沙地柏生物生态因子与天然药用产物鬼臼毒素含量关系研究
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摘要
沙地柏(Sabina vulgaris Ant.)为柏科(Cupressaceae)圆柏属常绿葡匐灌木,化学成分复杂多样,具有多种生物活性,是一种极具开发前景的植物资源。其主要次生产物鬼臼毒素类化合物抗肿瘤活性显著,杀虫作用方式独特,作用机理特殊,是一类重要的生理活性物质。本论文采用超临界CO2 流体萃取和高效液相色谱分析技术对沙地柏茎、叶中鬼臼毒素含量进行了分析测定,探讨生物生态因子对沙地柏中鬼臼毒素含量的影响。得到了以下结果:
(1)对鬼臼毒素的超临界CO2 流体萃取(SFE)工艺条件进行了研究。其萃取效果以鬼臼毒素萃取率和粗物质萃取率2 个评价指标予以衡量,鬼臼毒素含量测定采用高效液相色谱法(PAD 检测器)。萃取方法采用先静态萃取后动态萃取的方法,综合考虑了静态时间、压力、温度、CO2 流量四个因素对萃取效果的影响。确定萃取鬼臼毒素较优工艺条件为:压力6000 psi,温度35℃,静态时间15 min ,CO2 流量40 mL/g。
(2)对7 种不同生态环境条件下沙地柏中鬼臼毒素含量进行了测定。结果表明,生境对沙地柏茎中鬼臼毒素含量无显著影响,而对叶中鬼臼毒素含量有显著影响。在梁峁沙地柏中鬼臼毒素含量最高,人工林沙地柏中鬼臼毒素含量最低。
(3)对梁峁、固定沙地和人工林三个生境土壤中矿物质元素含量进行了测定。发现N(全N 和有效N)和Fe 的含量与沙地柏中鬼臼毒素的含量线性相关。当土壤中N和Fe 的含量高时,沙地柏中鬼臼毒素含量也比较高。
(4)对不同年龄沙地柏茎、叶中鬼臼毒素含量进行了测定。试验结果表明,沙地柏叶中鬼臼毒素含量高于茎中鬼臼毒素含量,按鬼臼毒素含量从高到低排序依次为:1年生叶>2 年生叶>1 年生茎>2 年生茎>3 年生茎。不同年龄茎、叶中鬼臼含量间均有显著差异。表明沙地柏中鬼臼毒素主要是积累于器官叶中,同时鬼臼毒素在沙地柏新生组织内的含量较老龄组织高。
(5)对我国沙地柏两个变种—榆林叉子圆柏和叉子圆柏茎、叶中鬼臼毒素含量进行了分析测定,结果显示,榆林叉子圆柏中鬼臼毒素含量高于叉子圆柏中鬼臼毒素含量。
(6)对未成熟果实和成熟果实中鬼臼毒素含量进行了测定,发现未成熟的果实中鬼臼毒素含量较成熟果实中鬼臼毒素含量高。
(7)季节变化对沙地柏中鬼臼毒素含量影响显著。鬼臼毒素在4 月、7 月、11 月出现了3 个含量高峰点,6 月和8 月时鬼臼含量降至最低点。说明沙地柏中鬼臼毒素主要积累在4 月、7 月和11 月份,而在6 月、8 月时含量较低。
The evergreen prostrate shrub Sabina vulgaris Ant. (syn.: Juniperus sabina Linnaeus) belonging to the genus Sabina ( family Cupressaceae) is widely distributed in the northwest of China. Much attention has been paid to chemical constituents, bioactives, biotic, ecological and physiological characteristics of this plant. One type of mainly secondary metabolites of the plant, podophyllotoxin and its congeners exhibit pronounced biological activity mainly as strong antiviral agents, antineoplastic drugs, and as insecticides. However, the influences of ontogeny and environment on the content of podophyllotoxin in S. vulgaris still remain unclear. In order to provid scientific basis for the further rational utilization of the S. vulgar and podophyllotoxin., therefore, in this paper the relationship between the biotic and ecological factors and the podophyllotoxin content in the plant were studied, using supercritical fluid extraction (SFE) and high performance chromatography (HPLC), and the results were described as following:
1. The conditions for extraction of podophyllotoxin from S. vulgaris with SFE were studied. The podophyllotoxin content was analyzed by HPLC with PAD detector. The extracting efficiency was evaluated by 2 indexes, i.e., total compound extraction ratio and podophyllotoxin extraction ratio. Using static extraction followed by dynamic extraction methods, the effects of different parameters, such as pressure, temperature, modifier volume and extraction time, on the supercritical fluid extraction (SFE) of podophyllotoxin were investigated. The results showed that, under a pressure of 6000 psi, temperature 35℃, flow-rate of CO2 40mL/g, and static extraction time of 15 min, extraction was more selective for the podophyllotoxin.
2. The podophyllotoxin content of S. vulgaris leaves and stems in 7 different habitats were examined. The results showed that the habitats exhibited significant influences on the content of podophyllotoxin in the leaves, while little influences on the content of podophyllotoxin in the stems, and the content of the podophyllotoxin of the plant was more higher in protruding lands while less lower in artificial woods
3. The mineral substances in soil of settled sands, protruding land and artificial woods were surveyed respectively, and there was a strong linear relationship between nitrogen and soil-available nitrogen and Fe contents and podophyllotoxin content, and increased nitrogen
and Fe content in the soil resulted in increase of podophyllotoxin content. 4. The content of the podophyllotoxin of S.vulgaris leaves and stems in different ages were studied. It was founded that there were notable differences in the content of podophyllotoxin among stems and leaves of different ages, and the contents were decreased in the following order: annual leaves > biennial leaves > annual stems > biennial stems > triennial stems, On the basis of these facts it may be concluded that the leaves and the younger tissues of S.vulgaris were rich in podophyllotoxin. 5. A comparison of podophyllotoxin content among 2 differences species showed that the content of podophyllotoxin among Sabina rulgaris Ant.Var.yulinensis was higher than that among Sabina rulgaris Ant.obviously. 6. The content of the podophyllotoxin of S.vulgaris in immature seeds and seeds were analyzed. The podophyllotoxin was found in higher content in immature seeds and in lower amount in seeds 7. The results also showed that there was notable variability in S.vulgaris with respect to podophyllotoxin content all year long. The content of the podophyllotoxin was relative higher during April, July and November while relative lower during June and August. It appears that the accumulation or production of podophyllotoxin in S.vulgaris was mainly during April, July and August in the year.
(1)对鬼臼毒素的超临界CO2 流体萃取(SFE)工艺条件进行了研究。其萃取效果以鬼臼毒素萃取率和粗物质萃取率2 个评价指标予以衡量,鬼臼毒素含量测定采用高效液相色谱法(PAD 检测器)。萃取方法采用先静态萃取后动态萃取的方法,综合考虑了静态时间、压力、温度、CO2 流量四个因素对萃取效果的影响。确定萃取鬼臼毒素较优工艺条件为:压力6000 psi,温度35℃,静态时间15 min ,CO2 流量40 mL/g。
(2)对7 种不同生态环境条件下沙地柏中鬼臼毒素含量进行了测定。结果表明,生境对沙地柏茎中鬼臼毒素含量无显著影响,而对叶中鬼臼毒素含量有显著影响。在梁峁沙地柏中鬼臼毒素含量最高,人工林沙地柏中鬼臼毒素含量最低。
(3)对梁峁、固定沙地和人工林三个生境土壤中矿物质元素含量进行了测定。发现N(全N 和有效N)和Fe 的含量与沙地柏中鬼臼毒素的含量线性相关。当土壤中N和Fe 的含量高时,沙地柏中鬼臼毒素含量也比较高。
(4)对不同年龄沙地柏茎、叶中鬼臼毒素含量进行了测定。试验结果表明,沙地柏叶中鬼臼毒素含量高于茎中鬼臼毒素含量,按鬼臼毒素含量从高到低排序依次为:1年生叶>2 年生叶>1 年生茎>2 年生茎>3 年生茎。不同年龄茎、叶中鬼臼含量间均有显著差异。表明沙地柏中鬼臼毒素主要是积累于器官叶中,同时鬼臼毒素在沙地柏新生组织内的含量较老龄组织高。
(5)对我国沙地柏两个变种—榆林叉子圆柏和叉子圆柏茎、叶中鬼臼毒素含量进行了分析测定,结果显示,榆林叉子圆柏中鬼臼毒素含量高于叉子圆柏中鬼臼毒素含量。
(6)对未成熟果实和成熟果实中鬼臼毒素含量进行了测定,发现未成熟的果实中鬼臼毒素含量较成熟果实中鬼臼毒素含量高。
(7)季节变化对沙地柏中鬼臼毒素含量影响显著。鬼臼毒素在4 月、7 月、11 月出现了3 个含量高峰点,6 月和8 月时鬼臼含量降至最低点。说明沙地柏中鬼臼毒素主要积累在4 月、7 月和11 月份,而在6 月、8 月时含量较低。
The evergreen prostrate shrub Sabina vulgaris Ant. (syn.: Juniperus sabina Linnaeus) belonging to the genus Sabina ( family Cupressaceae) is widely distributed in the northwest of China. Much attention has been paid to chemical constituents, bioactives, biotic, ecological and physiological characteristics of this plant. One type of mainly secondary metabolites of the plant, podophyllotoxin and its congeners exhibit pronounced biological activity mainly as strong antiviral agents, antineoplastic drugs, and as insecticides. However, the influences of ontogeny and environment on the content of podophyllotoxin in S. vulgaris still remain unclear. In order to provid scientific basis for the further rational utilization of the S. vulgar and podophyllotoxin., therefore, in this paper the relationship between the biotic and ecological factors and the podophyllotoxin content in the plant were studied, using supercritical fluid extraction (SFE) and high performance chromatography (HPLC), and the results were described as following:
1. The conditions for extraction of podophyllotoxin from S. vulgaris with SFE were studied. The podophyllotoxin content was analyzed by HPLC with PAD detector. The extracting efficiency was evaluated by 2 indexes, i.e., total compound extraction ratio and podophyllotoxin extraction ratio. Using static extraction followed by dynamic extraction methods, the effects of different parameters, such as pressure, temperature, modifier volume and extraction time, on the supercritical fluid extraction (SFE) of podophyllotoxin were investigated. The results showed that, under a pressure of 6000 psi, temperature 35℃, flow-rate of CO2 40mL/g, and static extraction time of 15 min, extraction was more selective for the podophyllotoxin.
2. The podophyllotoxin content of S. vulgaris leaves and stems in 7 different habitats were examined. The results showed that the habitats exhibited significant influences on the content of podophyllotoxin in the leaves, while little influences on the content of podophyllotoxin in the stems, and the content of the podophyllotoxin of the plant was more higher in protruding lands while less lower in artificial woods
3. The mineral substances in soil of settled sands, protruding land and artificial woods were surveyed respectively, and there was a strong linear relationship between nitrogen and soil-available nitrogen and Fe contents and podophyllotoxin content, and increased nitrogen
and Fe content in the soil resulted in increase of podophyllotoxin content. 4. The content of the podophyllotoxin of S.vulgaris leaves and stems in different ages were studied. It was founded that there were notable differences in the content of podophyllotoxin among stems and leaves of different ages, and the contents were decreased in the following order: annual leaves > biennial leaves > annual stems > biennial stems > triennial stems, On the basis of these facts it may be concluded that the leaves and the younger tissues of S.vulgaris were rich in podophyllotoxin. 5. A comparison of podophyllotoxin content among 2 differences species showed that the content of podophyllotoxin among Sabina rulgaris Ant.Var.yulinensis was higher than that among Sabina rulgaris Ant.obviously. 6. The content of the podophyllotoxin of S.vulgaris in immature seeds and seeds were analyzed. The podophyllotoxin was found in higher content in immature seeds and in lower amount in seeds 7. The results also showed that there was notable variability in S.vulgaris with respect to podophyllotoxin content all year long. The content of the podophyllotoxin was relative higher during April, July and November while relative lower during June and August. It appears that the accumulation or production of podophyllotoxin in S.vulgaris was mainly during April, July and August in the year.
引文
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