不同种类无机盐对灰毡毛忍冬“渝蕾1号”悬浮培养体系中细胞生长和绿原酸含量的影响

Effects of different species of inorganic salt on the chlorogenic acid content and the growth of cell in suspension cultures of Lonicera macranthoides Hand.-Mazz. “Yulei 1”

关键词:  灰毡毛忍冬“渝蕾1号”, 悬浮培养, 绿原酸, 高效液相色谱, 无机盐

Key words:  Lonicera macranthoides Hand. -Mazz. “Yulei 1”, suspension culture, chlorogenic acid, HPLC, inorganic salt

摘要:

为提高灰毡毛忍冬“渝蕾1号”悬浮培养体系中绿原酸的含量,该研究探讨了B₅培养基中不同浓度的无机盐对灰毡毛忍冬“渝蕾1号”悬浮培养细胞生物量及绿原酸含量的影响,通过在悬浮培养体系中添加不同浓度的无机盐,采用重量法测定灰毡毛忍冬“渝蕾1号”悬浮培养细胞的生物量及采用高效液相色谱法测定绿原酸的含量。结果表明:当硝态氮和铵态氮配比与B₅培养基中硝态氮和铵态氮配比一致时,即NO₃^- /NH₄⁺摩尔比值为13:1时,培养体系有利于细胞的生长和绿原酸的积累。当KNO₃浓度为3.5 g·L^-1时,细胞生物量达到最大,为19.26 g·L^-1; 当KNO₃在较低浓度(0.5 g·L^-1和1.5 g·L^-1)时,积累较多的绿原酸。NO₃^- 的两项研究结果均与对照浓度(2.5g·L^-1)有一定的差异。另外,对(NH₄)₂SO₄来说,在高于对照浓度0.134 g·L^-1,即浓度为0.268 g·L^-1时,生物量和绿原酸含量都达到了最大。P、Ca、Mg三种矿质元素的研究结果表明,当NaH₂PO₄·2H₂O浓度为0.10 g·L^-1、CaCl₂的浓度为0.20 g·L^-1时,细胞的生长和绿原酸的积累均可达到最大值; 而对Mg²⁺来说,低浓度促进细胞的生长,高浓度促进绿原酸的积累。兼顾细胞生物量和绿原酸含量两个指标,需选择适中的浓度。这些结果均与对照浓度有一定的差异。这说明灰毡毛忍冬“渝蕾1号”悬浮细胞所需无机盐的浓度与B₅培养基无机盐的浓度有一定的差异,选择适宜的浓度可促进其悬浮细胞的生长及次生代谢产物绿原酸的积累。该研究结果为绿原酸的工业化生产打下了基础。

Abstract:

In order to improve the content of chlorogenic acid in cell suspension cultures of Lonicera macranthoides Hand.-Mazz. “Yulei 1”, this paper discussed that the different species of inorganic salt had many effects on the chlorogenic acid content and growth of cell in suspension cultures of L. macranthoides Hand.-Mazz. “Yulei 1”. We weighed the biomass of the L. macranthoides Hand.-Mazz. “Yulei 1” cells and analyzed the content of chlrogenic acid by HPLC through the additions of different concentrations of inorganic salt in cell suspension cultures of L. macranthoides Hand.-Mazz. “Yulei 1”. The result showed that the optimal NO₃^-/NH₄⁺ ratio was consisted with NO₃^-/NH₄⁺ (nitrate nitrogen/ammonium nitrogen)ratio in B₅ medium. In other words, the suspension culture would be benefitial for the accumulation of chlorogenicacid and growth of cell when NO₃^-/NH₄⁺ (nitrate nitrogen/ ammonium nitrogen)ratio was 13:1. When single N source was added, the biomass of callus reached the maximum(19.26 g·L^-1)in 3.5 g·L^-1KNO₃. However, when KNO₃ concentration was at relatively low levels(0.5 and 1.5 g·L^-1), high chlorogenic acid production was accumulated. The two studies of NO₃^- had some differences from the control concentration(2.5 g·L^-1). What's more, while(NH₄)₂SO₄ concentration(0.268 g·L^-1)exceeded control concentration(0.134 g·L^-1), the highest growth of biomass of the L. macranthoides Hand.-Mazz. “Yulei 1” and the high chlorogenic acid production were observed. In the same way, different species and different concentrations of P, Ca and Mg might have different impacts on the chlorogenic acid content and growth of cell. The result showed that when the highest growth of biomass and the high chlorogenic acid production were accumulated, when NaH₂PO₄·2H₂O concentration was 0.10 g·L^-1. In addition, when the highest growth of biomass and the high chlorogenic acid production were accumulated, when CaCl₂ concentration was 0.20 g·L^-1. So far as Mg²⁺ was concerned, a low MgSO₄·7H₂O concentration could promote the growth of cell, while excessive MgSO₄·7H₂O concentration would promote the accumulation of chlorogenic acid. Taking the biomass growth and chlorogenic acid production into consideration, the optimal concentration of Mg was moderate. These results had some differences from the control. Our study demonstrated that inorganic salt concentration had some differences from inorganic salt concentration of B₅ in suspension cultures of L. macranthoides Hand.-Mazz. “Yulei 1”. This study suggested that the optimized conditions could improve biomass accumulation and chlorogenic acid production through the addition of moderate concentration of inorganic salt in cell suspension cultures of L. macranthoides Hand.-Mazz. “Yulei 1”. These results lays a foundation for large-scale production of chlorogenic acid by using the cell suspension cultures of L. macranthoides Hand.-Mazz. “Yulei 1”. This study also has practical significance to large-scale productions of chlorogenic acid.