萌发试验中,针叶水浸提液乙醚萃取相具有最强的抑制效应(IC50=5.84mg/mL),而正丁醇萃取相的抑制效应最弱(IC50>10mg/mL)；幼苗生长试验中,1.25mg/mL针叶水浸提液乙醚萃取相对幼苗生长具有显著抑制作用(p<0.05),而大于或等于2.5mg/mL正丁醇萃取相对幼苗生长具有显著抑制效应(p<0.05)。从三种有机萃取相的化学组成中,初步鉴定出了包括酚酸、长链脂肪酸、单宁酸、吲哚类和黄酮类物质在内的多种化合物。3,4-二羟基苯乙酮存在于乙醚萃取相中。
3.3,4-二羟基苯乙酮的自毒效应强于化感效应。3,4-二羟基苯乙酮处理液浓度为2.5mM,显著抑制天山云杉种子萌发(p<0.05);0.5mM即能显著抑制幼苗生长(p<0.05)。6个浓度的3,4-二羟基苯乙酮处理液对双子叶植物白萝卜、辣椒和黄瓜种子萌发无影响。处理液浓度在0.1mM、0.5mM、1mM,显著促进洋葱种子萌发(p<0.05)。处理液浓度在1mM和10mM,显著促进水稻种子萌发(p<0.05),10mM时,种子发芽势高达对照的166.69%。与对照相比,3,4-二羟基苯乙酮处理液浓度大于等于1mM,显著抑制白萝卜和黄瓜幼苗生长(p<0.05);处理液浓度大于等于10mM,显著抑制辣椒和洋葱幼苗生长(p<0.05),处理液浓度为0.5mM,显著促进洋葱幼苗生长；处理液浓度大于等于2.5mM,显著抑制水稻幼苗生长(p<0.05)。
Picea schrenkiana, the most important zonal vegetation of Tianshan Mountains in China, is an endemic species in Middle Asia and Mountains of Asia. In China, it is mainly distributed in the northern and southern slopes of Tianshan Mountains, and the northern slope of the western part of Kunlun Mountain, accounting for half of the woodland area in Xinjiang province with the area of528,400hm2. As the main tree species and the source of life of Tianshan Mountains and even the forest ecosystems of Xinjiang, P. schrenkiana plays an important role in water conservation. However, with the disturbance on forest ecosystems resulted from human activities, natural regeneration issue of P. schrenkiana is more prominent without fire interference relief. Previous results showed that autotoxicity might be the main cause of the issue for the current forest interference system. Therefore, our study tested the potential autotoxicity of aqueous extract from P. schrenkiana litters and leaves. Water extract from P. schrenkiana litters and leaves was partitioned against diethyl ether, ethyl acetate, and n-butanol sequentially. The organic fractions, original water extract and the residual water after the organic solvent extraction were tested for inhibitory activity on seed germination and seedling growth under climatic cabinate conditions. Furthermore, preliminary identification of the chemical composition of each organic fraction of P. schrenkiana litters and leaves were undertaken by GC-MS-MS analysis.3,4-dihydroxyacetophenone was also isolated and purified from diethyl ether fraction by Silica gel column chromatography and recrystallization. Autotoxicity and allelopathy effects of3,4-dihydroxyacetophenone were also bioassayed. The results were as following:
1. Among the five fractions, it showed that the original water extract, diethyl ether fraction, ethyl acetate fraction and n-butanol fraction of P. schrenkiana litters all exhibited different phytotoxic effect. In the seed germination experiments, diethyl ether fraction exhibited the strongest phytotoxicity and reduced seed germination at low concentration (IC50=5mg/mL) and n-butanol fraction had the least effect (IC50>10mg/mL). In the seedling growth experiments, diethyl ether fraction at concentration of1.25mg/mL had significant inhibitory effect on seedling growth (p<0.05). n-Butanol fraction at1.25mg/mL could slightly stimulate seedling growth; while the fraction at concentrations higher than or equal to2.5mg/mL all showed obviously inhibitory effect on seedling growth (p<0.05). Phemolics, long-chain fatty acids, tannic acids and indoles were preliminary identified in the three fractions of aqueous extracts from P. schrenkiana litters.3,4-dihydroxyacetophenone, which was identified but not reported as an allelochemical or autotoxin previously, was isolated and purified from the diethyl ether fraction.
2. Among the five fractions, it showed that the original water extract, diethyl ether fraction, ethyl acetate fraction and n-butanol fraction of P. schrenkiana leaves all exhibited different phytotoxic effect. In the seed germination experiments, diethyl ether fraction exhibited the strongest phytotoxicity and reduced seed germination at low concentration (IC50=5.84mg/mL) and n-butanol fraction had the least effect (IC50>10mg/mL). In the seedling growth experiments, diethyl ether fraction at concentration of1.25mg/mL had significant inhibitory effect on seedling growth (p<0.05); while n-butanol fraction at concentrations higher than or equal to2.5mg/mL could show obviously inhibitory effect on seedling growth (p<0.05). Phemolics, long-chain fatty acids, tannic acids, indoles and flavonoids were preliminary identified in the three fractions of aqueous extracts from P. schrenkiana leaves.3,4-dihydroxyacetophenone was also isolated and purified from the diethyl ether fraction.
3. Autotoxicity effect of3,4-dihydroxyacetophenone was stronger than its allelopathy effect.3,4-dihydroxyacetophenone treatment solution at concentration of2.5mM had significant inhibitory effect on seed germination of P. schrenkiana (p<0.05); at concentration of0.5mM could significant inhibitory effect on seedling growth of P. schrenkiana (p<0.05). Six different concentrations of3,4-dihydroxyacetophenone treatment solutions had no effect on seed germination of Raphanus sativus, Capsicum frutescens and Cucumis sativus. The treatment solutions at concentrations of0.1mM,0.5mM,1mM could significantly promote seed germination of Allium cepa.(p<0.05) The treatment solutions at concentrations of1mM,10mM could significantly promote seed germination of Oiyza sativa (p<0.05), and seed germination vigor was up to166.69%of control at lOmM. Compared with control, concentrations of3,4-dihydroxyacetophenone treatment solutions higher than or equal to1mM exhibited significant inhibitory effects on seedling growth of R. sativus and C. sativus (p<0.05). Concentrations at higher than or equal to10mM had significant inhibitory effects on seedling growth of C. frutescens and A. cepa (p<0.05), concentrations at0.5mM could significantly promote seedling growth of A. cepa. Concentrations at higher than or equal to2.5mM had significant inhibitory effects on seedling growth of C. frutescens and O. sativa (p<0.05).
引文
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