Jin Ye Rin, Oh Myung Jin, Yuk Heung Joo, An Hyun Joo, Kim Dong Seon
Herbal Medicine Research Division, Korea Institute of Oriental Medicine, Daejeon, Republic of Korea.
University of Science and Technology, Daejeon, Republic of Korea.
J Ginseng Res. 2021 Sep;45(5):539-545. doi: 10.1016/j.jgr.2021.02.005. Epub 2021 Feb 21.
Red ginseng polysaccharides (RGPs) have been acknowledged for their outstanding immunomodulation and anti-tumor activities. However, their studies are still limited by the complexity of their structural features, the absence of purification and enrichment methods, and the rarity of the analytical instruments that apply to the analysis of such macromolecules. Thus, this study is an attempt to establish a new mass spectrometry (MS)-based analysis procedure for RGPs.
Saponin pre-excluded powder of RG (RG-SPEP, 10 mg) was treated with 200 μL of distilled water and centrifuged for 5 h at 1000 rpm and 85 °C. Ethanol-based precipitation and centrifugation were applied to obtain RGPs from the heated extracts. Further, endo-carbohydrase treatments were performed to produce specific saccharide fragments. Solid-phase extraction (SPE) processes were implemented to purify and enrich the enzyme-treated RGPs, while matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) MS was employed for the partial structural analysis of the obtained RGPs.
Utilizing cellulase, porous graphitized carbon (PGC), hydrophilic interaction chromatography (HILIC), and MALDI-TOF/TOF MS, the neutral and acidic RGPs were qualitatively analyzed. Hex and Hex 18 (cellulose analogs) were determined to be novel neutral RGPs. Additionally, the [Unknown + Hex] species were also determined as new acidic RGPs. Furthermore, HexA (H) was determined as another form of the acidic RGPs.
Compared to the previous methods of analysis, these unprecedented applications of HILIC-SPE and MALDI-TOF/TOF MS to analyze RGPs proved to be fairly effective for fractionating and detecting neutral and acidic components. This new procedure exhibits great potential as a specific tool for searching and determining various polysaccharides in many herbal medicines.
红参多糖（RGPs）因其出色的免疫调节和抗肿瘤活性而受到认可。然而，对它们的研究仍受到其结构特征复杂性、缺乏纯化和富集方法以及适用于此类大分子分析的分析仪器稀缺的限制。因此，本研究试图建立一种基于质谱（MS）的RGPs分析新方法。
将10 mg红参皂苷预排除粉末（RG-SPEP）用200 μL蒸馏水处理，在1000 rpm和85°C下离心5小时。采用乙醇沉淀和离心从加热提取物中获得RGPs。此外，进行内切碳水化合物酶处理以产生特定的糖片段。实施固相萃取（SPE）过程以纯化和富集酶处理后的RGPs，同时采用基质辅助激光解吸/电离飞行时间/飞行时间（MALDI-TOF/TOF）质谱对所得RGPs进行部分结构分析。
利用纤维素酶、多孔石墨化碳（PGC）、亲水相互作用色谱（HILIC）和MALDI-TOF/TOF质谱对中性和酸性RGPs进行了定性分析。己糖和己糖18（纤维素类似物）被确定为新型中性RGPs。此外，[未知+己糖]物种也被确定为新的酸性RGPs。此外，己糖醛酸（H）被确定为酸性RGPs的另一种形式。
与以前的分析方法相比，HILIC-SPE和MALDI-TOF/TOF质谱在分析RGPs方面的这些前所未有的应用被证明对于分离和检测中性和酸性成分相当有效。这种新方法作为一种在许多草药中搜索和确定各种多糖的特定工具具有巨大潜力。
