天山云杉针叶水提取物自毒效应及自毒物质的分离鉴定

doi:10.3773/j.issn.1005-264x.2009.01.021

摘要/Abstract

摘要：

天山云杉(Picea schrenkiana)是亚洲中部山地的特有物种, 在中国仅见于新疆, 主要分布在天山南北坡和昆仑山西部北坡, 对天山山地的水源涵养、水土保持和林业生产具有不可或缺的重要性。云杉天然林自然更新不良或障碍和生产力下降等问题的出现已成为长期困惑林业工作者的一大难题。该文从植物间的化感与自毒作用研究着手, 分析云杉针叶水提液的乙醚、乙酸乙酯、正丁醇萃取物对自身种子萌发和幼苗生长影响实验。以种子发芽率、发芽势和发芽指数作为种子萌发参数, 以胚根、胚芽长度和实生苗干湿重的变化作为幼苗生长参数。研究结果表明: 种子萌发实验中乙醚萃取物具有最强的抑制效应(IC₅₀ = 5.84 mg·ml^-1), 而正丁醇萃取物的抑制效应最弱(IC₅₀> 10.00 mg·ml^-1); 幼苗生长实验中, 1.25 mg·ml^-1乙醚萃取物对幼苗生长具有显著抑制作用(p<0.05), 1.25 mg·ml^-1正丁醇萃取物能够促进其幼苗生长, 大于2.5 mg·ml^-1的正丁醇萃取物对幼苗生长具有显著抑制效应(p<0.05)。采用GC-MS-MS和NMR技术分析3种有机萃取物的化学组成, 鉴定并定量分析出了包括酚酸、长链脂肪酸、单宁酸和吲哚类物质在内的17种化合物。乙醚萃取物中存在的2-keto-4a-methyl-8-methoxy-2, 3, 4, 4a, 5, 6, 11, 12-ocahydrochrysene (在该文中, 将其命名为云杉酮)为一新的化感物质。该研究证明了天山云杉针叶中存在的化感与自毒物质是导致其种群天然更新障碍的一个最主要原因, 同时揭示了自毒作用发生的化学物质基础。

关键词: 自毒作用, 天山云杉, 提取物, 发芽指数

Abstract:

Aims Schrenk spruce (Picea schrenkiana) forest is an important zonal vegetation in Tianshan Mountain, northwestern China. In addition to being an important timber resource, P. schrenkiana has important soil and water conservation functions and makes up 98.6% of the total stand volume in the region. Schrenk spruce forest exhibits slow growth, regeneration obstacles, declining productivity and confusing size and age structure. Our aims were to 1) compare autotoxicities of three organic fractions from leaves and 2) identify autotoxins from leaves. Findings could improve understanding the mechanisms of P. schrenkiana autotoxicity.
Methods Fresh leaves were extracted with water and partitioned against diethyl ether, ethyl acetate and n-butanol sequentially. We tested the three organic fractions for inhibitory activity, using germination rate, vigor and index as germination parameters and radicle and plumule elongation and weight (wet and dry) change of seedlings as seedling growth parameters. We investigated the chemical nature of the three fractions using GC-MS-MS and NMR. The chemicals in the samples were estimated on the basis of ion-molecular messenger of MS-MS spectral of samples and MS-MS user library spectra of pure reference compounds. These findings were confirmed by comparing the retention time and mass spectral in the GC-MS-MS of samples with those of reference standards under the same conditions.
Important findings The diethyl ether fraction exhibited the strongest phytotoxicity, reducing seed germination at low concentration (IC₅₀ = 5.84 mg·ml^-1), and n-butanol had the weakest effect (IC₅₀ > 10.00 mg·ml^-1). The diethyl ether fraction had a significant inhibitory effect on seedling growth at a concentration of 1.25 mg·ml^-1, whereas, the n-butanol fraction at 1.25 mg·ml^-1 stimulated it and at 2.5 mg·ml^-1 inhibited it. Seventeen secondary metabolites were identified and quantified in the three fractions, including 2-keto-4a-methyl-8-methoxy-2,3,4,4a,5,6,11,12-ocahydro chrysene in the diethyl ether fraction (this metabolite had been found but not previously reported as an allelochemical). When we used identified components to prepare synthetic mixtures, it showed the same inhibitory effect on seed germination and seedling growth as did the three fractions.

Key words: autotoxicity, Picea schrenkiana, extracts, germination index

潘存德, 王强, 阮晓, 李兆慧. 天山云杉针叶水提取物自毒效应及自毒物质的分离鉴定. 植物生态学报, 2009, 33(1): 186-196. DOI: 10.3773/j.issn.1005-264x.2009.01.021

PAN Cun-De, WANG Qiang, RUAN Xiao, LI Zhao-Hui. BIOLOGICAL ACTIVITY AND QUANTIFICATION OF POTENTIAL AUTOTOXINS FROM THE LEAVES OF PICEA SCHRENKIANA. Chinese Journal of Plant Ecology, 2009, 33(1): 186-196. DOI: 10.3773/j.issn.1005-264x.2009.01.021

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https://www.plant-ecology.com/CN/Y2009/V33/I1/186

图/表 10

参考文献 31

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	浓度Concentrations (mg·ml^-1)	发芽率 Germination rate ( % )	发芽势 Germination vigor ( % )	发芽指数 Germination index
乙醚萃取物 Diethyl ether extract	1.25	74.37±6.43^B	57.73±5.37^C	30.35±1.71^C
	2.50	64.60±3.86^CD	45.87±5.40^DE	23.46±1.53^DE
	5.00	47.90±2.10^F	25.10±4.35^F	15.31±2.15^F
	10.00	26.67±6.86^H	12.17±4.05^G	5.38±0.83^H
乙酸乙酯萃取物 Ethyl acetate extracts	1.25	84.13±2.21^A	66.13±2.02^B	40.65±1.38^B
	2.50	71.93±3.43^B	55.00±2.85^C	29.29±0.88^C
	5.00	54.87±6.69^EF	41.80±5.43^DE	20.92±1.44^E
	10.00	39.40±2.94^G	26.47±3.32^F	12.15±1.24^G
正丁醇萃取物 n-butanol extract	1.25	88.83±3.07^A	74.07±2.06^A	44.79±1.42^A
	2.50	68.03±4.99^BC	55.13±2.78^C	24.86±2.67^D
	5.00	60.43±3.89^DE	47.67±4.93^D	22.95±2.44^DE
	10.00	51.80±6.78^F	39.43±1.72^E	14.83±0.99^FG
CK	CK	86.67±1.15^A	70.67±7.02^AB	43.49±2.13^AB

	浓度Concentrations (mg·ml^-1)	发芽率 Germination rate ( % )	发芽势 Germination vigor ( % )	发芽指数 Germination index
乙醚萃取物 Diethyl ether extract	1.25	74.37±6.43^B	57.73±5.37^C	30.35±1.71^C
	2.50	64.60±3.86^CD	45.87±5.40^DE	23.46±1.53^DE
	5.00	47.90±2.10^F	25.10±4.35^F	15.31±2.15^F
	10.00	26.67±6.86^H	12.17±4.05^G	5.38±0.83^H
乙酸乙酯萃取物 Ethyl acetate extracts	1.25	84.13±2.21^A	66.13±2.02^B	40.65±1.38^B
	2.50	71.93±3.43^B	55.00±2.85^C	29.29±0.88^C
	5.00	54.87±6.69^EF	41.80±5.43^DE	20.92±1.44^E
	10.00	39.40±2.94^G	26.47±3.32^F	12.15±1.24^G
正丁醇萃取物 n-butanol extract	1.25	88.83±3.07^A	74.07±2.06^A	44.79±1.42^A
	2.50	68.03±4.99^BC	55.13±2.78^C	24.86±2.67^D
	5.00	60.43±3.89^DE	47.67±4.93^D	22.95±2.44^DE
	10.00	51.80±6.78^F	39.43±1.72^E	14.83±0.99^FG
CK	CK	86.67±1.15^A	70.67±7.02^AB	43.49±2.13^AB

乙醚萃取物 Diethyl ether extract (mg·ml^-1)	干重 Dry weight (g)	乙酸乙酯萃取物 Ethyl acetate extract (mg·ml^-1)	干重 Dry weight (g)	正丁醇萃取物 n-butanol extract (mg·ml^-1)	干重 Dry weight (g)
1.25	0.041±0.003^A	1.25	0.041±0.003^A	1.25	0.041±0.002^A
2.50	0.041±0.001^A	2.50	0.041±0.002^A	2.50	0.040±0.002^A
5.00	0.042±0.002^A	5.00	0.040±0.002^A	5.00	0.042±0.002^A
10.00	0.041±0.002^A	10.00	0.042±0.001^A	10.00	0.040±0.002^A
CK	0.040±0.001^A	CK	0.040±0.001^A	CK	0.040±0.001^A

乙醚萃取物 Diethyl ether extract (mg·ml^-1)	干重 Dry weight (g)	乙酸乙酯萃取物 Ethyl acetate extract (mg·ml^-1)	干重 Dry weight (g)	正丁醇萃取物 n-butanol extract (mg·ml^-1)	干重 Dry weight (g)
1.25	0.041±0.003^A	1.25	0.041±0.003^A	1.25	0.041±0.002^A
2.50	0.041±0.001^A	2.50	0.041±0.002^A	2.50	0.040±0.002^A
5.00	0.042±0.002^A	5.00	0.040±0.002^A	5.00	0.042±0.002^A
10.00	0.041±0.002^A	10.00	0.042±0.001^A	10.00	0.040±0.002^A
CK	0.040±0.001^A	CK	0.040±0.001^A	CK	0.040±0.001^A

保留时间 Retention time (min)	化合物 Chemical constituents	相对含量Relative amount (%)	化学文摘登记号 CAS	前体物离子 Precursor ion (m/z)	定量物离子 Quantization ion (m/z)
9.76	对羟基苯甲酸p-hydroxybenzoic acid	8.19	99-96-7	267	223
13.53	云杉酮Schrenkianaone	20.32	102323-79-5	294	279
14.53	肉桂酸 t-cinnamic acid	9.98	140-10-3	205	159
15.98	脱落酸 ABA	30.47	14375-45-2	293	249
17.32	槲皮素 Quercetin	14.49	6151-25-3	587	466
—	其他未知成分Other unknown components	16.55