Research progress on separation and analysis methods of oligosaccharides in traditional Chinese medicine
With the creation of the 2012 roadmap for the field of sugar science, it has become the forefront of life sciences. More and more scientists recognize that polysaccharides, as essential information and functional molecules in life processes, play a crucial role in the growth, differentiation, and development of cells. The International Federation of Functional Glycomics (CFG) reported its research results to the National Institutes of Health in the United States, pointing out that the study of sugar chain structure and function in animals, plants, and microorganisms is an important direction for future glycobiology research, which will bring new hope for the research and treatment of human diseases. In 2018, the new drug for Alzheimer’s disease “manno oligosaccharide diacid (GV-971)” jointly developed by Ocean University of China, Chinese Academy of Sciences Shanghai Institute of Materia Medica and Shanghai Green Valley Pharmaceutical opened up a new path for Alzheimer’s drug research and development, and is expected to lead a new wave of sugar drug research and development. The successful development of GV-971 has made people more aware that carbohydrates are changing human understanding of life phenomena and diseases. The complex structure and diverse functions of sugar chains meet the standard requirements for the treatment of complex diseases, and sugar drugs are expected to lead the innovation frontier in the treatment of complex diseases. Oligosaccharides are also an important component of carbohydrate research. Oligosaccharides are a class of carbohydrates consisting of 2-10 identical or different monosaccharides connected by glycosidic bonds to form straight or branched chains. The main building blocks are five or six carbon sugars, with glucose, fructose, galactose, xylose, arabinose, and mannose being the most common. Modern research has shown that oligosaccharides have various pharmacological and physiological activities, such as lowering blood sugar, anti traumatic stress disorder, anti-tumor, antibacterial, immune enhancement, regulating gut microbiota, antidepressant, enhancing hematopoietic function, antiviral, etc. In terms of the diversity of traditional Chinese medicine resources and their potential pharmacological activities, the research on traditional Chinese medicine oligosaccharides has significant significance and development prospects. This article reviews the research progress of traditional Chinese medicine oligosaccharides in three aspects: extraction and purification, separation and analysis, and structural identification, in order to provide scientific references for the modern drug research, clinical application, and efficacy product creation of oligosaccharides.

This article reviews the research progress in the extraction, separation, purification, and structural characterization of traditional Chinese medicine oligosaccharides.

When extracting oligosaccharides, factors such as the solubility of the target oligosaccharide component in the solvent, the stability of oligosaccharides in the extraction solvent, oligosaccharide yield, oligosaccharide purity, extraction time, solvent dosage, solvent environmental friendliness and safety, and the ease of operation of the equipment used should be considered. At present, water and ethanol water solutions are commonly used as extraction solvents for oligosaccharides. When water is used as the extraction solvent, some polysaccharides and proteins are also extracted together, which poses certain difficulties for separation and purification. In addition, some oligosaccharides are also unstable in aqueous solutions. Therefore, another solvent for oligosaccharide extraction is ethanol water solution, but the disadvantage of this method is that it consumes a large amount of ethanol and is not an ideal solvent for drug extraction. From this, it can be seen that there are still limitations in the types of extraction solvents for oligosaccharides, and it is urgent to discover new solvent systems to meet the extraction requirements of different types of oligosaccharides. While exploring new extraction methods, the goal is to combine the advantages of traditional methods with new methods and equipment to explore a green, economical, and efficient extraction method with the aim of obtaining high-quality target oligosaccharides.

The main methods for separating oligosaccharides include membrane separation, liquid chromatography, and capillary electrophoresis. Conventional membrane separation technologies include microfiltration, ultrafiltration, nanofiltration, reverse osmosis, as well as electrodialysis and continuous electrodialysis combined with electrochemical technology. Membrane separation method has high separation efficiency, low energy consumption, and no pollution. It can achieve a simple and easy to control molecular level filtration process, and has the functions of separation, concentration, purification, and refining. It has its own unique advantages in compound separation, purification, and impurity removal. The disadvantage is that this technology has high equipment and membrane requirements, complex operation, and is also affected by various factors such as pressure, temperature, and time. The new membrane separation technology has put forward higher requirements for membrane performance, membrane flux, reduction of membrane fouling, reduction of pressure driven consumption, and even cost reduction, simplification of membrane manufacturing technology, and extension of the service life of individual membranes. At present, new membrane separation technologies have also emerged, including pervaporation membranes, liquid membranes, and dynamic membranes. Column chromatography is widely used in the separation and purification of oligosaccharides. According to different separation materials, mobile phases and separation principles, it can be divided into gel exclusion chromatography, hydrophilic interaction chromatography, anion exchange chromatography, graphitized carbon column chromatography, reverse phase high performance liquid chromatography, etc. The focus of this method is on separating materials, and suitable separation materials can be selected based on the properties of oligosaccharides to achieve the best separation and purification effect. Moreover, the separation materials of sugar compounds have always been a research hotspot and have been relatively mature in natural product applications. Therefore, the separation and purification of oligosaccharides will become faster, more efficient, and have more selectivity. The separation of oligosaccharides by electrophoresis can be achieved through four methods: pre column derivatization, direct and indirect UV detection without derivatization, laser-induced fluorescence detection, and electrode pulse amperometry detection. In addition, indirect UV detection can also identify non reducing oligosaccharides and aldonic acids that cannot be derivatized. At present, CE still has its own characteristics and advantages in drug analysis, and shows great potential for development. However, there are still multiple important bottleneck issues such as sensitivity, repeatability, qualitative ability, etc., which urgently need improvement and breakthroughs.
The structural identification of oligosaccharides generally uses UV and thin-layer chromatography to qualitatively identify sugars with fluorescence or color development. Infrared absorption spectroscopy is mainly used to identify the sugar ring skeleton and functional groups in oligosaccharides, and is generally used for preliminary characterization of oligosaccharide structures; Mass spectrometry is generally used to determine oligosaccharide chains and molecular weight, and is one of the important methods for identifying oligosaccharide structures; Nuclear magnetic resonance spectroscopy uses specific atomic nuclei to absorb only specific radio frequency radiation in a given magnetic field to form nuclear magnetic resonance signals for structural identification. The structural information of oligosaccharides can be obtained by the signals generated by the chemical shifts of various elements in sugar residues, and the relative content of residues can be obtained by integrating the signal peak area. The purity, molecular weight, connection position, and connection mode of oligosaccharides can be determined by GC, MS, NMR, and various methods. Oligosaccharides have complexity and diversity, and the above determination methods have certain requirements for the properties, purity, or dosage of polysaccharides. It is precisely because of the limitations of various structural characterization methods that the future development trend will inevitably be the combination of multiple methods, including the separation and analysis of oligosaccharides. Therefore, the continuous development of extraction methods, separation techniques, and structural characterization methods for oligosaccharides has profound significance for their development and application. A reasonable and feasible solution can definitely achieve efficient preparation, rapid separation, and precise characterization of oligosaccharides, making oligosaccharides in traditional Chinese medicine play a more important role in fields such as food and medicine.