CN-121975884-A - Dendrobium candidum oligosaccharide and preparation method and application thereof

Abstract

The invention discloses dendrobium candidum oligosaccharide, a preparation method and application thereof, and belongs to the technical field of preparation of functional components of natural products. According to the invention, the dendrobium candidum oligosaccharide (DOO 1) with the molecular weight of about 921 Da, the molar ratio of mannose to glucose of about 4.53:1 and the main glycosidic bond of → 4) -beta-D-Manp- (1 → is obtained by optimizing the technological conditions of beta-mannase for enzymolysis of dendrobium candidum polysaccharide, including substrate concentration, enzyme dosage, pH, temperature and time, and purifying. The prepared dendrobium candidum oligosaccharide not only remarkably improves the solubility and bioavailability, but also has good alpha-amylase inhibition activity and the effect of improving glucose uptake of insulin resistant cells, and further expands the functional application potential of dendrobium candidum.

Inventors

• LI YUAN
• LIANG ZONGSUO
• YANG DONGFENG
• ZHANG XIAODAN
• LI ZHENDA
• XIAO YI

Assignees

• 浙江理工大学绍兴生物医药研究院有限公司
• 宁波易中禾药用植物研究院有限公司

Dates

Publication Date

20260505

Application Date

20251223

Claims (6)

1. 1. The dendrobium candidum oligosaccharide is characterized in that the molecular weight of the dendrobium candidum oligosaccharide is 921 Da-931 Da, the monosaccharide composition of the dendrobium candidum oligosaccharide takes mannose and glucose as main components, the molar ratio of the mannose to the glucose is 3.92-5.16:1, and the main connection mode is → 4) -beta-D-2-O-Ac-Manp- (1 → 4) -beta-D-Manp- (1 → 4, 6) -beta-D-Manp- (1 → 4) -beta-D-Glcp- (1 →).
2. 2. A dendrobium candidum oligosaccharide as claimed in claim 1, wherein the dendrobium candidum oligosaccharide is mainly composed of seven hexose units, wherein three sites are acetylated.
3. 3. The method for preparing the dendrobium candidum oligosaccharide according to any one of claims 1-2, which is characterized by comprising the following steps: 1) Polysaccharide extraction, namely removing impurities from dried and crushed dendrobium candidum powder by using an organic solvent, extracting for 4 hours at 70 ℃ by using water as an extraction solvent, extracting for a plurality of times, merging supernatants, concentrating, precipitating with ethanol, deproteinizing by a Sevag method, and freeze-drying to obtain dendrobium candidum crude polysaccharide DOP; 2) Dissolving DOP in water to prepare a solution with the substrate concentration of 10-30mg/mL, regulating the pH value to 4.0-6.0, adding 10-50U/mL of beta-mannase, reacting for 3-7 hours at the temperature of 30-70 ℃ and the temperature of 150 rpm, heating to inactivate enzyme after the reaction is finished, centrifuging, and taking supernatant; 3) Crude extraction and purification, namely adding ethanol into the supernatant obtained in the step 2) to precipitate incompletely hydrolyzed polysaccharide, centrifuging to remove the supernatant, concentrating under reduced pressure, freeze-drying to obtain crude oligosaccharide DOO, performing DEAE-52 anion exchange column chromatography, eluting with deionized water, collecting target components, concentrating, and freeze-drying to obtain purified dendrobium candidum oligosaccharide DOO1.
4. 4. A method for preparing dendrobium candidum oligosaccharide as claimed in claim 3, wherein in the step 2), the substrate concentration is 26 mg/mL, the enzyme dosage is 42U/mL, the pH is 4.78, the reaction temperature is 50 ℃, and the reaction time is 6 hours.
5. 5. Use of the dendrobium candidum oligosaccharide of any one of claims 1-4 in the manufacture of a medicament for improving insulin resistance and/or preventing and treating type 2 diabetes.
6. 6. Use of the dendrobium candidum oligosaccharide of any one of claims 1-4 in the preparation of a prebiotic product for use in modulating intestinal flora and reducing potential pathogenic bacteria.

Description

Dendrobium candidum oligosaccharide and preparation method and application thereof Technical Field The invention belongs to the technical field of preparation of functional components of natural products, and particularly relates to dendrobium candidum oligosaccharide, a preparation method and application thereof. Background Diabetes, particularly type 2 diabetes, is centered on insulin resistance, and its incidence continues to rise. The existing oral hypoglycemic agents are easy to cause adverse reactions after long-term use, and safer treatment strategies are needed. Intestinal flora plays an important role in the occurrence and development of T2DM, unbalance of the intestinal flora can lead to abnormal glucose metabolism and reduced SCFA generation, and regulation of the flora becomes a potential auxiliary treatment means. Dendrobium candidum polysaccharide has the function of reducing blood sugar, but has the advantages of huge molecular weight, complex structure composition, high viscosity and difficult penetration of various barriers of organisms to directly exert the efficacy, thereby greatly limiting the application of the dendrobium candidum polysaccharide in food chemistry. Recent researches show that the functional oligosaccharide has the characteristics of small molecular weight, good solubility, definite structure and the like, and is widely applied to the fields of medicines and foods as a natural active carbohydrate with low heat and no toxicity. A large number of researches show that the compound has remarkable effects in the aspects of regulating immunity, resisting tumors and regulating metabolism. Oligosaccharides derived from seaweed, medicinal plants and edible fungi generally show good hypoglycemic and prebiotic activities, and show wide application prospects in diabetes prevention and treatment and intestinal health regulation. The oligosaccharide can be extracted from natural animals and plants, chemically synthesized or prepared by enzymolysis and acidolysis of natural polysaccharide. Among them, the direct extraction method has low efficiency and more impurities, and the chemical synthesis method has high yield but high cost and environmental pollution. At present, the preparation of oligosaccharide by enzymolysis or acidolysis of polysaccharide is widely adopted because of high efficiency, specificity and environmental friendliness. However, the dendrobium candidum oligosaccharide which has a definite structure, low molecular weight and dual activities of reducing blood sugar and prebiotics is prepared by a specific enzymolysis process, and is not reported. Disclosure of Invention Aiming at the problems existing in the prior art, the invention aims to provide dendrobium candidum oligosaccharide, a preparation method and application thereof, and the invention is realized by the following technical scheme: A dendrobium candidum oligosaccharide has a molecular weight of 921 Da-931 Da, the monosaccharide composition takes mannose and glucose as main components, the molar ratio of mannose to glucose is 3.92-5.16:1, and the main connection mode is → 4) -beta-D-2-O-Ac-Manp- (1 → 4) -beta-D-Manp- (1 → 4, 6) -beta-D-Manp- (1 → 4) -beta-D-Glcp- (1 → 4). Further, the dendrobium candidum oligosaccharide is mainly composed of seven hexose units, wherein three sites are acetylated. The preparation method of the dendrobium candidum oligosaccharide comprises the following steps: 1) Polysaccharide extraction, namely removing impurities from dried and crushed dendrobium candidum powder by using an organic solvent, extracting for 4 hours at 70 ℃ by using water as an extraction solvent, extracting for a plurality of times, merging supernatants, concentrating, precipitating with ethanol, deproteinizing by a Sevag method, and freeze-drying to obtain dendrobium candidum crude polysaccharide DOP; 2) Dissolving DOP in water to prepare a solution with the substrate concentration of 10-30mg/mL, regulating the pH value to 4.0-6.0, adding 10-50U/mL of beta-mannase, reacting for 3-7 hours at the temperature of 30-70 ℃ and the temperature of 150 rpm, heating to inactivate enzyme after the reaction is finished, centrifuging, and taking supernatant; 3) Crude extraction and purification, namely adding ethanol into the supernatant obtained in the step 2) to precipitate incompletely hydrolyzed polysaccharide, centrifuging to remove the supernatant, concentrating under reduced pressure, freeze-drying to obtain crude oligosaccharide DOO, performing DEAE-52 anion exchange column chromatography, eluting with deionized water, collecting target components, concentrating, and freeze-drying to obtain purified dendrobium candidum oligosaccharide DOO1. Further, in step 2), the substrate concentration was 26 mg/mL, the enzyme amount was 42U/mL, the pH was 4.78, the reaction temperature was 50℃and the reaction time was 6 hours. The application of the dendrobium candidum oligosaccharide in preparing medicaments for improving in