天葵抗TMV有效成分提取分离及其制剂研究
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摘要
烟草花叶病毒病是烟草种植业上的重要病害之一,由于植物病毒的绝对寄生性,使其难于防治,目前国内外缺少高效的抗病毒药剂。随着人们生活水平的提高,环境问题备受关注。抗病毒剂向低毒、低残、高效及环境和谐的方向发展是一种趋势。本文从天葵(Semiaquilegia adoxoides(DC)Makino)块根中提取分离抗烟草花叶病毒(Tobacco mosaic virus,TMV)的活性成分,为植物源抗病毒活性成分的筛选提供重要的理论依据与实践指导。
在本实验室以往研究的基础上,通过继续分离纯化天葵有效组分,采用半叶枯斑法进行活性追踪,筛选出天葵萃取物中抗TMV的活性组分TK和有效成分TK-1;经过剂型加工,初步研制出了20%TK水乳剂系列制剂。取得了一些有价值的试验结果。
1.本研究对天葵萃取物的化学成分进行了较为系统地预试验。结果表明:天葵初提物中含有糖类、有机酸、蒽醒、酚类、挥发油等成分。
2.采用半叶枯斑法测定了天葵萃取物抗TMV的生物活性。生物活性结果表明,天葵萃取物抗TMV的生物活性随处理时间不同而不同,抑制增殖作用结果说明TMV在接触心叶烟后的2h内是其侵染期,2h后TMV已经侵染并已开始大量增殖。治疗作用施药时间以接种后1h处理为最佳。天葵提取物与等量的TMV混合30min后处理可达到最佳钝化作用防治效果。
3.研究确定了天葵有效成分提取分离的一般方法和流程,即先将天葵根粉粗提物经石油醚、氯仿、乙酸乙酯、正丁醇、水依次萃取,然后采用薄层层析和柱层析相结合的方法分离。氯仿萃取物浸膏(B)经过氯仿.甲醇梯度洗脱(83:17)得有效组分B4-3,其保护和治疗作用是52.54%和48.69%;乙酸乙酯萃取物浸膏(C)经过氯仿-甲醇梯度洗脱(75:25).得有效成分C5-5,其保护和治疗作用为42.98%～62.07%。
4.进行了20%TK-1水乳剂系列制剂的研究,初步确定了2号水乳剂的配方及其质量控制指标。该水乳剂有效成分为TK-1,溶剂为二甲亚砜,乳化剂为OX-7513,分散剂为乙二醇,抗冻剂为羧甲基纤维素钠,助剂为pH调节剂、增稠剂、着色剂、气味调节剂。经剂型加工和质量检测,各项质量控制指标良好。
5.研究了2号水乳剂在烟叶上的抗TMV生物活性。结果表明,在心叶烟(Nicotiana ghutinosa)上的活体保护、治疗和钝化作用抑制率分别为62.05%、64.28%和68.64%;在普通烟K326(Nicotiana TabIeacum var)上抗TMV的防治效果为68.64%。2号水乳剂的ED_(50)为724.44mg/L,回归方程为y=1.0501x+2(r=0.9874~*)。
Tobacco mosaic virus (TMV) is one of the important diseases in agriculture productions, almost all of agronomic crops are endangered by TMV. It is difficult to control, because of its absolute parasitism. At present, the profitable antiviral medicament products are very rare in both here and abroad. With the development of the living standard, people have paid more and more attention to the environmental problem. Botanical pesticides are a better choice in the future agriculture, for its characteristics such as low toxicity, low residual, highly activity and environment compatibility. Through tracking biological activity, the active component of anti-TMV was gained from of Semiaquilegia adoxoides (DC) Makino (a kind of herbal medicine) earthnut by extraction and separation. It will maybe provide the important academic theory and practical direction for screening active components of antiviral agents.
On the base of the early studies, the anti-TMV active component TK and effective component TK-1 were screened from S. adoxoides extract by half-leaf dry spot method. The series of preparations of the natural product No. 2 emulsion oil in water (EW) against TMV was also made through formulation processing and detecting. The results are as follows:
1.In this study, the chemical composition of S. adoxoides was tested extensively. The results show that the S. adoxoides extract includes sugars, organic acids, peter anthracene, phenols and volatile oil.
2. In this study, biologic activity of S. adoxoides extracts against TMV was determined by adopting half-leaf dry spot method, the experiment operating condition of half-leaf dry spot method thus was established. The results of biologic activity show that biologic activity of S. adoxoides extracts against TMV varied with treatment time. The effect of inhibiting multiplication means that TMV invaded tobacco leaf within 2h; after 2h, TMV had invaded and propagated cosmically; the best treatment time was 1h after inoculation; S. adoxoides extract mixed for 30 min with the same volume of the TMV had the best effect.
3. In this study, the method of separating active ingredients from S. adoxoides was improved. The powders of S. adoxoides root were extracted and then purified by using TLC combined with column chromatographic analysis. The effective components B4-3 was gained by using chloroform-methanol (83: 17) to elute gradiently chloroform extract (B), and its protection effect and treatment effect was 52.54 percent and 48.69 percent, respectively. The active ingredients C5-5 was also gained by using chloroform-methanol (75: 25) to elute gradiently ethyl acetate extract (C), and protection effect and treatment effect of C5-5 was 42.98 % and 62.07 %, respectively.
4. In this study, the series of preparations of TK-1 EW against TMV were formulated. Prescription No. 2 EW and its quality control targets were confirmed primarily. This EW is consisted of effective component TK-1, solvent dimethylsulfoxide, emulsifier OX-7513, dispersant glycol, antifreeze cymene-fibrinuria, and additives such as pH regulator, thickener, coloring agent and odor-conditioning agent. After detection of the formulation processing and the quality, each quality control targets were accord with requests.
5. Biologic activity of No. 2 EW against TMV in tobacco was studied. The results show that, in vivid, protection, treatment and inhibition rate in heart shape tobacco (Nicotiana glutinosa) was 62.05%, 64.28%, and 68.64%, respectively; control effect in common tobacco K326 (Nicotiana Tableacum var) was 68.64%. ED_(50) of No. 2 EW was 724.44 mg/L, the regression equation was y = 1.0501x+2 (r = 0.9874).
在本实验室以往研究的基础上,通过继续分离纯化天葵有效组分,采用半叶枯斑法进行活性追踪,筛选出天葵萃取物中抗TMV的活性组分TK和有效成分TK-1;经过剂型加工,初步研制出了20%TK水乳剂系列制剂。取得了一些有价值的试验结果。
1.本研究对天葵萃取物的化学成分进行了较为系统地预试验。结果表明:天葵初提物中含有糖类、有机酸、蒽醒、酚类、挥发油等成分。
2.采用半叶枯斑法测定了天葵萃取物抗TMV的生物活性。生物活性结果表明,天葵萃取物抗TMV的生物活性随处理时间不同而不同,抑制增殖作用结果说明TMV在接触心叶烟后的2h内是其侵染期,2h后TMV已经侵染并已开始大量增殖。治疗作用施药时间以接种后1h处理为最佳。天葵提取物与等量的TMV混合30min后处理可达到最佳钝化作用防治效果。
3.研究确定了天葵有效成分提取分离的一般方法和流程,即先将天葵根粉粗提物经石油醚、氯仿、乙酸乙酯、正丁醇、水依次萃取,然后采用薄层层析和柱层析相结合的方法分离。氯仿萃取物浸膏(B)经过氯仿.甲醇梯度洗脱(83:17)得有效组分B4-3,其保护和治疗作用是52.54%和48.69%;乙酸乙酯萃取物浸膏(C)经过氯仿-甲醇梯度洗脱(75:25).得有效成分C5-5,其保护和治疗作用为42.98%～62.07%。
4.进行了20%TK-1水乳剂系列制剂的研究,初步确定了2号水乳剂的配方及其质量控制指标。该水乳剂有效成分为TK-1,溶剂为二甲亚砜,乳化剂为OX-7513,分散剂为乙二醇,抗冻剂为羧甲基纤维素钠,助剂为pH调节剂、增稠剂、着色剂、气味调节剂。经剂型加工和质量检测,各项质量控制指标良好。
5.研究了2号水乳剂在烟叶上的抗TMV生物活性。结果表明,在心叶烟(Nicotiana ghutinosa)上的活体保护、治疗和钝化作用抑制率分别为62.05%、64.28%和68.64%;在普通烟K326(Nicotiana TabIeacum var)上抗TMV的防治效果为68.64%。2号水乳剂的ED_(50)为724.44mg/L,回归方程为y=1.0501x+2(r=0.9874~*)。
Tobacco mosaic virus (TMV) is one of the important diseases in agriculture productions, almost all of agronomic crops are endangered by TMV. It is difficult to control, because of its absolute parasitism. At present, the profitable antiviral medicament products are very rare in both here and abroad. With the development of the living standard, people have paid more and more attention to the environmental problem. Botanical pesticides are a better choice in the future agriculture, for its characteristics such as low toxicity, low residual, highly activity and environment compatibility. Through tracking biological activity, the active component of anti-TMV was gained from of Semiaquilegia adoxoides (DC) Makino (a kind of herbal medicine) earthnut by extraction and separation. It will maybe provide the important academic theory and practical direction for screening active components of antiviral agents.
On the base of the early studies, the anti-TMV active component TK and effective component TK-1 were screened from S. adoxoides extract by half-leaf dry spot method. The series of preparations of the natural product No. 2 emulsion oil in water (EW) against TMV was also made through formulation processing and detecting. The results are as follows:
1.In this study, the chemical composition of S. adoxoides was tested extensively. The results show that the S. adoxoides extract includes sugars, organic acids, peter anthracene, phenols and volatile oil.
2. In this study, biologic activity of S. adoxoides extracts against TMV was determined by adopting half-leaf dry spot method, the experiment operating condition of half-leaf dry spot method thus was established. The results of biologic activity show that biologic activity of S. adoxoides extracts against TMV varied with treatment time. The effect of inhibiting multiplication means that TMV invaded tobacco leaf within 2h; after 2h, TMV had invaded and propagated cosmically; the best treatment time was 1h after inoculation; S. adoxoides extract mixed for 30 min with the same volume of the TMV had the best effect.
3. In this study, the method of separating active ingredients from S. adoxoides was improved. The powders of S. adoxoides root were extracted and then purified by using TLC combined with column chromatographic analysis. The effective components B4-3 was gained by using chloroform-methanol (83: 17) to elute gradiently chloroform extract (B), and its protection effect and treatment effect was 52.54 percent and 48.69 percent, respectively. The active ingredients C5-5 was also gained by using chloroform-methanol (75: 25) to elute gradiently ethyl acetate extract (C), and protection effect and treatment effect of C5-5 was 42.98 % and 62.07 %, respectively.
4. In this study, the series of preparations of TK-1 EW against TMV were formulated. Prescription No. 2 EW and its quality control targets were confirmed primarily. This EW is consisted of effective component TK-1, solvent dimethylsulfoxide, emulsifier OX-7513, dispersant glycol, antifreeze cymene-fibrinuria, and additives such as pH regulator, thickener, coloring agent and odor-conditioning agent. After detection of the formulation processing and the quality, each quality control targets were accord with requests.
5. Biologic activity of No. 2 EW against TMV in tobacco was studied. The results show that, in vivid, protection, treatment and inhibition rate in heart shape tobacco (Nicotiana glutinosa) was 62.05%, 64.28%, and 68.64%, respectively; control effect in common tobacco K326 (Nicotiana Tableacum var) was 68.64%. ED_(50) of No. 2 EW was 724.44 mg/L, the regression equation was y = 1.0501x+2 (r = 0.9874).
引文
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