Biosynthesis and Bioactivity of Chlorogenic Acids in Panax notoginseng Suspension Cells
-
摘要: 利用三七 (Panax. notoginseng) 悬浮细胞生物合成绿原酸类物质 (CGAs)，并对其生物活性进行了研究。对三七细胞中CGAs进行定性分析，优化了细胞培养条件以及6种诱导模式，测定了三七细胞CGAs的抗氧化及α-葡萄糖苷酶活性抑制能力。结果表明三七愈伤组织内含有4种CGAs；最优工艺条件为：B5 + 4.0 mg/L NAA + 0.2 mg/L 6-BA + 40 g/L蔗糖 + 2 g/L PVP，pH 7.5，15%接种量；经30 μmol/L 茉莉酸甲酯诱导3 d后，CGAs产量比原工艺提高了0.44倍 (684.07 mg/L)；三七细胞CGAs具有良好的抗氧化及α-葡萄糖苷酶活性抑制能力。Abstract: In this paper, Panax notoginseng (P. notoginseng) suspension cells were used for the production of chlorogenic acids (CGAs), of which bioactivities were evaluated. Firstly, CGAs in P. notoginseng suspension cells were identified by liquid chromatography-tandem mass spectrometry. Secondly, the suspension culture system and elicitation mode were optimized. At last, the antioxidant and α-glucosidase inhibitory activity of CGAs from P. notoginseng cells were determined. The main results were as follows: 1) Four CGAs were inferred in P. notoginseng cells. 2) The optimal condition was obtained (B5 + 4.0 mg/L NAA + 0.2 mg/L 6-BA + 40 g/L sucrose + 2 g/L PVP, pH 7.5 and 15% inoculation size). 3) By co-culture with 30 μmol/L methyl jasmonate for 3 days, the CGAs yield could reach up to 684.07 mg/L, which was 1.44 times that of control. 4) CGAs from P. notoginseng cells showed an excellent antioxidant capacity in radical-scavenging test and, moreover, certain inhibition on α-glucosidase activity. Thus, our results indicated that the production of CGAs by P. notoginseng cells have potential applications in healthy food and daily cosmetics.
-
图 1 三七愈伤组织中绿原酸 (CGAs) HPLC与UPLC-MS/MS分析
Figure 1. Detection of CGAs from P. notoginseng calli
图 2 三七愈伤组织中CGAs裂解过程
Figure 2. Deduced fragmentation pathways of CGAs from P. notoginseng calli
图 3 三七悬浮细胞培养体系的建立
Figure 3. Development of P. notoginseng suspension cell culture system
图 4 不同培养条件对三七悬浮细胞生物量及CGAs产量影响
Figure 4. Biomass and CGAs yield of P. notoginseng suspension cells grown under different conditions
图 6 不同诱导子对三七悬浮细胞生物量及CGAs产量的影响
Figure 6. Effects of different elicitors on cells biomass and CGAs yield of P. notoginseng suspension cells
图 7 三七细胞CGAs抗氧化能力评估及诱导前后HPLC图
Figure 7. Antioxidant activities of CGAs from P. notoginseng cells
Vc is positive control; CK is CGAs from P. notoginseng cells without elicitor; MeJA is CGAs from P. notoginseng cells with 30 μmol/L MeJA
图 8 三七细胞CGAs与抗氧化活性的效应关系分析
Figure 8. Spectrum efficacy relationship between CGAs from P. notoginseng cells and antioxidant activity
图 9 三七细胞CGAs对α-葡萄糖苷酶活性抑制
Figure 9. Inhibition of CGAs from P. notoginseng cells on α-glucosidase activity
表 1 三七悬浮细胞生长及CGAs产物合成动力学参数
Table 1. Kinetics parameters for cells growth and CGAs biosynthesis by P. notoginseng suspension cells
Medium system Cell growth CGAs biosynthesis μm/d−1 R2 α/mg s·L−1) β/(mg s·L−1·d−1) R2 Initial medium 0.22±0.01 0.98 −39.58±3.76 10.60±0.48 0.99 Optimized medium 0.37±0.01 0.97 −36.11±0.72 12.06±0.18 0.99 μm is the maximum specific growth rate; R2 is the correlation coefficient 
下载: 导出CSV
表 2 不同诱导子对三七悬浮细胞CGAs产量的影响
Table 2. Effects of different elicitors on CGAs yield in P. notoginseng suspension
Elicitors Optimum content CGAs yield/(mg·L−1) Increase coefficient MeJA 30 μmol/L 684.07 1.44 YE 0.1 g/L 598.19 1.26 SA 100 μmol/L 518.57 1.09 ANE 0 475.18 No effect MT 10 μmol/L 517.70 1.09 PDE 25 mg/L 622.07 1.31
下载: 导出CSV
-
[1] YANG Y G, JU Z C, YANG Y B, et al. Phytochemical analysis of Panax species: A review[J]. Journal of Ginseng Research, 2021, 45(1): 1-21. doi: 10.1016/j.jgr.2019.12.009 [2] TAN M M, CHEN M H, HAN F, et al. Role of bioactive constituents of Panax notoginseng in the modulation of tumorigenesis: A potential review for the treatment of cancer[J]. Frontiers in Pharmacology, 2021, 12: 738914. doi: 10.3389/fphar.2021.738914 [3] ZHANG X L, ZHANG B, ZHANG C Y, et al. Effect of Panax notoginseng saponins and major anti-obesity components on weight loss[J]. Frontiers in Pharmacology, 2021, 11: 601751. doi: 10.3389/fphar.2020.601751 [4] SANTANA-GALVEZ J, CISNEROS-ZEVALLOS L, JACOBO-VELAZQUEZ D A. Chlorogenic acid: Recent advances on its dual role as a food additive and a nutraceutical against metabolic syndrome[J]. Molecules, 2017, 22(3): 358. doi: 10.3390/molecules22030358 [5] 朱延胜, 魏明, 钱森和, 等. 紫山药多酚分离纯化及其对α-葡萄糖苷酶活性的抑制作用[J]. 食品与发酵工业, 2022: 1-10. [6] 王箴言, 夏晴, 王玉, 等. 桦褐孔菌不同多糖组分的体内、外抗氧化活性[J]. 中国食品学报, 2021, 21(8): 152-158. [7] KURNIAWAN M F, ANDARWULAN N, WULANDARI N, et al. Metabolomic approach for understanding phenolic compounds and melanoidin roles on antioxidant activity of Indonesia robusta and arabica coffee extracts[J]. Food Science and Biotechnology, 2017, 26(6): 1475-1480. doi: 10.1007/s10068-017-0228-6 [8] LIU Z, ZHU X, MOHSIN A, et al. Embryogenic callus induction, cell suspension culture, and spectrum-effect relationship between antioxidant activity and polyphenols composition of Siraitia grosvenorii cultured cells[J]. Industrial Crops and Products, 2022, 176: 114380. doi: 10.1016/j.indcrop.2021.114380 [9] JACOB A, MAHANTY B, THOMAS J. Dynamic modelling of growth and flavonoid production from Ocimum tenuiflorum suspension culture[J]. Bioprocess and Biosystems Engineering, 2020, 43(11): 2053-2064. doi: 10.1007/s00449-020-02394-6 [10] LI J X, LIU S J, WANG J, et al. Fungal elicitors enhance ginsenosides biosynthesis, expression of functional genes as well as signal molecules accumulation in adventitious roots of Panax ginseng C. A. Mey[J]. Journal of Biotechnology, 2016, 239: 106-114. doi: 10.1016/j.jbiotec.2016.10.011 [11] KOSE L P, GULCIN I, GOREN A C, et al. LC-MS/MS analysis, antioxidant and anticholinergic properties of galanga (Alpinia officinarum Hance) rhizomes[J]. Industrial Crops and Products, 2015, 74: 712-721. doi: 10.1016/j.indcrop.2015.05.034 [12] 王振宇, 杨斯月, 吕维, 等. 利用3种不同化学计量学方法分析枸杞子抗氧化部位的谱效关系[J]. 中国中药杂志, 2021, 46(13): 3377-3387. [13] 王闪. 酶法合成绿原酸酰化衍生物及其抗氧化和α-淀粉酶/葡萄糖苷酶抑制活性研究 [D]. 江苏无锡, 江南大学, 2021. [14] OUYANG H, LI J M, WU B, et al. A robust platform based on ultra-high performance liquid chromatography Quadrupole time of flight tandem mass spectrometry with a two-step data mining strategy in the investigation, classification, and identification of chlorogenic acids in Ainsliaea fragrans Champ[J]. Journal of Chromatography A, 2017, 1502: 38-50. doi: 10.1016/j.chroma.2017.04.051 [15] JAISWAL R, MULLER H, MULLER A, et al. Identification and characterization of chlorogenic acids, chlorogenic acid glycosides and flavonoids from Lonicera henryi L. (Caprifoliaceae) leaves by LC-MSn[J]. Phytochemistry, 2014, 108: 252-263. doi: 10.1016/j.phytochem.2014.08.023 [16] LIU L H, ZHANG J Y, ZHENG B J, et al. Rapid characterization of chlorogenic acids in Duhaldea nervosa based on ultra-high-performance liquid chromatography-linear trap quadropole-Orbitrap-mass spectrometry and mass spectral trees similarity filter technique[J]. Journal of Separation Science, 2018, 41(8): 1764-1774. doi: 10.1002/jssc.201701047 [17] ZHANG J Y, ZHANG Q, LI N, et al. Diagnostic fragment-ion-based and extension strategy coupled to DFIs intensity analysis for identification of chlorogenic acids isomers in Flos Lonicerae Japonicae by HPLC-ESI-MSn[J]. Talanta, 2013, 104: 1-9. doi: 10.1016/j.talanta.2012.11.012 [18] 王晓惠, 王泽建, 肖慈英, 等. 醉金香葡萄愈伤细胞悬浮培养基优化促进白藜芦醇的合成[J]. 华东理工大学学报(自然科学版), 2022, 48(2): 203-212. [19] 杜尚广, 盛文涛. 影响植物组织培养生产次生代谢产物的因素[J]. 园艺与种苗, 2018(11): 30-33+43. [20] 陈继光, 上官新晨, 尹忠平, 等. 青钱柳悬浮细胞的培养及其基质消耗的规律[J]. 现代食品科技, 2014, 30(1): 44-49+107. [21] RAMIREZ-ESTRADA K, VIDAL-LIMON H, HIDALGO D, et al. Elicitation, an effective strategy for the biotechnological production of bioactive high-added value compounds in plant cell factories[J]. Molecules, 2016, 21(2): 182. doi: 10.3390/molecules21020182 [22] SETHI S, JOSHI A, ARORA B, et al. Significance of FRAP, DPPH, and CUPRAC assays for antioxidant activity determination in apple fruit extracts[J]. European Food Research and Technology, 2020, 246(3): 591-598. doi: 10.1007/s00217-020-03432-z [23] 李清清, 余旭亚, 耿树香, 等. 复合核桃油的体外抗氧化活性[J]. 食品与发酵工业, 2020, 46(24): 31-36. [24] FAN Q, YANG R J, YANG F X, et al. Spectrum-effect relationship between HPLC fingerprints and antioxidant activity of Angelica sinensis[J]. Biomedical Chromatography, 2020, 34(2): 10. [25] 王帅, 赵冬雪, 韩成凤, 等. 6种活性多糖的结构、性质及其抗氧化活性的比较研究[J]. 食品研究与开发, 2021, 42(16): 7-15. [26] 张艳, 严晓波, 姚秋萍, 等. 绿原酸的提取分离及其在食品中的应用[J]. 现代食品, 2021(17): 19-22. [27] MA Y, LIU Y, YANG P, et al. The synthesis mechanism of chlorogenic acid in leaves of Eucommia ulmoides Oliver[J]. Applied Ecology and Environmental Research, 2020, 18(2): 2719-2725. doi: 10.15666/aeer/1802_27192725 [28] KONG D X, LI Y Q, BAI M, et al. Correlation between the dynamic accumulation of the main effective components and their associated regulatory enzyme activities at different growth stages in Lonicera japonica Thunb[J]. Industrial Crops and Products, 2017, 96: 16-22. doi: 10.1016/j.indcrop.2016.11.024 [29] LIU Z B, CHEN J G, YIN Z P, et al. Methyl jasmonate and salicylic acid elicitation increase content and yield of chlorogenic acid and its derivatives in Gardenia jasminoides cell suspension cultures[J]. Plant Cell Tissue and Organ Culture, 2018, 134(1): 79-93. doi: 10.1007/s11240-018-1401-1 [30] 陈日道, 刘晓, 邹建华, 等. 天山雪莲悬浮培养细胞中紫丁香苷、绿原酸和1, 5-二咖啡酰奎尼酸的生物合成调控[J]. 中国中药杂志, 2014, 39(12): 2275-2280. [31] XU A, ZHAN J C, HUANG W D. Combined elicitation of chitosan and ultraviolet C enhanced stilbene production and expression of chitinase and β-1, 3-glucanase in Vitis vinifera cell suspension cultures[J]. Plant Cell Tissue and Organ Culture, 2016, 124(1): 105-117. doi: 10.1007/s11240-015-0879-z [32] 廖斌, 徐小萍, 李珊珊, 等. 苯丙氨酸和茉莉酸甲酯对龙眼胚性悬浮细胞柯里拉京积累的影响[J]. 应用与环境生物学报, 2020, 26(2): 287-293. [33] 段永波, 鲁放, 崔婷婷, 等. 非生物诱导子茉莉酸甲酯和水杨酸对半夏悬浮细胞中生物碱代谢的影响[J]. 中国中医药信息杂志, 2017, 24(1): 87-90. doi: 10.3969/j.issn.1005-5304.2017.01.021 [34] LIU Z B, MOHSIN A, WANG Z J, et al. Enhanced biosynthesis of chlorogenic acid and its derivatives in methyl-jasmonate-treated Gardenia jasminoides cells: A study on metabolic and transcriptional responses of cells[J]. Frontiers in Bioengineering and Biotechnology, 2021, 8: 604957. doi: 10.3389/fbioe.2020.604957 [35] OBOH G, AGUNLOYE O M, ADEFEGHA S A, et al. Caffeic and chlorogenic acids inhibit key enzymes linked to type 2 diabetes (in vitro): A comparative study[J]. Journal of basic and clinical physiology and pharmacology, 2015, 26(2): 165-170. [36] 庞美蓉, 刘零怡, 高汪磊, 等. 绿原酸调节糖脂代谢的作用机制研究进展[J]. 中草药, 2015, 46(2): 305-312. -
点击查看大图
计量
- 文章访问数: 86
- HTML全文浏览量: 53
- PDF下载量: 7
- 被引次数: 0
