DataSheet_1_Suspension cell cultures of Panax vietnamensis as a biotechnological source of ginsenosides: growth, cytology, and ginsenoside profile assessment.docx

IntroductionPanax vietnamensis is a valuable medicinal plant and a source of a broad spectrum of biologically active ginsenosides of different structural groups. Overexploitation and low adaptability to planation cultivation have made this species vulnerable to human pressure and prompted the development of cell cultivation in vitro as a sustainable alternative to harvesting wild plants for their bioactive components. Despite high interest in biotechnological production, little is known about the main factors affecting cell growth and ginsenoside biosynthesis of this species under in vitro conditions. In this study, the potential of cell cultures of P. vietnamensis as a biotechnological source of ginsenosides was was assessed.MethodsSix suspension cell lines that were developed from different sections of a single rhizome through a multi-step culture optimization process and maintained for over 3 years on media with different mineral salt base and varying contents of auxins and cytokinins. These cell lines were evaluated for productivity parameters and cytological characteristics. Ginsenoside profiles were assessed using a combination of the reversed-phase ultra-high-performance liquid chromatography–Orbitrap–tandem mass spectrometry (UHPLC–Orbitrap–MS/MS) and ultra-performance liquid chromatography–time of flight–mass spectrometry (UPLC–TOF–MS).ResultsAll lines demonstrated good growth with a specific growth rate of 0.1–0.2 day−1, economic coefficient of 0.31–0.70, productivity on dry weight (DW) of 0.30–0.83 gDW (L·day)−1, and maximum biomass accumulation varying from 10 to 22 gDW L−1. Ginsenosides of the protopanaxadiol (Rb1, Rb2/Rb3, malonyl-Rb1, and malonyl-Rb2/Rb3), oleanolic acid (R0 and chikusetsusaponin IV), and ocotillol (vinaginsenoside R1) groups and their isomers were identified in cell biomass extracts. Chikusetsusaponin IV was identified in P. vietnamensis cell culture for the first time.DiscussionThese results suggest that suspension cell cultures of Vietnamese ginseng have a high potential for the biotechnological production of biomass containing ginsenosides, particularly of the oleanolic acid and ocotillol groups.

Panax vietnamensis，被誉为珍贵药用植物，并富含多种结构组分的广谱生物活性人参皂苷。由于过度采伐以及对人工种植的低适应性，该物种易受人类压力影响，进而推动了体外细胞培养技术的发展，作为从野生植物中提取生物活性成分的可持续替代方案。尽管对生物技术生产表现出浓厚兴趣，但对于影响该物种在体外条件下细胞生长和人参皂苷生物合成的关键因素，目前了解甚少。本研究旨在评估越南人参细胞培养作为人参皂苷生物技术来源的潜力。方法：通过多步骤培养优化过程，从单个根茎的不同部位培养出六种悬浮细胞系，并在含有不同矿物质盐基和不同含量生长素与细胞分裂素的培养基中维持超过三年。对这些细胞系进行了生产力参数和细胞学特性的评估。采用反相高效液相色谱- Orbitrap-串联质谱 (UHPLC-Orbitrap-MS/MS) 和超高效液相色谱-飞行时间-质谱 (UPLC-TOF-MS) 的组合方法对人参皂苷谱进行评估。结果：所有细胞系均表现出良好的生长状态，其比生长率为0.1-0.2天−1，经济系数为0.31-0.70，干重产量为0.30-0.83 gDW (L·day)−1，最大生物量积累量在10到22 gDW L−1之间。在细胞生物量提取物中鉴定出了原人参二醇（Rb1、Rb2/Rb3、琥珀酰-Rb1和琥珀酰-Rb2/Rb3）、橄榄酸（R0和齐墩果皂苷IV）和薯蓣皂苷元（乙酸人参皂苷R1）组及其同系物。齐墩果皂苷IV首次在Panax vietnamensis细胞培养中被鉴定。讨论：这些结果表明，越南人参的悬浮细胞培养在生物技术生产富含人参皂苷的生物量方面具有很高的潜力，尤其是橄榄酸和薯蓣皂苷元组。