Based on molecular docking and enzyme inhibition kinetics, explore the inhibitory activity of α – glucosidase in vitro extracted from Rhodiola rosea. diabetes is a common disease, which can be divided into two types: diabetes type 1 (T1DM) and diabetes type 2 (T2DM). T2DM accounts for more than 90% of diabetes patients, mainly caused by increased postprandial blood glucose due to insulin resistance and abnormal insulin secretion. Alpha glucosidase is one of the main enzymes that cause postprandial hyperglycemia, and inhibiting alpha glucosidase activity has become the main way to control postprandial hyperglycemia and treat T2DM. At present, the alpha glucosidase inhibitors commonly used in clinical practice are acarbose, miglitol, etc. Although they have high inhibitory activity, they are expensive and prone to a series of gastrointestinal reactions such as indigestion. Natural products derived from plants have become high-quality sources of therapeutic agents due to their low toxicity and side effects. Therefore, searching for safe and low adverse reaction alpha glucosidase inhibitors from natural active substances has become a research hotspot.

Rhodiola sachalinensis, as one of the traditional Chinese medicinal materials with food and medicine homology, contains salidroside, catechin, tyrosol and other active ingredients. At present, research at home and abroad mainly focuses on anti hypoxia, anti apoptosis, anti-tumor, relieving stress damage, etc. Some studies also show that it has a certain role in diabetes rodent models, and can alleviate hyperglycemia, hyperlipidemia, and urine sugar symptoms in diabetes mice by activating 5 ‘- adenosine monophosphate dependent protein kinase (AMPK) related signaling pathways. Recently, researchers have found that Rhodiola extract can significantly reduce postprandial blood glucose levels in mice, and its active ingredients exhibit a certain degree of inhibitory effect in vitro, but the specific inhibition mechanism is not clear. Our team’s preliminary research found that polyphenolic substances in Rhodiola rosea have certain in vitro alpha glucosidase inhibitory activity. Based on the above research background, this study combines enzyme inhibition kinetics, UHPLC-QE-MS, and molecular docking technology to explore the inhibitory effect of Rhodiola rosea extract on alpha glucosidase in a visualized manner. The research aims to provide theoretical reference for the development of hypoglycemic drugs and health foods, in order to promote the further development and utilization of Rhodiola rosea.

 

 

Alpha glucosidase exists in the brush border of the small intestine mucosa in the body, which can break down oligosaccharides or disaccharides into monosaccharides. Inhibiting its activity can slow down carbohydrate hydrolysis and glucose production rate, thereby reducing the absorption of glucose by the small intestine and postprandial blood glucose. Research shows that extracts from various plants such as star apple, elephant leg banana, black wolfberry, Schisandra chinensis, purple yam, and Guizhou dandelion all have inhibitory activity against alpha glucosidase. The half maximal inhibitory concentration (IC50) can be used to evaluate the inhibitory effect of inhibitors on enzymes. The smaller the IC50, the higher the inhibitory activity. The team’s previous research has shown that Rhodiola polyphenols have a good inhibitory effect on alpha glucosidase, with an IC50 of 2.83mg/mL. The RCE IC50 obtained in this experiment is 1.538mg/mL, which is not only lower than acarbose, but also lower than Rhodiola polyphenols. This may be due to the presence of flavonoids, organic acids and other substances in RCE. The types of enzyme inhibition by inhibitors can be divided into reversible inhibition and irreversible inhibition. The RCE and α – glucosidase obtained in this study are reversible inhibition, characterized by non covalent binding between the inhibitor and the enzyme, which can restore enzyme activity through physical methods. The reversible inhibition types can be further divided into four categories: competitive inhibition, non competitive inhibition, anti competitive inhibition, and mixed inhibition. The mixed inhibition types include competitive and non competitive mixed inhibition. The characteristic of this type is that an increase in inhibitor concentration will reduce the maximum reaction rate (Vmax) of the enzymatic reaction and increase the Michaelis constant (Km). This study shows that an increase in RCE concentration leads to a decrease in Vmax The increase in Km is consistent with the above characteristics, therefore the inhibition type of RCE on α – glucosidase is determined to be a mixed type of competitive and non competitive inhibition, indicating that Rhodiola extract is an effective natural α – glucosidase inhibitor. RCE is consistent with the inhibition types of α – glucosidase by polyphenols from Rhodiola rosea, polysaccharides from Schisandra chinensis, and polysaccharides from Scenedesmus macrophylla. However, it is necessary to clarify which compounds belong to competitive inhibition and which compounds belong to non competitive inhibition, and further exploration is needed. RCE has good α – glucosidase inhibitory activity, but further animal models are needed to verify whether the effect is significant in vivo. At present, there are many sources of alpha glucosidase, mainly including brewing yeast, Aspergillus niger, thermophilic Bacillus subtilis, and Leuconostoc enterica. The properties of α – glucosidase from different sources vary greatly, and the α – glucosidase used in this study is from brewing yeast. Therefore, further investigation is needed to determine whether RCE has the same inhibitory effect on enzymes from other sources.
UHPLC-QE-MS has the characteristics of speed and accuracy, and can be used for non target analysis of compounds in complex Chinese medicinal materials. This study analyzed RCE and identified 1245 compounds through comparison with the database, including 263 compounds in negative ion mode and 982 compounds in positive ion mode. The main solvents for extracting the active ingredients of Rhodiola rosea are ethanol and water. Compared with pure water, aqueous solutions of organic solvents can extract more compounds. The content of gallic acid in the aqueous extract is higher than that in the alcohol extract, while the content of tyrosol, chlorogenic acid, and tannins is lower than that in the water alcohol extract. Therefore, in order to extract more compounds, this experiment used 50% ethanol as the solvent for RCE extraction preparation. However, due to the single extraction solvent, the detected compounds may not necessarily be all compounds of Rhodiola rosea, and there are still some compounds that cannot be extracted or identified. Therefore, further comparative experiments with different extraction solvents are needed. Zakharenko’s research shows that the main active ingredients in Rhodiola rosea are salidroside, luteolin, catechin, quercetin, herbal extract, and catechin. This is consistent with the results of this study, as RCE contains high levels of luteolin and quercetin. Molecular docking can predict whether small molecule ligands can bind to receptor proteins and the binding forces involved. In this study, 20 compounds with high content obtained from UHPLC-QE-MS were docked with α – glucosidase. The results showed that 11 compounds could bind to α – glucosidase, including luteolin, salidroside, kaempferol, (-) – epicatechin 3-O-gallate, caffeic acid, L-malic acid, (+) – epicatechin, quercetin, (-) – erythrothro-anethole glycol 1-glucoside, 3-hydroxy-4,6-heptyne-1-yl 1-glucoside, and tyrosol. Among them, caffeic acid formed the most hydrogen bonds. It can bind to residues His-515, Arg-437, Glu-432, and His-348. (+) – Epicatechin has the best binding activity with Asn-443 and His-515, with the lowest binding energy (-17.08 kJ/mol); Docking PNPG with α – glucosidase revealed that Arg-437 and Glu-432 are its two binding sites. Caffeic acid shares the same binding sites with PNPG, Arg-437 and Glu-432, while L-malic acid, quercetin, and tyrosol share the same binding site with PNPG, Arg-437. Therefore, it is inferred that the inhibition types of caffeic acid, L-malic acid, quercetin, and tyrosol on α – glucosidase may be competitive inhibition, while the other six compounds are non competitive inhibition. However, further purification of the compounds and Lineweaver Burk reciprocal plots are needed to determine their inhibition types.
In summary, this study explored the inhibitory effect of RCE on α – glucosidase in a visualized manner through enzyme inhibition kinetics, UHPLCQE-MS, and molecular docking techniques. The inhibition type was a mixed type of competitive and non competitive inhibition, and the binding activity between (+) – epicatechin and α – glucosidase was the best in RCE. This article provides basic research for the development of RCE as a natural alpha glucosidase inhibitor and the utilization of Rhodiola resources.