A method for isolating and purifying chicory polysaccharides and their application in the preparation of drugs for treating sarcopenic obesity.

Abstract

This invention provides a method for isolating and purifying chicory polysaccharides and their application in the preparation of drugs for treating sarcopenic obesity, belonging to the field of polysaccharide extraction technology. The main chain of the chicory polysaccharide of this invention is primarily composed of →1)-β-D-Fruf-(2→ and →6)-α-D-Glcp-(1→) residues, with →1,6)-β-D-Fruf-(2→) residues scattered throughout as branch points. The side chain consists of terminal β-D-Fruf-(2→) units connected to the C-6 position of the furanofructose residues in the main chain. The therapeutic effects of chicory polysaccharides were evaluated in a db / db mouse model. The results showed that CGP-A effectively reduced insulin resistance and protected pancreatic function, alleviated systemic obesity in db / db mice and corrected lipid metabolism disorders, while also alleviating hepatic steatosis and reducing muscle atrophy in db / db mice.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide extraction technology, specifically to a method for separating and purifying chicory polysaccharides and their application in the preparation of drugs for treating sarcopenic obesity. Background Technology

[0002] Sarcoidemia (SO), characterized by a decrease in skeletal muscle mass and the accumulation of visceral fat, is a particularly debilitating complication of type 2 diabetes mellitus (T2DM). This dual pathological state triggers a vicious cycle of metabolic deterioration, significantly increasing risk. Traditional drug interventions often struggle to address both core issues of this syndrome simultaneously; for example, while glucagon-like peptide-1 receptor agonists (GLP-1 receptor agonists) can effectively control blood sugar and reduce weight, they may inadvertently accelerate muscle loss, thereby exacerbating the core characteristics of SO. Therefore, there is an urgent market need for safe, multi-functional drugs that can simultaneously reduce hepatic steatosis and muscle atrophy.

[0003] Natural polysaccharides, especially those derived from medicinal and edible plants, are attracting increasing attention as candidate drugs for managing complex metabolic disorders due to their low toxicity and multi-target regulatory potential. (Chicorydalis yanhusuo) Cichorium glandulosum Boiss. et Huet is a traditional medicinal herb widely used in the Xinjiang Uygur Autonomous Region, historically valued for its potent hepatoprotective and anti-diabetic properties. Current research on this plant primarily focuses on its small-molecule bioactive components, such as chicoric acid, lactucin, and lactucinol. Although the efficacy of chicory in folk medicine is widely recognized, the chemical properties of its high-molecular-weight polysaccharides (CGPs) in an SO2 context and their systemic therapeutic mechanisms remain largely unexplored. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for isolating and purifying chicory polysaccharides and their application in the preparation of drugs for treating sarcopenic obesity. The chicory polysaccharides provided by this invention have a novel structure and can improve sarcopenic obesity by regulating hepatic lipid metabolism and maintaining skeletal muscle homeostasis.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a chicory polysaccharide having the structure shown in Formula I: Formula I; In Equation I, a = 5~20, b = 5~20, and a+b = 25~34.

[0006] Preferably, the molecular weight of the chicory is 5kDa to 8kDa.

[0007] This invention provides a method for isolating and purifying the above-mentioned chicory polysaccharide, comprising the following steps: The root powder of *Chicorylia cuminata* was subjected to degreasing and decolorization treatment to obtain pretreated *Chicorylia cuminata* root powder. The pretreated chicory root powder was subjected to water extraction and alcohol precipitation, and the precipitate was collected to obtain a crude polysaccharide extract. The crude polysaccharide extract was dissolved in water, decolorized using AB-8 macroporous resin, and the water eluent was collected to obtain the decolorized crude polysaccharide extract. The proteins in the decolorized crude polysaccharide were removed using Sevag reagent, and the preliminarily purified polysaccharide extract was obtained by dialysis. The preliminary purified polysaccharide extract was separated and purified sequentially using a DEAE-52 cellulose column and a Sephadex G-100 column to obtain chicory polysaccharide.

[0008] Preferably, the degreasing and decolorizing treatment involves sequentially soaking the food in petroleum ether and ethanol for 18-24 hours each.

[0009] Preferably, the liquid-to-solid ratio of the water extraction is 30 mL / g, the temperature is 85℃, the water extraction is performed twice, and the extraction time for each cycle is 3 hours. The alcohol precipitation temperature is 4°C, the alcohol precipitation is performed twice, and the time for each precipitation is 8~24 hours.

[0010] Preferably, the Sevag reagent is a mixture of chloroform and n-butanol, wherein the volume ratio of chloroform to n-butanol is 4:1; The molecular weight cutoff for the dialysis is 3.5 kDa.

[0011] Preferably, the DEAE-52 cellulose column has dimensions of 2.6 cm × 40 cm; The eluent used in the DEAE-52 cellulose column was water and 0.1-0.5 M sodium chloride solution, with an elution rate of 1.0 mL / min.

[0012] Preferably, the Sephadex G-100 column has dimensions of 1.6 cm × 50 cm; The Sephadex G-100 column uses water as the eluent and has an elution rate of 0.3 mL / min.

[0013] This invention provides the application of commercially available chicory polysaccharide in the preparation of anti-oligomuscular obesity drugs.

[0014] The present invention provides an anti-sarcopenic obesity drug, comprising pharmaceutical excipients and active ingredients, wherein the active ingredients include the above-mentioned chicory polysaccharide.

[0015] This invention provides a chicory polysaccharide having the structure shown in Formula I. The main chain of the chicory polysaccharide is composed primarily of →1)-β-D-Fruf-(2→ and →6)-α-D-Glcp-(1→) residues, with →1,6)-β-D-Fruf-(2→) residues scattered throughout as branch points. The side chain consists of terminal β-D-Fruf-(2→) units attached to the C-6 position of the furanofructose residues in the main chain. This is a novel branched inulin-type fructan, named CGP-A. This invention elucidates the primary structure of CGP-A using a combination of methylation analysis and one-dimensional / two-dimensional nuclear magnetic resonance spectroscopy. Its therapeutic effect was then evaluated in a db / db mouse model, which reproduces the characteristics of obesity, impaired insulin sensitivity, and muscle atrophy in human SO. The results showed that CGP-A effectively alleviates insulin resistance and protects pancreatic function, relieves systemic obesity in db / db mice and corrects lipid metabolism disorders, while also alleviating hepatic steatosis and reducing muscle atrophy in db / db mice.

[0016] This invention provides a method for separating and purifying the above-mentioned chicory polysaccharide. The separation and purification method provided by this invention is simple to operate, has a high yield, and the obtained chicory polysaccharide has high uniformity and purity. Attached Figure Description

[0017] Figure 1 Gradient elution curves for the separation and purification of cellulose using a DEAE-52 column; Figure 2 Elution curves and photographs of purified CGP-A powder obtained by Sephadex G-100 column separation and purification; Figure 3 The preliminary results of CGP-A are characterized; Figure 4 The NMR spectrum of CGP-A; Figure 5 Results of CGP-A alleviating insulin resistance in db / db mice; Figure 6 CGP-A was used to alleviate lipid metabolism disorders and obesity in db / db mice. Figure 7 The results of CGP-A in alleviating hepatic steatosis and muscle atrophy in db / db mice. Detailed Implementation

[0018] This invention provides a chicory polysaccharide having the structure shown in Formula I: Formula I; In Formula I, a = 5~20, preferably 10~15, and specifically can be 5, 8, 10, 14, 16, 18 or 20; b = 5~20, preferably 10~15, specifically 5, 8, 10, 14, 16, 18 or 20; And a + b = 25~34.

[0019] In this invention, the main chain of the chicory polysaccharide is mainly composed of →1)-β-D-Fruf-(2→ and →6)-α-D-Glcp-(1→ residues, with →1,6)-β-D-Fruf-(2→ residues scattered in the middle as branch points, and the side chain is composed of terminal β-D-Fruf-(2→ units connected to the C-6 position of the furanofructose residue in the main chain.

[0020] In this invention, the molecular weight of the chicory is preferably 5kDa to 8kDa, and more preferably 6.722 kDa.

[0021] This invention provides a method for isolating and purifying the above-mentioned chicory polysaccharide, comprising the following steps: The root powder of *Chicorylia cuminata* was subjected to degreasing and decolorization treatment to obtain pretreated *Chicorylia cuminata* root powder. The pretreated chicory root powder was subjected to water extraction and alcohol precipitation, and the precipitate was collected to obtain a crude polysaccharide extract. The crude polysaccharide extract was dissolved in water, decolorized using AB-8 macroporous resin, and the water eluent was collected to obtain the decolorized crude polysaccharide extract. The proteins in the decolorized crude polysaccharide were removed using Sevag reagent, and the preliminarily purified polysaccharide extract was obtained by dialysis. The preliminary purified polysaccharide extract was separated and purified sequentially using a DEAE-52 cellulose column and a Sephadex G-100 column to obtain chicory polysaccharide.

[0022] This invention involves degreasing and decolorizing chicory root powder to obtain pretreated chicory root powder. In this invention, the degreasing and decolorizing treatment is preferably performed by sequentially soaking the powder in petroleum ether and ethanol. The preferred soaking temperature for petroleum ether is 20-25°C, and the preferred soaking time is 18-24 hours; the preferred soaking temperature for ethanol is 20-25°C, and the preferred soaking time is 18-24 hours.

[0023] This invention involves water extraction and alcohol precipitation of the pretreated chicory root powder, collecting the precipitate to obtain a crude polysaccharide extract. In this invention, the liquid-to-solid ratio of the water extraction is preferably 30 mL / g, the temperature is preferably 85℃, the water extraction is preferably performed twice, and the extraction time for each cycle is preferably 3 hours. After the water extraction, this invention preferably concentrates the obtained water extract under reduced pressure.

[0024] In this invention, the reagent used for alcohol precipitation is preferably ethanol, the temperature of alcohol precipitation is preferably 4°C, the number of alcohol precipitations is preferably 2, and the time for each alcohol precipitation is preferably 8-24 hours, more preferably 12-18 hours. After the first alcohol precipitation, the resulting alcohol precipitate is preferably concentrated under reduced pressure.

[0025] In this invention, the crude polysaccharide extract is dissolved in water, decolorized using AB-8 macroporous resin, and the water eluent is collected to obtain the decolorized crude polysaccharide extract. In this invention, the preferred type of AB-8 macroporous resin is S30931, purchased from Shanghai Yuanye Company, and its specific surface area is preferably 400-500 m² / g. 2 / g, with a pore size preferably of 10~14 nm; in this invention, the elution reagent for decolorizing the AB-8 macroporous resin is water, and the elution rate is preferably 2.0 mL / min.

[0026] This invention uses Sevag's reagent to remove proteins from decolorized crude polysaccharides, and after dialysis, a preliminary purified polysaccharide extract is obtained. In this invention, the Sevag's reagent is preferably a mixture of chloroform and n-butanol, with a preferred volume ratio of 4:1. In this invention, the preferred method for removing proteins from decolorized crude polysaccharides using Sevag's reagent is shaking followed by natural settling via a separatory funnel.

[0027] In this invention, the molecular weight cutoff for dialysis is preferably 3.5 kDa, and the dialysis time is preferably 24-48 h, more preferably 36 h.

[0028] This invention uses a DEAE-52 cellulose column and a Sephadex G-100 column sequentially to separate and purify the preliminarily refined polysaccharide extract to obtain chicory polysaccharide. In this invention, the DEAE-52 cellulose column preferably has a size of 2.6 cm × 40 cm; the eluent used for the DEAE-52 cellulose column is water and a 0.1–0.5 M sodium chloride solution, with an elution rate preferably of 1.0 mL / min. In this invention, the fraction collected after separation and purification using the DEAE-52 cellulose column is the aqueous fraction.

[0029] In this invention, the Sephadex G-100 column is preferably 1.6 cm × 50 cm in size; the eluent used in the Sephadex G-100 column is preferably distilled water, and the elution rate is preferably 0.3 mL / min.

[0030] This invention provides the application of the above-mentioned chicory polysaccharide in the preparation of anti-oligomuscular obesity drugs.

[0031] This invention provides a drug for treating sarcopenic obesity, comprising pharmaceutical excipients and an active ingredient, wherein the active ingredient includes the aforementioned chicory polysaccharide. In this invention, the excipients are preferably pharmaceutically acceptable excipients, carriers, or diluents.

[0032] The following detailed description, in conjunction with embodiments, illustrates the method for isolating and purifying chicory polysaccharides provided by the present invention and their application in the preparation of drugs for treating sarcopenic obesity. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1 The roots of the chicory were harvested from an organic plantation in Hotan City, Xinjiang Uygur Autonomous Region, China.

[0034] The method for isolating and purifying chicory polysaccharides comprises the following steps: First, dried chicory root (1.0 kg) was ground into a fine powder. The powder was pretreated with petroleum ether and 80% ethanol for degreasing and decolorization. The petroleum ether soaking temperature was 25℃ for 18–24 h, and the 80% ethanol soaking temperature was 25℃ for 24 h. Then, it was extracted twice with hot water at 85℃ at a liquid-to-solid ratio of 30 mL / g, with each cycle lasting 3 h. After concentration under reduced pressure, ethanol was added to a final concentration of 80%, and precipitation was carried out overnight at 4℃. The mixture was then concentrated under reduced pressure and precipitated again at 4℃ by adding four times its volume of anhydrous ethanol for 24 h to obtain the crude polysaccharide extract CGP (18.74 g).

[0035] The crude polysaccharide extract CGP was dissolved in water and further decolorized using AB-8 macroporous resin with water as the eluent at a rate of 2.0 mL / min. The eluent was collected and mixed with Sevag reagent (chloroform / n-butanol, volume ratio 4:1), shaken, and allowed to stand naturally in a separatory funnel to remove proteins. Subsequently, dialysis was performed using a 3.5 kDa molecular weight cutoff membrane for 48 h to obtain a preliminary purified polysaccharide extract (12.58 g).

[0036] For further purification, 2.0 g of the preliminarily purified polysaccharide extract was dissolved in distilled water and loaded onto a DEAE-52 cellulose column (2.6 cm × 40 cm). Elution was performed first with distilled water at a flow rate of 1.0 mL / min, followed by elution with progressively increasing concentrations of sodium chloride solution (0.1, 0.2, 0.3, 0.4, and 0.5 M). Fractions (4 mL / tube) were collected, and the carbohydrate content in each tube was monitored at 625 nm using the phenol-sulfuric acid method. The major fraction eluted with distilled water was collected, concentrated, eluted with deionized water using a Sephadex G-100 column (1.6 cm × 50 cm), and freeze-dried to obtain chicory polysaccharide CGP-A (156.52 mg).

[0037] Structural characterization The following methods were used to characterize the results of chicory polysaccharide.

[0038] ① Total neutral sugar content The total sugar content of CGP-A was quantified using the phenol-sulfuric acid colorimetric method. In short, D-glucose standard solutions (0–80 μg / mL) were prepared to construct a standard curve (R²). 2 >0.99). The absorbance was recorded at 490 nm using a UV-2600 spectrophotometer (Shimadzu, Japan).

[0039] ② Glucuronite level The uronic acid content was determined by the m-hydroxybiphenyl method, and the absorbance was measured at 525 nm.

[0040] ③ Ultraviolet spectroscopy The ultraviolet absorption spectrum of CGP-A (0.5 mg / mL in deionized water) was obtained in the wavelength range of 200–400 nm using a UV-2600 spectrophotometer.

[0041] ④ Molecular weight distribution The molecular weight distribution of CGP-A was determined using high-performance gel permeation chromatography (HPGPC) on a Shimadzu LC-20AR system equipped with a RID-20A differential refractive index detector. Separation was performed on a TSK-GEL GMPWXL column (7.8 mm × 300 mm, 13 μm, Tosoh, Japan). Purified water was used as the mobile phase at a flow rate of 0.6 mL / min, and the column temperature was 35 °C. The injection volume was 10 μL. Calibration curves were constructed using a series of dextran standards with molecular weights ranging from 5.2 to 3870.0 kDa.

[0042] ⑤ Monosaccharide composition The monosaccharide composition of CGP-A was determined using a Thermo ICS 5000+ system (Thermo Fisher Scientific, USA) via high-performance anion exchange chromatography-pulse amperometric detection (HPAEC-PAD). In short, approximately 5 mg of sample was hydrolyzed in a sealed tube with 2M trifluoroacetic acid (TFA) at 60°C for 1 hour. The hydrolysate was dried under a nitrogen stream, followed by multiple washing and drying cycles (2–3 times) with methanol to ensure complete removal of residual TFA. The final residue was redissolved in deionized water and filtered through a 0.22 μm microporous membrane before analysis.

[0043] Chromatographic separation was performed on a Dionex CarboPac PA20 column (150 × 3.0 mm, 10 μm) at 30 °C with an injection volume of 5 μL. The mobile phase, consisting of ultrapure water (A), 0.1 M NaOH (B), and 0.1 M NaOH containing 0.2 M NaOAc (C), was delivered at a flow rate of 0.5 mL / min. The gradient elution program was set as follows: 0 minutes A / B / C (95:5:0, V / V), 26 minutes A / B / C (85:5:10, V / V), 42 minutes A / B / C (85:5:10), 42.1 minutes A / B / C (60:0:40), 30.1 minutes A / B / C (20:0:80, V / V), 46 minutes A / B / C (20:0:80, V / V), 46.1 minutes A / B / C (0:100:0, V / V), 52 minutes A / B / C (60:40:0, V / V), 52.1 minutes A / B / C (95:5:0:0, V / V), and 60 minutes A / B / C (95:5:0, V / V). Qualitative and quantitative analyses were performed using 13 monosaccharide standards, including fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, mannuronic acid, and guluronic acid.

[0044] Quantitative analysis was achieved using an external standard calibration curve, with a correlation coefficient R0. 2 >0.99. All data were processed using Chromeleon 7.2 software. Molar ratios were calculated according to their respective calibration equations, which take into account the different electrochemical response factors of individual monosaccharides.

[0045] ⑥ Fourier transform infrared spectroscopy (FT-IR) Fourier transform infrared spectroscopy analysis was performed on a Nicolet iZ10 spectrophotometer (Thermo Fisher Scientific, USA). The sample (2 mg) was prepared as a potassium bromide tablet (200 mg), and the scanning range was 4000–400 cm⁻¹. -1 .

[0046] ⑦ Scanning electron microscopy (SEM) analysis The surface morphology of CGP-A was observed using a scanning electron microscope (JCM-7000 NeoScope, Japan). After gold sputtering coating, the target sample was observed at magnifications of 500×, 2000×, and 15000× using an electron beam accelerating voltage of 5.0 kV.

[0047] ⑧ Glycosidic bond analysis The glycosidic bond linkage mode of CGP-A was characterized by methylation analysis according to the partially methylated aldose acetates (PMAAs) method. The procedure is briefly described below: 1 mg of CGP-A was dissolved in 1 mL of dimethyl sulfoxide (DMSO), followed by the addition of 30 mg of sodium hydroxide (NaOH) and incubation for 30 min. Methylation was initiated by adding 250 μL of iodomethane (CH3I) and reacting for 1 h under a nitrogen atmosphere and in the dark, followed by the addition of another 250 μL of CH3I and a further 1 h reaction. The reaction was terminated with 1 mL of water, and the methylated product was extracted with 2 mL of dichloromethane (CH2Cl2). The organic phase was washed three times with water and dried under a nitrogen stream.

[0048] The dried methylated sample was then hydrolyzed with 1 mL of 2 M trifluoroacetic acid (TFA) at 121 °C for 120 min. After evaporation at 30 °C, the hydrolysis product was reduced with 1 mL of freshly prepared 1 M sodium borodeuteride (NaBD4, soluble in ammonia) under magnetic stirring at room temperature for 2.5 h. The reduction reaction was terminated with 300 μL of acetic acid and dried under a nitrogen stream. To ensure complete removal of borates, the residue was washed twice with 2 mL of 5% (v / v) methanol-acetic acid solution at 40 °C and dried, followed by two washes with methanol. Then, acetylation was carried out with 1.5 mL of acetic anhydride (Ac2O) at 100 °C for 2.5 h. The resulting partially methylated aldose acetates (PMAAs) were extracted with 1 mL of CH2Cl2, washed three times with water, and the organic layer was collected for gas chromatography-mass spectrometry (GC-MS) analysis.

[0049] Gas chromatography-mass spectrometry (GC-MS) analysis was performed on an Agilent 7890A-5977B system equipped with an HP-5MS capillary column (30 m × 0.25 mm × 0.25 µm). Helium was used as the carrier gas at a flow rate of 1.0 mL / min. The column oven temperature program was as follows: 50 °C for 1.0 min, increased to 130 °C at 50 °C / min, then increased to 230 °C at 3 °C / min and held for 2 min. The injection port temperature was 260 °C, and the split ratio was 10:1. Mass spectrometry analysis was performed using an electron impact (EI) ion source with an electron energy of 70 eV. The ion source temperature and quadrupole temperature were maintained at 230 °C and 150 °C, respectively. Mass spectra were recorded in full scan mode (m / z 30–600). Connection types were identified by comparing mass fragments of partially methylated aldose acetates (PMAAs) with the CCRC database and standard reference data.

[0050] 9. Nuclear Magnetic Resonance (NMR) Analysis To achieve complete deuteration exchange, the CGP-A sample (30 mg) was dissolved in 0.5 mL of deuterated water (D₂O) and lyophilized three times. The sample was then redissolved in 0.5 mL of D₂O. ¹H, ¹³C, ¹³C DEPT-135, ¹H-¹H COSY, HSQC, NOESY, TOCSY, and HMBC NMR spectra were acquired using a Bruker 600 MHz NMR spectrometer (Bruker GmbH, Germany) equipped with a cryogenic probe. The resulting proton and carbon chemical shifts were used to elucidate the glycosidic bond linkage sequence and structural features of CGP-A.

[0051] Structural characterization results: (1) The gradient elution curve for separation and purification of cellulose using the DEAE-52 column is shown below. Figure 1 As shown in the figure, the component eluted with 0 M NaCl solution is the main peak. This component was collected and purified using a Sephadex G-100 column. The resulting elution curve and photograph of the purified CGP-A powder are shown in the figure. Figure 2 As shown in the figure, the elution curve exhibits a single, symmetrical, and sharp peak, indicating that the target polysaccharide has high homogeneity and purity. After freeze-drying, the purified CGP-A appears as a white, loose, and fluffy powder.

[0052] (2) Preliminary results of CGP-A characterization are as follows Figure 3 As shown, Figure 3 In the image, (A) is the UV-Vis spectrum, (B) is the HPGPC spectrum, (C) is the ion chromatogram of the mixed standard and CGP-A, (D) is the FT-IR spectrum, (E) is the SEM image of CGP-A at different magnifications, and (F) is the GC-MS chromatogram.

[0053] Chemical composition analysis showed that CGP-A consisted of 95.23% total sugar and 0.82% uronic acid, with no protein detected. The absence of absorption peaks at 260 nm or 280 nm in the ultraviolet spectrum confirmed that CGP-A does not contain nucleic acids or proteins. Figure 3 HPGPC analysis showed that CGP-A exhibited a single, sharp, and symmetrical peak, with a residence time of 16.535 minutes. Figure 3 The presence of B in the figure indicates its high homogeneity and purity. Based on the standard calibration curve, the weight-average molecular weight (Mw) of CGP-A was calculated to be 6.722 kDa. Furthermore, monosaccharide composition analysis showed that CGP-A consists of fructose and glucose in a molar ratio of 5.25:1 (…). Figure 3 C). The FT-IR spectrum of CGP-A shows typical polysaccharide characteristic absorption bands (C). Figure 3 (D in the text). 3396.71 cm -1The strong and broad peaks at 2933.15 and 2891.71 cm⁻¹ are attributed to the OH stretching vibration. -1 The absorption bands at these locations correspond to the stretching vibrations of the methylene (-CH2-) and methine (-CH-) groups, respectively. 1646.71 cm⁻¹ -1 The signal at these locations originates from OH bending vibrations, indicating the presence of bound water in the polysaccharide matrix. (1456.91 and 1418.55 cm⁻¹) -1 The peak at 1338.19 cm⁻¹ belongs to the shear vibration of -CH₂-, while the peak at 1338.19 cm⁻¹ belongs to the shear vibration of -CH₂-. -1 The spectral bands at 1273.34 and 1216.97 cm⁻¹ are attributed to CH bending. -1 Asymmetric stretching vibrations of COC were observed at 1133.13 cm⁻¹. -1 1029.85 cm -1 The peaks at these locations are correlated with the angular vibrations of the OH group in alcohols. Notably, the peaks at 933.28 and 869.19 cm⁻¹ are also significant. -1 The characteristic absorption band at this location indicates that both β- and α-glycosidic bonds exist in CGP-A.

[0054] SEM imaging revealed the surface morphology of CGP-A ( Figure 3 (E in the image). At lower magnification, the polysaccharide exhibits an irregular, smooth, sheet-like stacked morphology with a porous structure, reflecting a highly ordered interchain hydrogen bond network. At higher magnification, the surface reveals fine cracks and irregular protrusions. The formation of these microcracks may be related to dehydration and drying-induced stress during sample preparation.

[0055] Figure 3 F in the figure shows the total ion current chromatography (TIC) chromatogram of the PMAA derivative. Specific glycosidic bond linkage modes can be clearly identified by cross-referencing its retention characteristics and mass spectrometry fragmentation patterns with standard indicators. The methylation analysis results of CGP-A are shown in Table 1: Table 1. Methylation analysis results of CGP-A

[0056] As shown in Table 1, CGP-A has four different linking bonds: 2-Fruf, 1,2-Fruf, 1,6-Glcp, and 1,2,6-Fruf. Specifically, the 2-Fruf residues are identified by a double peak at 15.55 and 15.77 minutes, with a molar ratio of 15.84%. The major backbone linking bond, 1,2-Fruf, exhibits a characteristic double-shoulder peak at 19.78 and 19.88 minutes, accounting for 71.36% of the total carbohydrate content. In addition, the 1,6-Glcp residues (20.76 minutes) and a minor branch point, 1,2,6-Fruf (23.62 minutes), account for 7.10% and 5.70%, respectively.

[0057] (3) NMR analysis of CGP-A Based on the above research data, CGP-A is a fructan, and the obtained spectrum is consistent with the characteristics of fructans. To accurately determine the structural architecture of CGP-A, NMR spectra were obtained, such as... Figure 4 As shown, Figure 4 In the middle, (A) is 1 H NMR spectrum, (B) is 13 C10 NMR spectrum, (C) is 13 C is the DEPT-135 NMR spectrum, (D) is the COSY spectrum, (E) is the HSQC spectrum, (F) is the NOESY spectrum, (G) is the TOCSY spectrum, (H) is the HMBC spectrum, and (I) is a schematic diagram of the glycosidic bond linkage mode in CGP-A.

[0058] Since fructose is a ketose, its anomeric center is located at C-2, a position without a attached proton; therefore, in 1 No anomeric proton resonances were observed in the 1H NMR spectrum. The proton signals of the D-Fruf residues (H-1, H-3, H-4, H-5, and H-6) were mainly distributed in the δ 3.60–4.30 range, which is consistent with the typical NMR spectrum of inulin-type fructans. Figure 4 As shown in A, 1 The H NMR signal is concentrated between δ 3.0 and 5.5. In the anomeric region (δ 4.3–5.4), a relatively weak signal is observed only at δ 5.43, which, according to literature, is attributed to the anomeric proton of the (α-D-Glcp-(1→) residue. 13 C NMR spectrum ( Figure 4In residue B), a significant anomeric carbon signal was observed at δ 103–104, corresponding to C-2 of the D-Fruf residue. The resonance peak at δ 92.12 was attributed to the anomeric carbon (C-1) of the →6)-α-D-Glcp-(1→ residue. Based on the molar ratios obtained by GC-MS, the signals at δ 103.18, 103.02, and 103.64 were attributed to C-2 of residues A, B, and C, respectively. Furthermore, the signals at δ 60–63, 60–64, 74–76, 76–78, and 80–82 were attributed to C-1, C-6, C-4, C-3, and C-5 positions of the D-Fruf residue, respectively. 13 C DEPT-135 spectrum ( Figure 4 The C-1 residue in the D-Fruf spectrum showed a negative signal (inverted peak) between δ 60 and 65, corresponding to the methylene group (CH2), which confirmed the presence of C-1 and C-6 in the D-Fruf residues. The disappearance of the carbon signal between δ 103 and 104 in the DEPT-135 spectrum further confirmed that they are quaternary carbons (C-2), which is consistent with the structural characteristics of fructan.

[0059] The anodic signal at δ 5.43 / 92.12 (H-1 / C-1) is attributed to residue →6)-α-D-Glcp-(1→). Starting from residue D at H-1 at δ 5.43, the 1H-1H COSY spectrum was used ( Figure 4 The H-2 to H-6 signals were identified sequentially from residue D. The chemical shifts of H-2 to H-6 of residue D were assigned δ 3.54, 3.76, 3.46, 3.83, and 3.71 / 3.63, respectively. The corresponding carbon shifts (C-2 to C-6) were determined by HSQC spectroscopy (HSQC). Figure 4 The values ​​of δ (E) were determined to be 71.20, 72.38, 69.15, 72.07, and 62.52, respectively. This was achieved by integrating 1H-1H COSY (… Figure 4 D in the middle) and HSQC ( Figure 4In the COSY spectrum, the C-1 signals at δ 62.51, 60.12, and 62.51 are attributed to →1)-β-D-Fruf-(2→ (residue A), β-D-Fruf-(2→ (residue B, terminal), and →1,6)-β-D-Fruf-(2→ (residue C, branch), respectively), and their corresponding H-1 signals are identified at δ 3.96 / 3.68, 3.94 / 3.71, and 3.94 / 3.63, respectively. The C-3 signals of residues A, B, and C are observed at δ 76.92, 76.62, and 76.72, respectively, and their associated H-3 signals are located at δ 4.25, 4.19, and 4.24 in the HSQC spectrum. The H-4 signals of residues A, B, and C are determined by the δ 160.61 in the COSY spectrum. The cross-peaks at 4.25 / 4.10, 4.19 / 4.11, and 4.24 / 4.11 were identified and subsequently assigned to their respective C-4 signals. NOESY ( Figure 4 F) and TOCSY ( Figure 4 The correlation of G in the data further validated the complete proton assignment, and the final 13C assignment of all residues is summarized in Table 2.

[0060] Table 2. Chemical shift assignments (δ) of CGP-A for 1H and 13C NMR.

[0061] Using HMBC spectroscopy ( Figure 4 The correlation at δ 5.43 / 103.64 (D-H1 / C-C2) elucidated the sequence and glycosidic bond connections between residues. The correlation at δ 5.43 / 103.64 (D-H1 / C-C2) indicates that the O-1 of residue D is linked to the C-2 of residue C. The cross-peak at δ 3.94 / 103.18 (C-H1 / C-C2) suggests a connection between the O-1 of residue C and the C-2 of residue A, while the signal at δ 3.94 / 103.64 (C-H1 / C-C2) indicates a C-1 to C-2 connection within residue C. Furthermore, the correlations at δ 3.68 / 103.18 (A-H1 / A-C2) and δ 3.68 / 103.02 (A-H1 / B-C2) demonstrate the connections of residue A to itself and the terminal residue B, respectively. Based on these results, combined with monosaccharide composition and methylation data, CGP-A was identified as a novel branched inulin-type fructan. Its main chain is primarily composed of →1)-β-D-Fruf-(2→ and →6)-α-D-Glcp-(1→) residues, interspersed with →1,6)-β-D-Fruf-(2→) residues serving as branch points. The side chain consists of terminal β-D-Fruf-(2→) units attached to the C-6 position of the furanofructose residues in the main chain. Figure 4 (I in the middle).

[0062] Performance testing The biological activities of the obtained Cichorium glandulosum polysaccharide were tested by the following methods: ① Animal experiments Ten 7-week-old male db / m mice were designated as the normal control group (db / m). Sixty 7-week-old male db / db mice were randomly divided into a model group (db / db), a positive control group (db / db + liraglutide, 0.25 mg / kg, Novo Nordisk, Denmark), a high-dose CGP-A group (db / db + CGP-AH, 200 mg / kg), and a low-dose CGP-A group (db / db + CGP-AL, 100 mg / kg) (n = 10). Liraglutide is a glucagon-like peptide-1 (GLP-1) receptor agonist approved by the US Food and Drug Administration (FDA) and is clinically used to treat type 2 diabetes (T2DM) and manage obesity. As a positive control, it was administered at a pharmacologically equivalent dose of 0.25 mg / kg. After a one-week adaptation period, the mice in the CGP-A treatment groups were given CGP-A dissolved in sterile saline by oral gavage daily. The mice in the positive control group were injected subcutaneously with liraglutide daily. The intervention continued for 8 weeks, during which all animals had free access to food and water. Body weight and fasting blood glucose (FBG) were recorded regularly throughout the experiment. All male db / m and db / db mice were purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd. (Nanjing, China; license number: SCXK (Su) 2023-0009) and were housed under specific pathogen-free (SPF) conditions with a 12-hour light / dark cycle, a temperature of 22 ± 2°C, and a humidity of 50 - 60%. The mice had free access to standard rodent feed and water. All experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals. All animal procedures were approved by the Institutional Animal Care and Use Committee of Xinjiang Medical University, Xinjiang Uygur Autonomous Region (IACUC-20251115-30).

[0063] ② Body composition analysis and grip strength test A Bruker minispec LF90II body composition analyzer (Bruker, Karlsruhe, Germany) was used to analyze body composition (lean body mass and fat mass). To evaluate muscle function, a digital dynamometer was used to quantify grip strength. Specifically, when the mouse grasped the metal grid with its front paws, its tail was pulled horizontally backward at a constant speed until it let go. The peak force (g) was recorded.

[0064] ③ Glucose homeostasis test To assess oral glucose tolerance (OGTT), mice were fasted for 12 hours before oral glucose administration (2.0 g / kg). Blood glucose concentrations were monitored at 0, 30, 60, 90, and 120 minutes after gavage. For the insulin tolerance test (ITT), mice were fasted for 4 hours before intraperitoneal injection of insulin (0.75 U / kg). Blood glucose levels were measured at 0, 30, 60, 90, and 120 minutes after injection. The area under the curve (AUC) for both tests was calculated.

[0065] ④ Biological sample collection After the final intervention, mice were fasted overnight, anesthetized, and blood was collected via the retroorbital sinus to separate serum. The liver, skeletal muscles (gastrocnemius and soleus), epididymal adipose tissue (EAT), perirenal adipose tissue (PAT), mesenteric adipose tissue (MAT), pancreas, and colon were carefully dissected, weighed, and stored at -80°C or immersed in 4% paraformaldehyde for histological examination.

[0066] ⑤ Biochemical analysis Serum biochemical parameters, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBiL), total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and free fatty acids (FFA), were quantified using commercially available assay kits. Serum insulin (INS) concentration was also measured using an enzyme-linked immunosorbent assay (ELISA) kit. All tests were performed strictly according to the manufacturer's protocols.

[0067] ⑥ Histological and immunofluorescence analysis Hematoxylin and eosin (H&E) staining was performed on collected liver, colon, gastrocnemius muscle, and adipose tissue (MAT, PAT, EAT). The cross-sectional area (CSA) of adipocytes and muscle fibers was then quantified. Further specialized histological techniques, including Oil Red O staining, were used to visualize lipid deposition in the liver and muscle tissues. Additionally, the ultrastructural morphology of muscle fibers and mitochondria was analyzed using transmission electron microscopy (TEM).

[0068] ⑦ Western blot analysis Protein expression levels of myosin heavy chain I (MyHC I), myogenic differentiation factor (MyoD), muscle ring finger protein 1 (MuRF1), and atrophic F-box protein (Atrogin-1) were detected by Western blotting. Protein abundance was normalized to β-actin levels, and band intensity was quantified using ImageJ software.

[0069] ⑧Statistical Analysis Statistical analysis and data visualization were performed using IBM SPSS Statistics 23.0 (IBM, USA). Experimental values ​​are expressed as mean ± standard deviation (SD). Student's t-test was used to assess comparisons between two groups, while differences among multiple groups were determined using one-way ANOVA followed by Tukey's post-hoc test. A p-value <0.05 was considered statistically significant.

[0070] The test results are as follows: (a) CGP-A improves insulin resistance in db / db mice To evaluate the therapeutic potential of CGP-A, db / db mice were administered two doses of this polysaccharide or liraglutide for 8 weeks. The results of CGP-A reducing insulin resistance in db / db mice were as follows: Figure 5 As shown, Figure 5 In the figures, (A) is a graphical overview of the animal study protocol and treatment schedule; (B) shows fasting blood glucose levels monitored from week 8 to week 16, and the final fasting blood glucose level at week 16; (C) shows serum insulin levels measured at the end of the study; (D) shows the HOMA-IR index; (E) shows the oral glucose tolerance test (OGTT) and the corresponding area under the curve (AUC); (F) shows the insulin tolerance test (ITT) and the corresponding area under the curve (AUC); and (G) shows representative H&E staining of pancreatic islet tissue (scale bar = 100 μm). Data are expressed as (x ± SD) (n = 6 or 10). Compared with the db / m group, △△ P<0.01; compared with the db / db group, Compared with the liraglutide group, ## P<0.01, #P<0.05.

[0071] like Figure 5 As shown in B, the db / db mice in the model group exhibited persistent and severe hyperglycemia (>25.0 mmol / L) throughout the study period. Although administration of CGP-A failed to restore blood glucose to normal levels, it resulted in a significant dose-dependent reduction in fasting blood glucose (FBG) compared to the untreated db / db group (P<0.01). Specifically, CGP-AH showed a more pronounced hypoglycemic effect than CGP-AL, while liraglutide remained the most effective treatment for lowering fasting blood glucose throughout the intervention period. Furthermore, the db / db mice exhibited severe hyperinsulinemia and a sharply elevated HOMA-IR index, indicating severe insulin resistance. Figure 5(C, D in the original text). CGP-A intervention, especially at high doses, significantly reduced serum insulin levels and the HOMA-IR index (P<0.01). The effect of CGP-A on systemic glucose management was further evaluated using the oral glucose tolerance test (OGTT) and the insulin tolerance test (ITT). In the OGTT ( Figure 5 Compared with the model group, CGP-AH significantly suppressed glycemic shift and enhanced the hypoglycemic response to exogenous insulin (P<0.01). Admittedly, although CGP-A significantly improved these parameters, its recovery effect was not as pronounced as that of the liraglutide group. Consistent with these metabolic improvements, H&E staining of the pancreas (…) Figure 5 The results (G) showed that hypertrophic and fragmented islets in db / db mice were partially restored after CGP-A administration, with the CGP-AH group maintaining better islet structural integrity than the CGP-AL group. Overall, these results indicate that although CGP-A is less effective at lowering blood glucose than liraglutide, it can effectively reduce insulin resistance and protect pancreatic function.

[0072] (ii) CGP-A alleviates lipid metabolism disorders and obesity in db / db mice CGP-A alleviated lipid metabolism disorders and obesity in db / db mice, as shown in the following results. Figure 6 As shown. Figure 6 In the table, (A) shows the weight changes from week 8 to week 16 and the final weight at week 16; (B) shows the fat / body weight ratio measured by TD-NMR; (C) shows the lean body mass / body weight ratio measured by TD-NMR; (D) shows the weight of epididymal white adipose tissue (PAT) normalized to body weight; (E) shows the weight of inguinal white adipose tissue (EAT) normalized to body weight; (F) shows the serum total cholesterol (TC) level; (G) shows the triglyceride (TG) level; (H) shows the low-density lipoprotein cholesterol (LDL-C) level; (I) shows the high-density lipoprotein cholesterol (HDL-C) level; (J) shows the free fatty acid (FFA) level; and (K) shows representative H&E staining (scale bar = 100 μm) and corresponding mean adipocyte cross-sectional area (CSA) of epididymal white adipose tissue (MAT), epididymal white adipose tissue (PAT), and inguinal white adipose tissue (EAT). Data are expressed as (x ± SD) (n = 6 or 10). Compared to the db / m group, △△ P<0.01; compared with the db / db group, Compared with the liraglutide group, ## P<0.01, # P<0.05.

[0073] like Figure 6As shown in Figure A, the db / db model group exhibited rapid and sustained weight gain (P < 0.01 compared to the db / m group). CGP-A intervention significantly suppressed weight gain from week 12, resulting in a significant reduction in endpoint weight compared to the untreated db / db group. TD-NMR analysis further confirmed that CGP-A treatment significantly reduced the fat / body weight ratio while preserving the lean body mass ratio. Figure 6 (B, C). Consistent with improved body composition, administration of CGP-A significantly reduced the weight of epididymal white adipose tissue (PAT) and inguinal white adipose tissue (EAT). Figure 6 (D, E in the text). Serum lipid profiles showed that db / db mice suffered from severe lipid metabolism disorders, which were alleviated by CGP-A in a dose-dependent manner. Figure 6 Specifically, CGP-A treatment significantly reduced levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and free fatty acids (FFA), while significantly increasing high-density lipoprotein cholesterol (HDL-C) levels (P<0.05). Although liraglutide exhibited the strongest lipid-regulating ability, CGP-AH was comparable in its effectiveness in reducing triglyceride (TG) and free fatty acid (FFA) levels. Histological analysis of white adipose tissue ( Figure 6 The results showed that CGP-A effectively attenuated adipocyte hypertrophy in the epididymal white adipose tissue (MAT), epididymal white adipose tissue (PAT), and inguinal white adipose tissue (EAT). Quantitative analysis of adipocyte cross-sectional area (CSA) confirmed that CGP-A intervention significantly reduced adipocyte size in these adipose deposits (P<0.01). Overall, these findings indicate that CGP-A can effectively alleviate systemic obesity and correct lipid metabolism disorders in db / db mice.

[0074] (III) CGP-A reduces hepatic steatosis and muscle atrophy in db / db mice This invention evaluated the protective effects of CGP-A on liver and muscle tissue to assess its role in obesity-related diseases. The results of CGP-A alleviating hepatic steatosis and muscle atrophy in db / db mice are as follows: Figure 7 As shown, Figure 7In the images, (A) shows a representative photograph of the liver, H&E staining of liver tissue, and Oil Red O staining (scale bar = 100 μm); (B) shows liver weight / body weight; (C) shows serum ALT level; (D) shows serum AST level; (E) shows serum TBIL level; (F) shows grip strength; (G) shows body weight-normalized gastrocnemius muscle weight; (H) shows body weight-normalized soleus muscle weight; (I) shows representative H&E staining (scale bar = 100 μm), transmission electron microscopy (scale bar = 5.0 μm), and Oil Red O staining (scale bar = 100 μm) of the gastrocnemius muscle; (J) shows the average cross-sectional area of ​​muscle fibers; (K) shows the immunoblotting analysis of MyHC I, MyoD, Atrogin-1, and MuRF1 in the gastrocnemius muscle; and (L) shows the quantification of protein expression. Data are expressed as (x ± SD) (n = 6 or 10). Compared with the db / m group, △△ P<0.01; compared with the db / db group, Compared with the liraglutide group, ## P<0.01, # P<0.05.

[0075] like Figure 7 As shown in Figure A, the livers of db / db model mice appeared pale and enlarged, and H&E and Oil Red O staining revealed severe hepatic steatosis and lipid droplet accumulation. CGP-A treatment, especially in the CGP-AH group, significantly reduced liver weight. Figure 7 (B) and reduced lipid deposition. Consistently, the sharply elevated serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) levels in the db / db group were significantly reduced after CGP-A administration (P<0.01, B). Figure 7 The C~E values ​​indicate that liver function has improved.

[0076] Regarding muscle health, db / db mice exhibit typical symptoms of SO, including a significant decrease in grip strength ( Figure 7 The ratio of F in the gastrocnemius muscle to the soleus muscle decreased. Figure 7 G,H). CGP-A intervention, especially CGP-AH, significantly increased grip strength and partially restored muscle mass. Histological analysis ( Figure 7 The results (I) showed that the myofiber fibers of db / db mice were sparse and small, with high intramuscular lipid infiltration (Oil Red O staining) and ultrastructural damage to mitochondria / sarcomeres (transmission electron microscopy). CGP-A treatment effectively increased the average myofiber cross-sectional area (P<0.01, Figure 7The J in the formula reduced lipid accumulation and preserved the integrity of myofibrils and mitochondria. To further explore the molecular mechanism, the expression of markers of muscle synthesis and degradation was analyzed. Figure 7 In the db / db model group, the expression of myogenic markers MyHC I and MyoD was significantly decreased, while the levels of atrophy-related ubiquitin ligases Atrogin-1 and MuRF1 were increased (P<0.01). Notably, CGP-AH treatment significantly upregulated the expression of MyHC I and MyoD while inhibiting the levels of Atrogin-1 and MuRF1. These results indicate that CGP-A effectively alleviates hepatic steatosis and reduces muscle atrophy in db / db mice by balancing muscle protein synthesis and degradation.

[0077] In summary, this invention systematically characterized the structure of CGP-A, a novel branched-chain inulin-type fructan derived from chicory, and demonstrated its significant efficacy in improving SO in db / db mice. These findings elucidate the structure and activity of CGP-A and position it as a novel polysaccharide for the holistic management of SO and related metabolic syndromes.

[0078] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A chicory polysaccharide, characterized in that, It has the structure shown in Equation I: Formula I; In Equation I, a = 5~20, b = 5~20, and a+b = 25~34.

2. The chicory polysaccharide according to claim 1, characterized in that, The molecular weight of the chicory is 5kDa~8kDa.

3. The method for isolating and purifying chicory polysaccharides according to claim 1 or 2, characterized in that, Includes the following steps: The root powder of *Chicorylia cuminata* was subjected to degreasing and decolorization treatment to obtain pretreated *Chicorylia cuminata* root powder. The pretreated chicory root powder was subjected to water extraction and alcohol precipitation, and the precipitate was collected to obtain a crude polysaccharide extract. The crude polysaccharide extract was dissolved in water, decolorized using AB-8 macroporous resin, and the water eluent was collected to obtain the decolorized crude polysaccharide extract. The proteins in the decolorized crude polysaccharide were removed using Sevag reagent, and the preliminarily purified polysaccharide extract was obtained by dialysis. The preliminary purified polysaccharide extract was separated and purified sequentially using a DEAE-52 cellulose column and a Sephadex G-100 column to obtain chicory polysaccharide.

4. The separation and purification method according to claim 3, characterized in that, The degreasing and decolorizing treatment involves sequentially soaking the food in petroleum ether and ethanol for 18-24 hours each.

5. The separation and purification method according to claim 3, characterized in that, The liquid-to-solid ratio of the water extraction was 30 mL / g, the temperature was 85℃, the water extraction was performed twice, and the extraction time for each cycle was 3 hours. The alcohol precipitation temperature is 4°C, the alcohol precipitation is performed twice, and the time for each precipitation is 8~24 hours.

6. The separation and purification method according to claim 3, characterized in that, The Sevag reagent is a mixture of chloroform and n-butanol, with a volume ratio of chloroform to n-butanol of 4:

1. The molecular weight cutoff for the dialysis is 3.5 kDa.

7. The separation and purification method according to claim 3, characterized in that, The dimensions of the DEAE-52 cellulose column are 2.6 cm × 40 cm; The eluent used in the DEAE-52 cellulose column was water and 0.1-0.5 M sodium chloride solution, with an elution rate of 1.0 mL / min.

8. The separation and purification method according to claim 3, characterized in that, The Sephadex G-100 column measures 1.6 cm × 50 cm; The Sephadex G-100 column uses water as the eluent and has an elution rate of 0.3 mL / min.

9. The use of the chicory polysaccharide according to claim 1 or 2 or the chicory polysaccharide obtained by any one of claims 3 to 8 in the preparation of anti-sarcopenic obesity drugs.

10. A drug for treating sarcopenic obesity, characterized in that, It includes pharmaceutical excipients and active ingredients, wherein the active ingredients include the chicory polysaccharide as described in claim 1 or 2 or the chicory polysaccharide obtained by any one of claims 3 to 8.

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