August 15, 2024 longcha9

Purification, physicochemical properties, and antioxidant activity of oligosaccharides from fresh Rehmannia glutinosa
Rehmannia glutinosa Libosch., a plant in the family Scrophulariaceae and genus Rehmannia, is the root tuber of Rehmannia glutinosa Libosch. It is a commonly used traditional Chinese medicine and is usually used in three forms (fresh Rehmannia, raw Rehmannia, and cooked Rehmannia). The fresh root of Rehmannia glutinosa is called fresh Rehmannia glutinosa. Fresh Rehmannia glutinosa has a cold nature, a sweet and bitter taste, and has the effects of lowering blood sugar, nourishing the liver and kidneys. Its special effects have been discussed in medical history and have been clinically proven. Fresh Rehmannia glutinosa has the custom of being eaten fresh in Henan folk culture, such as cold dishes and stir fried dishes. The sugar compounds in Rehmannia glutinosa occupy a large proportion of the total area, and its oligosaccharides are another type of active component discovered after glycosides and polysaccharides. It is known that Rehmannia glutinosa oligosaccharides have pharmacological activities such as blood tonifying and blood sugar lowering, and are the effective ingredients of Rehmannia glutinosa. Due to the difficulty in storage and transportation, the development and application of fresh Rehmannia glutinosa are limited to a certain extent. Currently, research is mostly focused on raw and cooked Rehmannia glutinosa, and there is relatively little research on the fresh Rehmannia glutinosa system. Jia et al.’s research results show that the content of raffinose in fresh Rehmannia glutinosa is 26.45%, which is significantly higher than that in raw Rehmannia glutinosa (9.16%) and cooked Rehmannia glutinosa (5.98%), and has potential research value. This experiment used fresh freeze-dried tablets as raw materials to prepare oligosaccharides using both ultrasound and hot water methods. After further purification, two groups of oligosaccharide fragments (GRCP and GRSP) were obtained, and their physicochemical properties and antioxidant capacity were systematically and comprehensively analyzed. The aim is to provide theoretical basis for the development and utilization of fresh Rehmannia glutinosa.

Using fresh Rehmannia glutinosa freeze-dried tablets as raw materials, the oligosaccharides contained in them were extracted and purified using ultrasound and hot water methods, resulting in two sets of fragments, GRCP and GRSP. The carbohydrate contents of the two are 901.39 and 948.06mg/g, respectively. Both have low impurities and high purity, indicating successful purification, which is much higher than the previous conclusion that the proportion of raffinose in the extracted sugars is 26.45%. This experiment comprehensively studied the extraction, purification, and physicochemical properties of raffinose in fresh Rehmannia glutinosa. The results showed that the main functional groups in GRCP and GRSP were the same, and the main components were oligosaccharides, mainly raffinose. Thermal analysis showed that their structures were relatively stable below 205.5 and 209.0 ℃, respectively. The monosaccharide composition of the two fragments is the same, but the proportions are different. SEM observation shows that their appearance and morphology have certain differences, indicating that different extraction methods have a certain influence on the structure of oligosaccharides. The activity assay results indicate that both groups of fragments have certain antioxidant activity. The mass spectrometry results also indicate that the main component of GRCP and GRSP is stachyose, which is a functional oligosaccharide composed of 2 molecules of alpha galactose, 1 molecule of alpha glucose, and 1 molecule of beta fructose. It has the effects of regulating gut microbiota balance and immune regulation, and is an excellent functional food ingredient. Oligosaccharides are oligosaccharides formed by connecting 2-10 monosaccharides through glycosidic bonds. The commonly used method for extracting oligosaccharides is water extraction and alcohol precipitation, which involves precipitation with 80% ethanol by volume to remove polysaccharides and reduce their interference, which is beneficial for further purification. Wu used 75% ethanol to extract fresh Rehmannia oligosaccharides, and took the supernatant for subsequent extraction. Meanwhile, ethanol with a volume fraction of around 80% is often used for precipitation to obtain polysaccharides. As Wang et al. found, polysaccharides of different molecular weights can be obtained by precipitation with ethanol at volume fractions of 30%, 60%, and 80%, respectively, to obtain polysaccharides from Pleurotus ostreatus. Yang et al. used 80% ethanol to precipitate polysaccharides and purified them, ultimately obtaining polysaccharides from Botrytis cinerea and jujube, respectively. Ye et al. obtained polysaccharides of Hedyotis diffusa from the 90% ethanol precipitation of the extraction solution. In this experiment, four times the volume of the extraction solution was added to anhydrous ethanol (with a final concentration of 80%) for alcohol precipitation. The precipitate was further purified, and the main component of the final purified product was raffinose, indicating that oligosaccharides were present in 80% of the ethanol precipitate, not all of which were polysaccharides. The reason for this result may be that stachyose is encapsulated and heavily precipitated by 80% ethanol, or that stachyose has limited solubility in 80% ethanol and can partially precipitate. However, the specific reasons still need further research. In the preliminary experiment, the alcohol precipitate was dialyzed using a 3500Da dialysis bag, and it was found that the yield of sugar substances in the 3500Da dialysis bag was only 0.61%, indicating that the majority of substances in the alcohol precipitate have small molecular weights. So in the later stage, a 500~1000Da dialysis bag is used to separate the purified substance from other small molecule substances and impurities, which is a simple and effective method to improve sample purity. This study provides a theoretical basis for the systematic research and further rational development and utilization of fresh Rehmannia oligosaccharides, especially raffinose resources.

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