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CN-121975028-A - Spirulina polysaccharide, preparation method and evaluation method for in vitro simulation of intestinal post-digestion immunomodulation

CN121975028ACN 121975028 ACN121975028 ACN 121975028ACN-121975028-A

Abstract

The invention discloses spirulina polysaccharide, a preparation method and an evaluation method for in-vitro simulated intestinal digestion post-immunoregulation of spirulina polysaccharide, wherein PSP-1 is prepared by steps of hot water ultrasonic extraction of spirulina powder, deproteinization and decoloration of macroporous resin, DEAE-52 anion exchange chromatography and the like. The weight average molecular weight of the spirulina is about 320kDa, and the spirulina is composed of fucose, rhamnose, galactose, glucose, xylose, glucuronic acid and the like, and is structurally different from the spirulina polysaccharide which has been reported. The immunoregulatory activity of PSP-1 for directly stimulating macrophage RAW264.7 and THP-1 is studied, and an intestinal epithelial cell-macrophage co-culture model is further established. The spirulina polysaccharide PSP-1 can promote macrophages to release inflammatory factors such as IL-6, TNF-alpha, IL-1 beta and the like by direct stimulation and intestinal cell post-stimulation. The method provides an evaluation model which is closer to the real physiological state of the human body for researching the immunocompetence of the dietary polysaccharide.

Inventors

  • HUANG RIMING
  • Wu nanshou
  • Zou Lingshan
  • Yu Jieting
  • MO YONGHUI
  • HUANG YAONAN
  • LIU QIN
  • GAO JIEXIN
  • ZHANG RONGXIN
  • ZHENG QIANWANG

Assignees

  • 华南农业大学

Dates

Publication Date
20260505
Application Date
20251223

Claims (10)

  1. 1. A spirulina polysaccharide is characterized in that the molecular weight of the spirulina polysaccharide is 3.2 multiplied by 10 5 ±0.15×10 5 Da, and the spirulina polysaccharide consists of fucose, rhamnose, galactose, glucose, xylose and glucuronic acid, wherein the molar ratio of the fucose to the galactose to the glucose to the xylose to the glucuronic acid=0.075:0.721:0.036:0.041:0.041:0.086.
  2. 2. The spirulina polysaccharide according to claim 1, wherein said spirulina polysaccharide comprises Xylp-(1→、Rhap-(1→、→2)-Rhap-(1→、Glcp-(1→、→3)-Rhap-(1→、→3)-Fucp-(1→、→2,3)-Rhap-(1→、→4)-Galp-(1→、→4)-Glcp-(1→ sugar residues.
  3. 3. The spirulina polysaccharide according to claim 1, wherein the molecular weight of the spirulina polysaccharide is 324741Da.
  4. 4. A process for preparing the spirulina polysaccharide according to any one of claims 1 to 3, characterized by comprising the steps of: (1) Performing hot water extraction, namely performing ultrasonic extraction on spirulina powder by heating water, wherein the extraction temperature is 80-90 ℃ to obtain water extract; (2) Filtering and centrifuging the extracting solution, and collecting supernatant; (3) Passing the supernatant through macroporous resin, removing proteins and pigments, and collecting eluent; (4) Concentrating the eluent, dialyzing for the first time, and freeze-drying the content of the dialysis bag to obtain crude polysaccharide PSP; (5) Dissolving crude polysaccharide PSP in water, performing column separation by using a DEAE-52 anion exchange chromatographic column, eluting by using water and 0.1-1.2M sodium chloride solution in sequence, detecting a sugar peak, collecting sugar-containing fractions, freeze-drying, and collecting different fractions of spirulina polysaccharide; (6) Performing secondary dialysis on the spirulina polysaccharide with 0.6M fraction, purifying with ultrafiltration tube, and lyophilizing to obtain spirulina polysaccharide PSP-1.
  5. 5. The method according to claim 4, wherein the method comprises any one or more of the following [1] to [17 ]: [1] air drying spirulina, and pulverizing to obtain spirulina powder; [2] In the step (1), the liquid-material ratio of the spirulina powder to the hot water leaching is 1:20-30 g/mL; [3] The water used in the step (1) is distilled water or tertiary water; [4] In the step (1), the ultrasonic time is 30 minutes, and the hot water leaching time is 1.5-2.5 hours; [5] repeating the leaching operation in the step (1) one or more times, and combining to obtain the water extract; [6] Filtering in the step (2) by a sieve or gauze with the mesh number of 100-600 meshes to obtain filtrate; [7] The centrifugation condition in the step (2) is 8000rpm-12000rpm, and the centrifugation time is 10-15min, so as to obtain supernatant; [8] the macroporous resin in the step (3) is NKA-9; [9] the wet volume BV ratio of the supernatant to the macroporous resin in the step (3) is 1:2-5; [10] concentrating the filtrate in the step (4) to obtain filtrate, and performing rotary evaporation under reduced pressure at 60-70 ℃ until the volume of the filtrate is 10% -15%; [11] the molecular weight cut-off of the dialysis bag for the first dialysis in the step (4) is 2800-3200Da; [12] the water used in the first dialysis in the step (4) is tap water or 1-3 grade water, and the dialysis time is 48-96 hours; [13] In the step (5), the crude polysaccharide PSP is dissolved in water and then filtered by a microporous filter membrane; [14] In the step (5), the crude polysaccharide PSP is dissolved into 5-10mg/mL to obtain a sample solution, and the volume ratio BV of the sample solution to the DEAE-52 anion exchange chromatography bed is 1:2-5; [15] the concentration of the sodium chloride solution in the step (5) is 0.1M, 0.3M, 0.6M, 0.9M and 1.2M in sequence; [16] The molecular weight cut-off of the dialysis bag for the second dialysis in the step (6) is 2800-8000Da; [17] the ultrafiltration purification in step (6) uses a ultrafiltration tube having a molecular weight cut-off of 30kda and 50kda in sequence.
  6. 6. A spirulina polysaccharide produced by the method of any one of claims 4-5.
  7. 7. A spirulina pharmaceutical formulation comprising the spirulina polysaccharide of any one of claims 1 to 3 or the spirulina polysaccharide of claim 6, and optionally a pharmaceutically acceptable adjuvant.
  8. 8. Use of the spirulina polysaccharide according to any one of claims 1 to 3 or the spirulina polysaccharide according to claim 6 or the metabolites of the spirulina pharmaceutical formulation according to claim 7 after oral intestinal metabolism for the preparation of a medicament, food and/or health product for enhancing immunity.
  9. 9. The method of claim 8, wherein the metabolite triggers the IkB/NF-kB signaling pathway by activating the TLR4 receptor on the surface of the macrophage, thereby enhancing immunity.
  10. 10. A method for regulating immunity after intestinal digestion of spirulina polysaccharide in vitro is characterized by comprising the following steps: (1) Preparing spirulina polysaccharide PSP-1; (2) Preparing intestinal epithelial cell conditioned medium HIEC-CM, culturing HIEC-6 cells to form a cell monolayer, completely fusing, adding spirulina polysaccharide PSP-1, continuously culturing, collecting cell culture solution, centrifuging to obtain HIEC-6 cell supernatant, and mixing HIEC-6 cell supernatant with complete medium at a volume ratio of 1:1 to prepare conditioned medium HIEC-CM; (3) Inducing THP-1 monocyte into macrophage to obtain induced THP-1 macrophage; (4) Culturing the induced THP-1 macrophage with conditioned medium HIEC-CM; (5) The difference in molecular weight of spirulina polysaccharide PSP-1 and metabolites produced by HIEC-6 cells stimulated by spirulina polysaccharide PSP-1, and the difference in immunological activity to THP-1 macrophages were examined.

Description

Spirulina polysaccharide, preparation method and evaluation method for in vitro simulation of intestinal post-digestion immunomodulation Technical Field The invention relates to the technical field of polysaccharides, in particular to a spirulina polysaccharide, a preparation method of the spirulina polysaccharide and an in-vitro simulated intestinal digestion immune regulation evaluation method based on intestinal epithelial cell-macrophage co-culture. Background Spirulina (spirorina), belonging to the genus of cyanobacteria (Cyanophyta), oscillatoriales (Oscillatoriales), oscillatoriaceae (Oscillatoriaceae) and Spirulina, is a kind of the oldest lower prokaryote on the earth, and is named as a spiral form. The spirulina polysaccharide (Polysaccharides ofSpirulinaplatensis) is a water-soluble natural product separated and extracted from spirulina, is a main existence form of carbohydrate in algae, has special biological activity, such as immunity improving, anti-tumor, antioxidant and anti-radiation effects. Chinese patent 201510312393.4 reports that spirulina polysaccharide consists of D-glucuronic acid, D-acetamido galactose, L-fucose and galactose, the ratio of the four monosaccharides is 3.5-4.5:3.5-4.5:1.7-2.3:1 in terms of mole ratio, and the weight average molecular weight range of the spirulina polysaccharide is 5000-18000 Da. Chinese patent 202211297733.7 also reports a high-purity low-molecular spirulina polysaccharide, an extraction process, a detection method and application thereof, wherein the low-molecular spirulina polysaccharide consists of five monosaccharides of D-glucuronic acid (D-GlcA), alpha-D-rhamnose (alpha-D-Rha), beta-D-rhamnose (beta-D-Rha), beta-D-glucose (beta-D-Glc) and beta-D-galactose (beta-D-Gal). The molecular weight was 45.3kDa. However, the novel spirulina polysaccharide consisting of different monosaccharides is separated from spirulina powder, and the composition and the glycosidic bond connection mode of the novel spirulina polysaccharide are different from those of the prior reports. Studies have shown that differences in monosaccharide composition, linkage and spatial configuration of polysaccharides can significantly affect their biological functions, such as immunomodulatory, antioxidant, antitumor, etc. Therefore, the polysaccharide of the invention is obviously different from the spirulina polysaccharide reported in the literature in structure, and is expected to have new characteristics and application potential in terms of biological activity. For the research part of polysaccharide pharmaceutical activity, the prior art research on spirulina polysaccharide immunocompetence is mostly focused on the direct stimulation effect of crude extract or purified components on a single immune cell model. However, this in vitro direct stimulation model has significant limitations in that when spirulina polysaccharides are ingested by the human body as food or oral drugs, the polysaccharides contained therein first undergo the digestive absorption process of the gastrointestinal tract, and it is difficult to directly contact and stimulate immune cells (such as macrophages) under the lamina propria of the intestinal tract in a complete form. The existing research cognition stays in the direct action mode of polysaccharide-immune cells, and neglects the indirect immunoregulation path of polysaccharide-intestinal epithelial cells-immune cells which is more in accordance with physiological reality. Also, it is currently unclear why the final active form of spirulina polysaccharide after digestion, absorption and metabolism by intestinal epithelial cells is, whether it is a crude polysaccharide, a digested fragment, or a cellular metabolite. Disclosure of Invention In order to solve the technical problems, the scheme for solving the technical problems is as follows: In a first aspect of the invention, there is provided a spirulina polysaccharide having a molecular weight of 3.2x10 5±0.15×105 Da, consisting of fucose, rhamnose, galactose, glucose, xylose and glucuronic acid, the molar ratio of monosaccharides being fucose to rhamnose to galactose to glucose to glucuronic acid=0.075:0.721:0.036:0.041:0.041:0.086. Further, the spirulina polysaccharide comprises Xylp-(1→、Rhap-(1→、→2)-Rhap-(1→、Glcp-(1→、→3)-Rhap-(1→、→3)-Fucp-(1→、→2,3)-Rhap-(1→、→4)-Galp-(1→、→4)-Glcp-(1→ sugar residues. Further, the molecular weight of the spirulina polysaccharide is 324741Da. A numerical value of "about" or "approximately" with respect to molecular weight means ± 5% of the numerical value, but explicitly include the exact numerical value. For example, "about 3.2×10 5 refers to from 3.05×10 5 to 3.5×10 5, but also specifically includes 3.2×10 5 Da. The invention also provides a preparation method of the spirulina polysaccharide, which comprises the following steps: (1) Performing hot water extraction, namely performing ultrasonic extraction on spirulina powder by heating water, wherein the extraction