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.

引言：Panax vietnamensis 为一种珍贵的药用植物，是广泛生物活性的人参皂苷的来源，这些皂苷隶属于不同的结构类别。由于过度采挖以及适应人工栽培的低适应性，该物种对人类压力十分脆弱，进而催生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的兴趣日益浓厚，但关于该物种在体外条件下影响细胞生长和人参皂苷生物合成的关键因素，知之甚少。在本研究中，评估了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：通过多步骤的培养优化过程，从单个根茎的不同部位发展出六种悬浮细胞系，并在含有不同矿物质盐基和不同含量的生长素与细胞分裂素的培养基上维持了超过三年的培养。这些细胞系被评估了生产参数和细胞学特征。人参皂苷谱图通过反相超高效液相色谱- 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）的组合进行评估。结果：所有细胞系均表现出良好的生长状况，具有0.1-0.2天-1的特定生长率，经济系数为0.31-0.70，干重（DW）生产率为0.30-0.83 gDW（L·天）-1，最大生物量积累量为10至22 gDW L-1。在细胞生物量提取物中鉴定出原人参二醇（Rb1，Rb2/Rb3，丙二酰-Rb1，以及丙二酰-Rb2/Rb3）、橄榄酸（R0和齐墩果酸皂苷IV）以及落新妇苷（乙酸人参皂苷R1）组及其同系物。首次在Panax vietnamensis细胞培养中鉴定出齐墩果酸皂苷IV。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含有人参皂苷的生物量方面具有很高的潜力，尤其是橄榄酸和落新妇苷组。