Sulfated zingiber officinale leaf polysaccharide, and preparation method and use thereof

Abstract

The application discloses sulfated Zingiber strioatum leaf polysaccharide, the total sugar content of the sulfated Zingiber strioatum leaf polysaccharide is 15.52 ± 4.99, the uronic acid content is 16.8 ± 3.26, the sulfate group content is 18.06 ± 0.93, and the sulfate group degree of substitution is 1.67; the sulfated Zingiber strioatum leaf polysaccharide belongs to heteropolysaccharide and is composed of fucose (0.98%), rhamnose (1.98%), arabinose (16.1%), galactose (14.04%), glucose (27.71%), xylose (28%), galacturonic acid (10.09%) and glucuronic acid (1.1%); the average molecular weight of the sulfated Zingiber strioatum leaf polysaccharide is 238 kDa, the particle size is 533.4 nm, and the potential is -53.9 mV. The sulfated Zingiber strioatum leaf polysaccharide provided by the application has excellent antioxidant activity and has inhibitory activity on α -glucosidase, α -amylase and pancreatic lipase, and can be applied to the preparation of antioxidant, sugar-reducing and lipid-regulating functional products.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry and biomedicine technology, specifically relating to a sulfated ginger leaf polysaccharide and its preparation method, as well as its application in the preparation of products with antioxidant, hypoglycemic and lipid-regulating functions. Background Technology

[0002] Ginger with Firewood Zingiber mioga Zingiber officinale (also known as ginger ginseng), a plant belonging to the ginger family (Zingiberaceae), has extremely high nutritional value in its flower buds and is hailed as "Asian ginseng." The medicinal history of Zingiber officinale has a long history, first recorded in the Eastern Jin Dynasty's supernatural tales, *Sou Shen Ji*. Subsequently, ancient medical books such as *Ming Yi Bie Lu* and *Ben Cao Gang Mu* recorded Zingiber officinale, gradually forming traditional medicinal theories regarding its effects on promoting blood circulation and regulating menstruation, relieving cough and phlegm, reducing swelling and detoxifying, and strengthening the stomach and resolving stagnation. Food and Health (2023, 35: 64). Modern pharmacological studies have confirmed that torch ginger is rich in various bioactive components such as terpenes, flavonoids, and polysaccharides. Among them, curcumin is a high-value compound with a very high market price. These active components endow torch ginger with significant pharmacological effects such as analgesia, anti-inflammation, immunomodulation, and anti-tumor activity. Molecules , 2022, 27:2826; Journal of Food Composition and Analysis ,2023,116:105050).

[0003] As an economic crop with both edible and medicinal uses, *Gynostemma pentaphyllum* is widely cultivated in twelve provinces in Southwest and East my country, yielding significant economic benefits. For example, *Gynostemma pentaphyllum* produced in Rudong, Jiangsu Province, is a national geographical indication agricultural product, with an average yield of up to 20,000 yuan per mu (approximately 0.16 acres). China Jiangsu Network (August 3, 2021). However, ginger lily plants have large leaves and a high biomass ratio. Currently, most industrial models only utilize the flower buds, and the leaves are often discarded as agricultural waste after harvest. This not only causes a huge waste of resources but also becomes a bottleneck restricting the green and whole-plant utilization of ginger lily. At present, there are few reports on the high-value reuse of waste ginger lily leaves, and in-depth development is urgently needed to enhance the added value of the industry.

[0004] The applicant found in previous research that the extraction of polysaccharides from ginger leaves using an ultrasound-assisted microwave method yielded a yield of 6.22 ± 0.14%, and that the polysaccharides exhibited certain antioxidant and anticoagulant activities. Ultrasonics Sonochemistry , 2023, 101: 106718). However, natural ginger leaf polysaccharides are biological macromolecules, and their direct application often suffers from problems such as low bioavailability and difficulty in absorption by the body. Cell Investigation(2026, 2: 100063). Furthermore, existing research is mainly limited to the antioxidant and anticoagulant activities of *Zingiber officinale* leaf polysaccharides; no reports have been found regarding their pharmacological effects on metabolic diseases such as hypoglycemia and lipid regulation. Given that the potential medicinal value of *Zingiber officinale* leaf polysaccharides has not been fully explored, it is urgent to further enhance their activity and expand their application areas through structural modification. This is also of great significance for realizing the transformation of agricultural waste into valuable resources and increasing the added value of the industry. Summary of the Invention

[0005] The purpose of this invention is to further improve the antioxidant capacity of *Zingiber officinale* leaf polysaccharide and expand its application scope. This invention provides a sulfated *Zingiber officinale* leaf polysaccharide, which exhibits excellent antioxidant activity. Compared with the unsulfated *Zingiber officinale* leaf polysaccharide, it can efficiently scavenge DPPH free radicals (94.94 ± 3.64% at 0.6 mg / mL), hydroxyl free radicals (91.94 ± 2.64% at 7 mg / mL), and superoxide anion free radicals (91.24 ± 1.64% at 3 mg / mL). Simultaneously, it was surprisingly found that the sulfated *Zingiber officinale* leaf polysaccharide has the potential to lower blood sugar and regulate blood lipids, and can effectively inhibit… α -Glucosidase (at 10 mg / mL, the inhibition rate reached 74.07 ± 1.77%) α - Amylase (inhibition rate reached 68.27 ± 1.45% at 10 mg / mL) and pancreatic lipase (inhibition rate reached 81.19 ± 2.00% at 10 mg / mL).

[0006] According to a first aspect of the present invention, the present invention provides a sulfated ginger leaf polysaccharide, wherein the sulfated ginger leaf polysaccharide is obtained by sulfated ginger leaf polysaccharide with aminosulfonic acid; wherein the degree of sulfate substitution of the sulfated ginger leaf polysaccharide is 0.16 to 1.67.

[0007] According to a second aspect of the present invention, a method for preparing sulfated ginger leaf polysaccharide is provided, comprising the following steps: placing ginger leaf polysaccharide and aminosulfonic acid in an organic solvent and heating to react for a certain time; cooling to room temperature, quenching the reaction with an alkaline solution, adjusting the pH of the reaction solution to neutral, precipitating with ethanol to obtain a precipitate, redissolving the precipitate with distilled water, dialyzing, and freeze-drying to obtain sulfated ginger leaf polysaccharide with different degrees of substitution. Specifically, the steps may be: weighing 1.0 g of ginger leaf polysaccharide and dissolving it in 70 mL of... N , NDifferent masses of aminosulfonic acid were added to dimethylformamide, stirred and mixed, and then reacted at 80℃ for 3 h. After the reaction was completed, the mixture was cooled to room temperature, and the pH was adjusted to neutral using 4 mol / L NaOH solution. Anhydrous ethanol was then added for alcohol precipitation, and the precipitate was collected after standing. The precipitate was redissolved in water, placed in a dialysis bag, and dialyzed three times to remove residual solvent, small molecules, and inorganic salt impurities. After dialysis, the solution was freeze-dried to obtain sulfated ginger leaf polysaccharide samples with different degrees of substitution.

[0008] Preferably, the mass ratio of the ginger leaf polysaccharide to aminosulfonic acid is 1:1 to 1:8, and more preferably 1:4; Preferably, the sulfated ginger leaf polysaccharide has a sulfate group substitution degree of 0.16 to 1.67; Preferably, the mass ratio of the *Zingiber officinale* leaf polysaccharide to sulfamic acid is 1:4, and the degree of sulfate substitution of the obtained sulfated *Zingiber officinale* leaf polysaccharide is 1.67. In some embodiments, the prepared sulfated *Zingiber officinale* leaf polysaccharide has a total sugar content of 15.52 ± 4.99%, a uronic acid content of 16.8 ± 3.26%, and a sulfate content of 18.06 ± 0.93%; the obtained sulfated *Zingiber officinale* leaf polysaccharide is composed of fucose (0.98%), rhamnose (1.98%), arabinose (16.1%), galactose (14.04%), glucose (27.71%), xylose (28%), galacturonic acid (10.09%), and glucuronic acid (1.1%); the average molecular weight of the sulfated *Zingiber officinale* leaf polysaccharide is 238 kDa; the particle size of the sulfated *Zingiber officinale* leaf polysaccharide is 533.4 nm, and the potential is -53.9 mV; Preferably, the polysaccharide used in the preparation of sulfated ginger leaf polysaccharide is purified ginger leaf polysaccharide (i.e., purified ginger leaf polysaccharide), and the preparation method of the purified ginger leaf polysaccharide includes the following steps: 1) Extraction of crude polysaccharides from ginger leaves The leaves of *Ginger lanceolata* were dried, pulverized, and sieved to obtain *Ginger lanceolata* leaf powder. The powder was mixed with petroleum ether at a ratio of 1:10 (g / mL), defatted for 24 h, and then filtered. The resulting powder was dried again to obtain a pretreated sample. 20 g of the pretreated sample was accurately weighed and added to distilled water at a ratio of 1:30 (g / mL). After soaking for 4 h, ultrasonic-microwave synergistic extraction was performed: first, treatment was carried out at 60℃ and 500 W ultrasonic power for 10 min, followed by treatment at 500 W microwave power for 5 min. The extract was cooled and filtered, and the filtrate was concentrated under reduced pressure to 1 / 5 of its original volume. Three times the volume of anhydrous ethanol was added to the concentrate, and the mixture was precipitated at 4℃ for 12 h. The precipitate was collected and freeze-dried to obtain crude polysaccharide from *Ginger lanceolata* leaves, with an extraction rate of 6.34 ± 0.26%. Wherein, crude polysaccharide extraction rate (%) = m / M × 100; where m is the mass (g) of crude polysaccharide from ginger leaves and M is the mass (g) of defatted ginger leaf powder. 2) Preparation of purified polysaccharides from ginger leaves The crude polysaccharide from *Ginger oleracea* leaves was subjected to three deproteinization treatments using the Sevag method. The Sevag reagent was prepared by mixing chloroform and n-butanol at a volume ratio of 4:1. After removing residual Sevag reagent, the deproteinized solution was placed in a dialysis bag and dialyzed with distilled water for 72 h (changing the water every 12 h). The dialysate was then freeze-dried to obtain the deproteinized polysaccharide.

[0009] 100 mg of deproteinized polysaccharide was accurately weighed and dissolved in 20 mL of deionized water. The solution was then loaded onto a diethylaminoethyl (DEAE) cellulose-52 column (2.6 cm × 60 cm). 0.5 mol / L NaCl solution was used as the eluent, and the flow rate was controlled at 1 mL / min. 5 mL of eluent was collected from each tube. The eluent was analyzed using the phenol-sulfuric acid method. The eluent corresponding to the polysaccharide elution peak was collected, combined, concentrated, dialyzed, and freeze-dried to obtain purified polysaccharide from ginger leaves, named PWZMLs. The total sugar content of PWZMLs was determined to be 72.73 ± 1.34%. According to another aspect of the present invention, the present invention provides the use of the sulfated ginger leaf polysaccharide in the preparation of products with antioxidant, hypoglycemic, and lipid-regulating (triglyceride) functions; it can be used to prepare preparations such as tablets, capsules, and oral liquids.

[0010] This invention modifies *Ginger lanceolata* leaf polysaccharides by sulfation, preparing four kinds of sulfated *Ginger lanceolata* leaf polysaccharides. Compared with the prior art, the advantages of this invention are as follows: ① The introduction of sulfate groups altered the chemical characteristics of *Ginger indicum* leaf polysaccharide. After multiple activity comparison screenings, the free radical scavenging ability of sulfated polysaccharide SPWZMLs-3 was significantly enhanced compared with that of unmodified *Ginger indicum* leaf polysaccharide, and its in vitro antioxidant activity was substantially improved.

[0011] ②After sulfation modification, it was surprisingly found that the polysaccharide in ginger leaves had a positive effect on... α -glucosidase, α - The inhibitory effects on amylase and pancreatic lipase were enhanced; among the four sulfated modified products, SPWZMLs-3 showed better inhibitory effects on the above enzymes than the other modified products and the raw polysaccharide, and sulfated modification brought a clear activity gain to the ginger leaf polysaccharide.

[0012] ③SPWZMLs-3 has a loose, porous microstructure with interwoven fibers and sheets. This structure provides more substrate binding sites, which is beneficial for its in vitro activity. This polysaccharide can be compounded with pharmaceutically acceptable excipients to prepare tablets, capsules, and oral liquids, thus expanding the formulation applications of Ginger Leaf Polysaccharide. Attached Figure Description

[0013] Figure 1 The scavenging capacity of *Ginger oleracea* leaf polysaccharides (PWZMLs) and their sulfates (SPWZMLs-1 ~ 4) against DPPH free radicals; Figure 2 The scavenging capacity of *Ginger indicus* leaf polysaccharides (PWZMLs) and their sulfates (SPWZMLs-1~4) against hydroxyl radicals; Figure 3 The scavenging capacity of *Ginger oleracea* leaf polysaccharides (PWZMLs) and their sulfates (SPWZMLs-1~4) against superoxide anion free radicals; Figure 4 The effects of *Ginger oleracea* leaf polysaccharides (PWZMLs) and their sulfates (SPWZMLs-1~4) on... α -Inhibitory activity of glucosidase; Figure 5 The effects of *Ginger oleracea* leaf polysaccharides (PWZMLs) and their sulfates (SPWZMLs-1~4) on... α - Inhibitory activity of amylase; Figure 6 The inhibitory activity of ginger leaf polysaccharides (PWZMLs) and their sulfates (SPWZMLs-1~4) on pancreatic lipase; Figure 7. Monosaccharide composition chromatogram of SPWZMLs-3 (peaks in the standard: 1-fucose; 2-rhamnose; 3-arabinose; 4-galactose; 5-glucose; 6-xylose; 7-mannose; 8-fructose; 9-galacturonic acid; 10-glucuronic acid). Figure 8 Molecular weight distribution of SPWZMLs-3; Figure 9 Infrared spectrum of SPWZMLs-3; Figure 10 Scanning electron microscope image of SPWZMLs-3. Detailed Implementation

[0014] The leaves of the ginger were collected from the ginger planting base in Rudong County, Nantong City, Jiangsu Province. α -glucosidase, α - Amylase and pancreatic lipase were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; monosaccharide standards and T-series dextran standards were purchased from Sinopharm Chemical Reagent Co., Ltd. Monosaccharide composition was determined using a Dionex ICS-5000 ion chromatograph; infrared spectroscopy was performed using a PerkinElmer Spotlight 200i Fourier transform infrared microscope system; and microstructure was observed using a Hitachi Regulus 8100 ultra-high resolution field emission scanning electron microscope (SEM).

[0015] Example 1: Extraction of crude polysaccharides from ginger leaves

[0016] The leaves of *Ginger lanceolata* were dried, pulverized, and sieved to obtain *Ginger lanceolata* leaf powder. The powder was mixed with petroleum ether at a ratio of 1:10 (g / mL), defatted for 24 h, and then filtered. The resulting powder was dried again to obtain a pretreated sample. 20 g of the pretreated sample was accurately weighed and added to distilled water at a ratio of 1:30 (g / mL). After soaking for 4 h, ultrasonic-microwave synergistic extraction was performed: first, treatment was carried out at 60℃ and 500 W ultrasonic power for 10 min, followed by treatment at 500 W microwave power for 5 min. The extract was cooled and filtered, and the filtrate was concentrated under reduced pressure to 1 / 5 of its original volume. Three times the volume of anhydrous ethanol was added to the concentrate, and the mixture was precipitated at 4℃ for 12 h. The precipitate was collected and freeze-dried to obtain crude polysaccharide from *Ginger lanceolata* leaves, with an extraction rate of 6.34 ± 0.26%. Wherein, crude polysaccharide extraction rate (%) = m / M × 100; where m is the mass (g) of crude polysaccharide from ginger leaves and M is the mass (g) of defatted ginger leaf powder.

[0017] Example 2: Preparation of purified polysaccharides from ginger leaves

[0018] The crude polysaccharide from *Ginger oleracea* leaves was subjected to three deproteinization treatments using the Sevag method. The Sevag reagent was prepared by mixing chloroform and n-butanol at a volume ratio of 4:1. After removing residual Sevag reagent, the deproteinized solution was placed in a dialysis bag and dialyzed with distilled water for 72 h (changing the water every 12 h). The dialysate was then freeze-dried to obtain the deproteinized polysaccharide.

[0019] 100 mg of deproteinized polysaccharide was accurately weighed and dissolved in 20 mL of deionized water. The solution was then loaded onto a diethylaminoethyl (DEAE) cellulose-52 column (2.6 cm × 60 cm). 0.5 mol / L NaCl solution was used as the eluent, and the flow rate was controlled at 1 mL / min. 5 mL of eluent was collected from each tube. The eluent was analyzed using the phenol-sulfuric acid method. The eluent corresponding to the polysaccharide elution peak was collected, combined, concentrated, dialyzed, and freeze-dried to obtain purified *Gynostemma pentaphyllum* leaf polysaccharide, named PWZMLs. The total sugar content of PWZMLs was determined to be 72.73 ± 1.34%.

[0020] Example 3 Preparation of sulfated tamarisk leaf polysaccharide

[0021] Weigh 1.0 g of purified polysaccharide from ginger leaves (PWZMLs) and dissolve it in 70 mL of water. N , N Different masses of aminosulfonic acid (SA) were added to dimethylformamide, stirred and mixed, and then reacted at 80℃ for 3 h. After the reaction was complete, the mixture was cooled to room temperature, and the pH was adjusted to neutral using 4 mol / L NaOH solution. Anhydrous ethanol was then added for alcohol precipitation, and the precipitate was collected after standing. The precipitate was redissolved in water, placed in a dialysis bag, and dialyzed three times to remove residual solvent, small molecules, and inorganic salt impurities. After dialysis, the solution was freeze-dried to obtain sulfated ginger leaf polysaccharide samples with different degrees of substitution. According to the mass ratio of PWZMLs to SA, they were numbered sequentially as: SPWZMLs-1 (1:1), SPWZMLs-2 (1:2), SPWZMLs-3 (1:4), and SPWZMLs-4 (1:8).

[0022] Example 4: Compositional Analysis of Sulfated Firefly Leaf Polysaccharide

[0023] The total sugar content of the samples was determined using the phenol-sulfuric acid method (the standard equation for glucose is Y = 11.583X - 0.0121, Rg). 2= 0.9992); Protein content was determined using the Coomassie Brilliant Blue method (bovine serum albumin standard equation: Y = 8.8771X + 0.0081, R² = 0.9996); Glucuronic acid content was determined using the m-hydroxybiphenyl method (galacturonic acid standard equation: Y = 9.2643X - 0.0117, R² = 0.9994); Sulfate content was determined using the barium chloride-gelatin turbidimetric method (potassium sulfate standard equation: Y = 1.7571X - 0.0008, R² = 0.9997); The degree of sulfate substitution (DS) was calculated as: DS = 1.62 × S / (32 - 1.02 × S), where S is the percentage of sulfate content. The particle size and zeta potential of the samples were determined using a Zetasizer Nano ZS Malvern nanoparticle size and zeta potential distributor.

[0024] As shown in Table 1, sulfation modification reduced the total sugar content of *Pleurotus ostreatus* leaf polysaccharides (PWZMLs). This is because during the sulfation reaction, the sulfate groups replace the hydroxyl groups on the polysaccharide chains, altering the original structure of the polysaccharides and making some sugar residues undetectable by the phenol-sulfuric acid method. Simultaneously, unstable glycosidic bonds are easily broken during the reaction, and the resulting small-molecule oligosaccharides and monosaccharides are lost during dialysis purification, further reducing the total sugar content of the sample. Carbohydrate Polymers , 2021, 272:118526).

[0025] The protein content of the sulfation-modified sample was lower than that of PWZMLs. This is because the high temperature and acidic reagents of the sulfation reaction can denature and release bound proteins in the polysaccharide through hydrolysis. Simultaneously, sulfation modification breaks easily cleaved glycopeptide bonds, allowing the released free proteins to be removed in subsequent purification steps. International Journal of Biology Macromolecules , 2020, 159: 108-116).

[0026] After sulfation modification, the uronic acid content of sulfated polysaccharides is lower than that of PWZMLs. This may be because the sulfate group first binds to the hydroxyl group at the C2 or C3 position of the uronic acid, and through steric hindrance or charge repulsion, the activity of the carboxyl group is reduced, resulting in a decrease in content. Trends in Food Science & Technology (2018, 74: 147-157). After sulfation modification, the sulfate content of each sample was significantly increased, confirming that the sulfation modification reaction described in this invention can effectively achieve polysaccharide modification. Among them, the SPWZMLs-3 sample with a ratio of 1:4 had the highest sulfate content, which was 18.06 ± 0.93%, corresponding to a sulfate substitution degree of 1.67 ± 0.16.

[0027] The average particle size of PWZMLs was 1343 nm. After sulfation modification, the particle size of polysaccharides in all samples decreased significantly. The particle sizes of SPWZMLs-1, SPWZMLs-2, SPWZMLs-3, and SPWZMLs-4 were 976.6 nm, 535.5 nm, 533.4 nm, and 590.7 nm, respectively. The principle is that the introduction of negatively charged sulfate groups into the polysaccharide molecular chains increases the intramolecular electrostatic repulsion, promoting the depolymerization and breakage of the polysaccharide chains, resulting in uniform dispersion and ultimately reducing the particle size. Ultrasonics Sonochemistry (2024, 103: 106803), among which SPWZMLs-3 showed the most significant reduction in particle size. All polysaccharide samples had negatively charged molecular surfaces. The Zeta potentials of PWZMLs, SPWZMLs-1, SPWZMLs-2, SPWZMLs-3, and SPWZMLs-4 were -35.8 mV, -43.2 mV, -45.9 mV, -53.6 mV, and -52.7 mV, respectively. Compared to PWZMLs, the sulfated modified samples showed a significantly increased negative potential, mainly because the sulfate groups greatly increased the negative charge density on the polysaccharide molecular surface, and the conformational changes of the polysaccharides further exposed negative charge sites (…). Materials Science and Engineering (C, 2021, 128: 112338), among which SPWZMLs-3 showed the most significant increase in negative potential.

[0028] The particle size and zeta potential of polysaccharides directly affect their aqueous solution stability, solubility, and bioactivity. Appropriately reducing particle size and increasing zeta potential helps improve the solubility and solution stability of polysaccharides, thereby enhancing their bioactivity and delivery performance. International Journal of Biological Macromolecules (2023, 253: 126636). Among them, SPWZMLs-3 has excellent particle size characteristics, the highest surface negative potential, sulfate content and degree of substitution, and the best overall modified structural characteristics. Combined with existing research theories, this unique structural advantage makes SPWZMLs-3 have better potential biological activity and functional product application value.

[0029] Table 1. Compositional analysis results of PWZMLs and their sulfates

[0030] Note: Different lowercase letters in the same column indicate statistically significant differences. P < 0.05 or P < 0.01); ND indicates not detected.

[0031] Example 5: Determination of the antioxidant activity of sulfated ginger leaf polysaccharide

[0032] 1. DPPH free radical scavenging power

[0033] Transfer 2 mL of sample solutions of different concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 0.6 mg / mL) into test tubes, add 2 mL of 0.1 mmol / L DPPH-ethanol solution, mix well, and react in the dark for 30 min. Measure the absorbance at 517 nm (A1). Use anhydrous ethanol instead of DPPH-ethanol solution as a control, and record the absorbance as A2. Use anhydrous ethanol instead of the sample solution as a blank control, and record the absorbance as A0. Calculate the DPPH free radical scavenging rate using the following formula.

[0034] DPPH radical scavenging rate (%) = [(A0 – A1 + A2) / A0] × 100

[0035] like Figure 1 As shown, *Zingiber officinale* leaf polysaccharide and its sulfated derivative exhibited certain scavenging ability against DPPH free radicals in the concentration range of 0–0.6 mg / mL, and the scavenging activity gradually increased with increasing concentration. When the concentration reached 0.6 mg / mL, SPWZMLs-3 showed the highest scavenging rate, reaching 94.94 ± 3.64%. The IC50 of the sulfated polysaccharide… 50 The values ​​were all lower than those of PWZMLs, with SPWZMLs-3 having the lowest IC50 value for scavenging DPPH radicals. 50 The lowest value was 0.11 ± 0.02 mg / mL. These results indicate that sulfation modification enhances the scavenging ability of PWZMLs against DPPH radicals to some extent. This may be because the introduction of the sulfate group increases the number of hydrogen atom donors and strengthens the electron delocalization effect, thereby enabling the polysaccharide to scavenge DPPH radicals more efficiently.

[0036] 2. Hydroxyl radical scavenging power

[0037] Transfer 1 mL of sample solutions of different concentrations (1, 2, 3, 4, 5, 6, 7 mg / mL) into test tubes, and add 1 mL of 6 mmol / L ferrous sulfate solution, 1.0 mL of 6 mmol / L salicylic acid-ethanol solution, and 1.0 mL of 6 mmol / L hydrogen peroxide solution in sequence. Mix well and react at 37℃ for 30 min. Then measure the absorbance at 510 nm (A1). Use deionized water instead of hydrogen peroxide solution as a control group and measure the absorbance, which is recorded as A2. Use deionized water instead of the sample solution as a blank control and measure the absorbance, which is recorded as A0. Calculate the hydroxyl radical scavenging rate using the following formula.

[0038] Hydroxyl radical scavenging rate (%) = [(A0 – A1 + A2) / A0] × 100

[0039] like Figure 2As shown, within the concentration range of 0–7 mg / mL, sulfated polysaccharides exhibited significantly higher scavenging rates of hydroxyl radicals than PWZMLs, and the activity increased with increasing concentration. When the concentration reached 7 mg / mL, SPWZMLs-3 showed the highest scavenging rate, reaching 91.94 ± 2.64%. The IC50 of SPWZMLs-3 for scavenging hydroxyl radicals was [not specified in the original text]. 50 The value was 2.7 ± 0.32 mg / mL, significantly lower than that of PWZMLs and other sulfates. The results indicate that sulfation modification enhances the ability of PWZMLs to scavenge hydroxyl radicals to some extent. This may be because the oxygen atom in the sulfate group forms a hydrogen bond network with the hydroxyl groups of the polysaccharide, promoting proton transfer and charge separation, providing more active hydrogen atoms, and thus increasing the polysaccharide's ability to scavenge hydroxyl radicals.

[0040] 3. Superoxide anion radical scavenging power

[0041] Transfer 1 mL of sample solution of different concentrations (0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg / mL) into a test tube, add 3 mL of 0.05 mol / L Tris-HCl buffer, mix well, and incubate in the dark for 20 min; then add 1 mL of 25 mmol / L pyrogallol solution, continue incubation for 5 min, and then add 0.5 mL of 1 mol / L hydrochloric acid to terminate the reaction. Measure the absorbance at 325 nm (A1); use deionized water instead of Tris-HCl buffer and pyrogallol solution as a control group, and record the absorbance as A2; use deionized water instead of sample solution as a blank control group, and record the absorbance as A0; the superoxide anion radical scavenging rate is calculated according to the following formula.

[0042] Superoxide anion radical scavenging rate (%) = [1 – (A1 – A2) / A0] × 100

[0043] like Figure 3 As shown, within the concentration range of 0–3 mg / mL, sulfated polysaccharides significantly enhanced the scavenging rate of superoxide anion radicals, and their scavenging activity was higher than that of PWZMLs. At a concentration of 3 mg / mL, the scavenging rate of SPWZMLs-3 reached 91.24 ± 1.64%. The IC50 of SPWZMLs-3 for scavenging superoxide anion radicals was [not specified in the original text]. 50The value was 0.56 ± 0.02 mg / mL, significantly lower than that of PWZMLs and other sulfates. These results indicate that sulfation modification enhances the scavenging ability of PWZMLs against superoxide anion radicals to some extent. This may be because the strong electron-withdrawing effect of the sulfate group makes adjacent carbon atoms in the polysaccharide molecule more easily polarized, promoting electron transfer to superoxide anions, thereby enhancing the polysaccharide's scavenging ability against superoxide anions.

[0044] Example 6: Determination of the hypoglycemic activity of sulfated ginger leaf polysaccharide

[0045] 1. α -Glucosidase inhibitory activity

[0046] In a 96-well plate, 50 μL of test sample solutions with different mass concentrations (0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 mg / mL) were respectively mixed with 40 μL of 0.5 U / mL [amount missing]. α - Mix with glucosidase, vortex to mix well, and incubate at 37°C for 30 min; then add 100 μL of 4 mM 4-nitrophenyl- β - D The substrate solution of galactopyranoside (PNPG) was thoroughly mixed and incubated for another 10 min. The reaction was terminated by adding 50 μL of sodium carbonate solution, and the absorbance was measured at a wavelength of 405 nm and recorded as A1. α - The glucosidase inhibition rate is calculated according to the following formula.

[0047] α - Glucosidase inhibition rate (%) = [1 - (A1 – A2) / (A3 – A4)] × 100

[0048] In the formula: A2 represents none α - Absorbance of glucosidase, A3 is the absorbance without sample, A4 is the absorbance without sample and without enzyme.

[0049] like Figure 4 As shown, within the concentration range of 0 ~ 10 mg / mL, sulfated polysaccharides have the effect on... α The inhibitory effect of SPWZMLs-3 on glucosidase increases with increasing concentration, and its inhibitory activity is higher than that of PWZMLs. At a concentration of 10 mg / mL, SPWZMLs-3 inhibits... α The inhibitory effect of β-glucosidase was the strongest, with an inhibition rate of 74.07 ± 1.77%, significantly higher than that of PWZMLs (56.88 ± 1.94%) and other sulfated polysaccharides (60.24 ± 1.78% ~ 67.15 ± 1.24%). These results indicate that sulfation modification can enhance the inhibitory effect of PWZMLs on β-glucosidase. αThe ability to inhibit glucosidase can potentially delay the breakdown of oligosaccharides into glucose.

[0050] 2. α -Amylase inhibitory activity

[0051] In a 96-well plate, 30 μL of the test sample solution with different mass concentrations (0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 mg / mL), 30 μL of 1% soluble starch aqueous solution, and 30 μL of 1 U / mL [agent / solvent] were added sequentially. α -Amylase solution, shake well and incubate at 37℃ for 15 min; add 50 μL of 0.4 mol / L hydrochloric acid solution to terminate the reaction, then add 100 μL of iodine reagent (prepared by dissolving 5 mmol / L I2 in 5 mmol / L KI solution), and measure the absorbance at 600 nm wavelength, denoted as A1; α - The amylase inhibition rate is calculated according to the following formula.

[0052] α -Amylase inhibition rate (%) = [1 - (A1 – A2) / (A3 – A4)] × 100

[0053] In the formula: A2 represents none α - Absorbance of amylase, A3 is absorbance without sample, A4 is absorbance without sample and without enzyme.

[0054] like Figure 5 As shown, within the concentration range of 0 ~ 10 mg / mL, sulfated polysaccharides have the effect on... α The inhibitory effect of SPWZMLs-3 on amylase increases with increasing concentration, and its inhibitory activity is higher than that of PWZMLs. At a concentration of 10 mg / mL, SPWZMLs-3 inhibits the activity of PWZMLs-3 on amylase. α -Amylase inhibition was strongest, with an inhibition rate of 68.27 ± 1.45%, significantly higher than that of PWZMLs (44.23 ± 2.22%) and other sulfated polysaccharides (41.48 ± 2.68% ~ 56.49 ± 2.09%). These results indicate that sulfation modification can further enhance the inhibitory activity of PWZMLs. α -Amylase inhibition ability. α -Amylase mainly catalyzes the hydrolysis of macromolecular starch into oligosaccharides. The polysaccharide obtained by sulfation modification of ginger leaves can enhance the inhibitory effect on this enzyme, potentially hindering the degradation of macromolecular starch and reducing the formation of oligosaccharide intermediates.

[0055] Example 7: Determination of the inhibitory activity of sulfated ginger leaf polysaccharide on pancreatic lipase.

[0056] Take 50 μL of the test sample solution at different mass concentrations (0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 mg / mL), add 50 μL of 50 mM PBS buffer solution and 50 μL of 10 mg / mL pancreatic lipase solution, mix thoroughly, and then add 50 μL of 0.5 mM 4-nitrobenzene laurate substrate solution. React at 37 °C for 20 min, and measure the absorbance at a wavelength of 405 nm, denoted as A3. The pancreatic lipase inhibition rate is calculated according to the following formula.

[0057] Pancreatic lipase inhibition rate (%) = [1 - (A3 – A4) / (A1 – A2)] × 100

[0058] In the formula: A1 is the absorbance without sample, A2 is the absorbance without sample and without pancreatic lipase, and A4 is the absorbance without pancreatic lipase.

[0059] like Figure 6 As shown, within the concentration range of 0–10 mg / mL, the inhibitory effect of sulfated polysaccharides on pancreatic lipase increased with increasing concentration, and their inhibitory activity was higher than that of PWZMLs. At a concentration of 10 mg / mL, SPWZMLs-3 exhibited the strongest inhibitory effect on pancreatic lipase, with an inhibition rate of 81.19 ± 2.00%, significantly higher than that of PWZMLs (61.51 ± 2.63%) and other sulfated polysaccharides (66.19 ± 1.72% ~ 73.24 ± 2.43%). These results indicate that pancreatic lipase can catalyze the hydrolysis of triglycerides into fatty acids and monoglycerides that can be absorbed by the intestine; the sulfate-modified *Zingiber officinale* leaf polysaccharide can enhance the inhibitory effect on this enzyme, reduce the degree of triglyceride hydrolysis, and reduce the formation of absorbable fat products, showing promising application prospects for lipid-lowering.

[0060] Example 8: Further structural characterization of SPWZMLs-3

[0061] Given that SPWZMLs-3 possesses superior antioxidant activity as well as potential for lowering blood sugar and lipids, further structural characterization analysis was conducted.

[0062] 1. Monosaccharide composition

[0063] Take 5 mg of SPWZMLs-3 and place it in a 5 mL test tube. Add 1 mL of 2 mol / L trifluoroacetic acid and place it in a 120℃ oven for 2 h for hydrolysis. After hydrolysis, dilute to 50 mL with distilled water and filter through a 0.45 μm microporous membrane to obtain the sample solution. The injection volume of the sample solution is 20 μL. Instrument and chromatographic conditions: A Dionex ICS-5000 ion chromatograph with a pulsed amperometric detector was used. The column was CarboPac PA20 (6.5 μm, 3 mm × 150 mm). Gradient elution mode was used. The mobile phase conditions were as follows: 0-21 min, 98% water + 2% 250 mM NaOH; 21.1-30 min, 93% water + 2% 250 mM NaOH + 5% 1 M NaAc; 30.1-50 min, 20% water + 80% 250 mM NaOH; the flow rate was set to 0.5 mL / min.

[0064] like Figure 7 As shown, by comparing with standard monosaccharides, SPWZMLs-3 is a heteropolysaccharide, composed of fucose (0.98%), rhamnose (1.98%), arabinose (16.1%), galactose (14.04%), glucose (27.71%), xylose (28%), galacturonic acid (10.09%), and glucuronic acid (1.1%); among which xylose (28.00%), glucose (27.71%), arabinose (16.1%), galactose (14.04%), and galacturonic acid (10.09%) are its main monosaccharide components.

[0065] 2. Molecular weight

[0066] SPWZMLs-3 was prepared into a 2 mg / mL sample solution, filtered through a 0.45 μm microporous membrane before injection, with an injection volume of 20 μL. The analytical instrument used was a Waters 1525 high-performance liquid chromatograph equipped with a 2414 differential refractive index detector and an Empower3 workstation; the chromatographic column was an Ultrahydrogel. TM Linear (2μm, 7.8mm × 300mm). A series of standard solutions of dextran with different molecular weights were prepared using dextran as standard substances. A third-order calibration curve for the determination of polysaccharide molecular weight was established based on retention time and corresponding molecular weight. Chromatographic conditions: mobile phase: 0.1M NaNO3; flow rate: 0.9mL / min; column temperature: 45℃.

[0067] like Figure 8As shown, SPWZMLs-3 is a heterogeneous polysaccharide, mainly containing two molecular weight distribution peaks. Peak 1 has a molecular weight of 260.1 kDa, accounting for 91.5%, and is the main peak of the polysaccharide; Peak 2 has a molecular weight of 0.4 kDa, accounting for 8.5%. Calculations show that the average molecular weight of SPWZMLs-3 is 238 kDa.

[0068] 3. Infrared Spectroscopy

[0069] Weigh 2 mg of dried SPWZMLs-3 sample, mix thoroughly with 100 mg of dried potassium bromide powder, grind, and compress into tablets. Infrared spectroscopy was performed using a Spotlight 200i Fourier transform infrared microscope system with a spectral scanning range of 4000 cm⁻¹. -1 ~ 400cm -1 .

[0070] like Figure 9 As shown, SPWZMLs-3 at 3435 cm -1 1630 cm -1 1401 cm -1 and 1021 cm -1 There are absorption peaks at these locations, and these absorption peaks are characteristic absorption peaks of polysaccharides. Among them, the 3435 cm⁻¹ peak is... -1 The relatively broad absorption peaks on the left and right are characteristic peaks of the OH stretching vibration of polysaccharides; 1630 cm⁻¹ -1 The peak at 1401 cm⁻¹ is related to the asymmetric stretching vibration of C=O; -1 The absorption peak at 1021 cm⁻¹ is caused by the symmetric stretching vibration of COO⁻ and is a characteristic infrared absorption peak of uronic acid; -1 The peak at this point is the COC stretching vibration peak of the glycosidic bond. SPWZMLs-3 has a characteristic wavenumber of 1243 cm⁻¹. -1 1144 cm -1 810 cm -1 and 583 cm -1 All exhibited distinct absorption peaks, corresponding to the S=O asymmetric stretching vibration, S=O / SO symmetric stretching vibration, COS stretching vibration, and O=S=O symmetric stretching vibration signals, respectively. Infrared spectroscopy further confirmed that the SPWZMLs-3 structure conforms to the characteristics of sulfated polysaccharides.

[0071] 4. Morphological Analysis

[0072] Take an appropriate amount of SPWZMLs-3 sample and evenly adhere it to a stage with double-sided conductive adhesive using a cotton swab. Place the sample in an ion sputtering instrument for foil pretreatment. After pretreatment, observe the microstructure of the sample using a Hitachi Regulus 8100 ultra-high resolution field emission scanning electron microscope.

[0073] like Figure 10 As shown, at a magnification of ×500, PWZMLs-3 exhibits a microstructure of interwoven lamellar and fibrous structures. The sample contains a large number of wrinkled and curled lamellar structures, mixed with slender fibers, and the surface of the lamellars is covered with pores and cracks of varying sizes.

[0074] Example 9 Preparation of SPWZMLs-3 tablets

[0075] Weigh 1.0g of SPWZMLs-3 and mix it evenly with 1.0g of starch and 1.5g of dextrin. Add 0.1g of tartaric acid and 2mL of 50% (v / v) ethanol to the powder mixture, mix thoroughly, and granulate through an 18-mesh sieve. Dry at 60℃, and after drying, granulate again through an 18-mesh sieve. Add 0.1g of magnesium stearate and mix evenly. After tableting, SPWZMLs-3 tablets are obtained.

[0076] Example 10 Preparation of SPWZMLs-3 capsules

[0077] Weigh 1.0g of SPWZMLs-3 and spray in 95% (v / v) ethanol at a material-to-liquid ratio of 1:1, and mix thoroughly. Add 10% starch slurry to prepare a soft mass, granulate it through an 18-mesh sieve, dry it in a 60℃ oven, and then granulate it again through an 18-mesh sieve. Under conditions where the relative humidity is below 65%, fill the granules into empty No. 3 capsule shells to obtain SPWZMLs-3 capsules.

[0078] Example 11 Preparation of SPWZMLs-3 oral solution

[0079] Weigh 1.0g SPWZMLs-3 and add it to 100mL of purified water. Stir at room temperature until the sample is completely dissolved. Then add 0.2g xylitol and 0.1g sodium benzoate in sequence and stir until the excipients are completely dissolved. Perform instantaneous sterilization on the resulting liquid and then dispense it into oral liquid bottles at a specification of 10mL / bottle. Seal the bottles to obtain SPWZMLs-3 oral liquid.

[0080] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A sulfated ginger leaf polysaccharide, wherein the sulfated ginger leaf polysaccharide is obtained by sulfated ginger leaf polysaccharide with aminosulfonic acid; wherein the degree of sulfate substitution of the sulfated ginger leaf polysaccharide is 0.16 to 1.

67.

2. The sulfated ginger leaf polysaccharide according to claim 1, characterized in that: The degree of sulfate substitution of the ginger leaf polysaccharide is 1.67 ± 0.16; And / or, the infrared spectrum of the sulfated ginger leaf polysaccharide is shown in Figure 9; And / or, the sulfated ginger leaf polysaccharide is composed of fucose (0.98%), rhamnose (1.98%), arabinose (16.1%), galactose (14.04%), glucose (27.71%), xylose (28%), galacturonic acid (10.09%) and glucuronic acid (1.1%); And / or, the total sugar content of sulfated ginger leaf polysaccharide is 15.52 ± 4.99%, the uronic acid content is 16.8 ± 3.26%, and the sulfate group content is 18.06 ± 0.93%; And / or, the average molecular weight of the sulfated ginger leaf polysaccharide is 238 kDa; And / or, the sulfated ginger leaf polysaccharide has a particle size of 533.4 nm and a potential of -53.9 mV.

3. A method for preparing sulfated ginger leaf polysaccharide according to claim 1, comprising the following steps: placing ginger leaf polysaccharide and aminosulfonic acid in an organic solvent and heating to react for a certain time; cooling to room temperature, quenching the reaction with an alkaline solution, adjusting the pH of the reaction solution to neutral, precipitating with ethanol to obtain a precipitate, redissolving the precipitate with distilled water, dialyzing, and freeze-drying to obtain sulfated ginger leaf polysaccharide with different degrees of substitution.

4. The method according to claim 3, characterized in that: The mass ratio of the ginger leaf polysaccharide to aminosulfonic acid is 1:1 to 1:

8.

5. The method according to claim 4, characterized in that: The sulfated ginger leaf polysaccharide has a sulfate group substitution degree of 0.16 to 1.

67.

6. The method according to claim 5, characterized in that: The mass ratio of the *Gingeria glabra* leaf polysaccharide to aminosulfonic acid is 1:4, and the degree of sulfate substitution of the obtained sulfated *Gingeria glabra* leaf polysaccharide is 1.

67.

7. The method according to claim 5, characterized in that: The obtained sulfated ginger leaf polysaccharide is composed of fucose (0.98%), rhamnose (1.98%), arabinose (16.1%), galactose (14.04%), glucose (27.71%), xylose (28%), galacturonic acid (10.09%), and glucuronic acid (1.1%). The total sugar content of the sulfated ginger leaf polysaccharide is 15.52 ± 4.99%, the uronic acid content is 16.8 ± 3.26%, and the sulfate group content is 18.06 ± 0.93%. The average molecular weight of the sulfated ginger leaf polysaccharide is 238 kDa. The particle size of the sulfated ginger leaf polysaccharide is 533.4 nm, and the potential is -53.9 mV.

8. The use of the sulfated ginger leaf polysaccharide according to claim 1, wherein the sulfated ginger leaf polysaccharide is prepared by any of the preparation methods in claims 3-7, for use in the preparation of antioxidant functional products.

9. The use of sulfated ginger leaf polysaccharide according to claim 1, wherein the sulfated ginger leaf polysaccharide is prepared by any of the preparation methods in claims 3-7, for use in the preparation of foods, health products or pharmaceuticals for lowering blood sugar and regulating lipids.

10. The use according to claims 8-9, characterized in that... The dosage forms of the antioxidant, hypoglycemic and lipid-regulating functional products are tablets, capsules or oral liquids.

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