Sedum sarmentosum polysaccharide extraction process

Abstract

The invention discloses a stringy stonecrop herb polysaccharide extraction process which comprises the following steps: drying and crushing stringy stonecrop herb, adding water according to a material-liquid ratio of (1: 20)-(1: 60), extracting, concentrating, and carrying out alcohol precipitation to obtain crude polysaccharide; deproteinizing, dialyzing and freeze-drying; carrying out gradient elution and purification to obtain sedum sarmentosum polysaccharide SSBP; an extraction process is optimized through a response surface method, optimal conditions are selected, and the yield of crude polysaccharide reaches 19.06%; according to structural characterization, SSBP is acidic polysaccharide, the molecular weight is 7.484 kDa, 36.12% of uronic acid is contained, monosaccharide is composed of rhamnose, arabinose, galactose, galacturonic acid and glucuronic acid, and the molar ratio of rhamnose to arabinose to galactose to galacturonic acid to glucuronic acid is 12.85: 19.13: 18.98: 35.73: 3.35; an in-vitro activity experiment shows that the SSBP has good antioxidant activity and remarkable anti-inflammatory activity, the release of RAW264.7 macrophage NO induced by LPS and the secretion of IL-6, IL-1beta and TNF-alpha can be inhibited in a dose-dependent manner, and the inhibition rates under the concentration of 1.2 mg / mL respectively reach 74.41%, 92.53%, 95.27% and 95.73%; and a new scheme is provided for deep development of sedum sarmentosum polysaccharide and preparation of anti-inflammatory and anti-oxidation products.

Description

Technical Field

[0001] This invention belongs to the field of plant active ingredient extraction technology, specifically relating to a process for extracting polysaccharides from Sedum sarmentosum. Background Technology

[0002] Sedum sarmentosum is a perennial herbaceous plant belonging to the Crassulaceae family. The whole plant is used medicinally; it is cool in nature, sweet and slightly sour in taste, and enters the liver, gallbladder, and small intestine meridians. It has the effects of clearing heat and detoxifying, promoting diuresis and relieving jaundice. First recorded in the Qing Dynasty's *Compendium of Materia Medica Supplement*, it is one of the commonly used medicinal materials in traditional Chinese medicine for treating acute and chronic hepatitis. Modern pharmacological research shows that Sedum sarmentosum possesses various biological activities such as liver protection and enzyme reduction, anti-inflammation, antioxidant, immunomodulatory, and anti-tumor effects, showing broad prospects for medicinal development. Currently, drugs developed based on Sedum sarmentosum are mainly compound preparations, including compound Sedum sarmentosum syrup, Sedum sarmentosum tea syrup, liver-protecting tablets, liver-nourishing granules, Sedum sarmentosum granules, and compound Sedum sarmentosum capsules, mainly used as adjunctive treatment for acute and chronic hepatitis. These preparations mostly use crude extracts of Sedum sarmentosum or in compound form, primarily targeting small molecule components such as flavonoids and cyanogenic glycosides.

[0003] From a chemical composition perspective, the active components of *Sedum sarmentosum* mainly include flavonoids (such as quercetin, kaempferol, isorhamnetin, and luteolin), triterpenoids, alkaloids, cyanogenic glycosides (mainly sedum glycoside), and polysaccharides. Flavonoids are the main active substances in *Sedum sarmentosum*, exhibiting anti-inflammatory, anti-fibrotic, antioxidant, and anti-angiogenic activities; sedum glycoside is one of the effective components for liver protection and enzyme reduction. In recent years, with the widespread confirmation of the pharmacological activities of polysaccharides in immunomodulation, antitumor, and antioxidant fields, research on *Sedum sarmentosum* polysaccharides has gradually attracted attention. Existing research shows that *Sedum sarmentosum* polysaccharides are arabinogalactoglucan composed of monosaccharides such as arabinose, glucose, and galactose. The crude polysaccharide is obtained through conventional methods such as hot water extraction, ethanol precipitation, and Sevage protein removal, followed by purification by ion exchange chromatography and gel filtration chromatography. Preliminary studies indicate that it possesses antioxidant, immunomodulatory, and antitumor effects.

[0004] Regarding patent technology, a search revealed relevant authorized patents. CN111892662B discloses a homogeneous polysaccharide from *Sedum sarmentosum*, its preparation method, and its uses. The method involves hot water extraction and alcohol precipitation to obtain crude polysaccharide, followed by elution with distilled water using a DE-52 anion exchange cellulose column to obtain a water-washed fraction, and then purification by gel chromatography with 0.2M NaCl solution to obtain homogeneous polysaccharide CPCW. This polysaccharide has a purity of over 99%, a molecular weight of 1-30 kDa, and its monosaccharide composition consists of arabinose, glucose, and galactose. It has been shown to have antitumor effects by inhibiting the proliferation of Huh-7 liver cancer cells. CN111297935B discloses an extract from *Sedum sarmentosum*, its preparation method, and its applications, mainly involving the ethyl acetate and ethanol fractions of *Sedum sarmentosum*. Its components include organic acids, flavonoids, triterpenoids, and alkaloids, and it has anti-influenza virus activity. This patent does not involve the extraction and application of polysaccharide components.

[0005] However, existing technologies related to Sedum sarmentosum polysaccharides still have significant shortcomings. Regarding purification methods, CN111892662B uses distilled water washing to elute the ion exchange column, obtaining neutral polysaccharide components. It does not address the acidic polysaccharide components that might be obtained through salt gradient elution, limiting the comprehensive development and utilization of polysaccharides with different charge properties in Sedum sarmentosum. In terms of process optimization, existing technologies have not systematically optimized extraction process parameters, resulting in unclear yield parameters and a lack of process parameter basis for industrial transformation. Regarding activity studies, existing technologies mainly focus on antitumor activity, lacking systematic research on the anti-inflammatory activity of Sedum sarmentosum polysaccharides, which is closely related to the traditional medicinal properties of Sedum sarmentosum, namely "clearing heat and detoxifying." Furthermore, existing technologies do not explicitly employ a dedicated protein removal step, which may affect the purity and activity stability of the polysaccharides.

[0006] Therefore, it is necessary to develop a new method for extracting Sedum sarmentosum polysaccharides to obtain polysaccharide components with novel structures and well-defined activities, and to systematically evaluate their anti-inflammatory and antioxidant activities, so as to provide new technical solutions for in-depth research and industrial development of Sedum sarmentosum polysaccharides.

[0007] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a process for extracting polysaccharides from Sedum sarmentosum.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A process for extracting polysaccharides from Sedum sarmentosum includes the following steps:

[0011] 1) Wash the whole plant of Sedum sarmentosum, dry it in an oven and then crush it for later use;

[0012] 2) Mix Sedum sarmentosum powder with distilled water, heat to extract, then centrifuge, collect the supernatant and concentrate;

[0013] 3) Add anhydrous ethanol in proportion, let stand overnight, centrifuge, collect the precipitate and redissolve, freeze dry to obtain Sedum sarmentosum crude polysaccharide CSSBP;

[0014] The yield of crude polysaccharides is calculated according to formula 1-1:

[0015]

[0016] In the formula: M1: mass of crude polysaccharide (m), M2: mass of raw material (m);

[0017] 4) Reconstitute CSSBP, mix with Sevage reagent to remove impurities; then treat with running water through a dialysis bag and then with static water, collect the dialysate and freeze dry;

[0018] 5) Dissolve the freeze-dried sample in water, centrifuge, and purify the supernatant by column chromatography; then elute with gradients, collect, freeze, and dry to obtain Sedum sarmentosum polysaccharide.

[0019] The SSBP is an acidic polysaccharide containing uronic acid, with a total sugar content of 85%~87%, a uronic acid content of 35%~37%, a protein content of 1.5%~1.8%, and a molecular weight of 7~8kDa.

[0020] Preferably, the monosaccharide composition of the SSBP includes rhamnose (Rha), arabinose (Ara), galactose (Gal), galacturonic acid (Gal-UA), and glucuronic acid (Glc-UA), with a molar ratio of (12~13):(19~20):(18~19):(35~36):(3~4).

[0021] Preferably, the infrared spectrum of the SSBP has characteristic absorption peaks at 3415 cm⁻¹, 2936 cm⁻¹, 1743 cm⁻¹, 1611 cm⁻¹, and 833 cm⁻¹, wherein the absorption peaks at 1743 cm⁻¹ and 1611 cm⁻¹ correspond to esterified carboxyl groups and free carboxyl groups, respectively, indicating the presence of uronic acid units; the absorption peak at 833 cm⁻¹ indicates the presence of α-glycosidic bonds.

[0022] Preferably, the drying temperature in step 1) is 37°C, and the Sedum sarmentosum powder is passed through a 50-mesh sieve.

[0023] Preferably, in step 2), the ratio of the powder of *Sedum sarmentosum* to distilled water is 1:20 to 1:60 (g / mL).

[0024] Preferably, the extraction temperature in step 2) is 60-100℃.

[0025] Preferably, the extraction time in step 2) is 1-5 hours, and the volume is concentrated to 1 / 10 of the original volume by rotary evaporation at 65°C.

[0026] Furthermore, the preferred conditions are a material-to-liquid ratio of 1:40, mixing with distilled water, and extraction at 100°C for 3 hours.

[0027] Preferably, in step 3), the ratio of anhydrous ethanol added is 1:4 by volume, and the standing condition is 4°C.

[0028] Preferably, in step 4), the Sevage reagent is a mixture of n-butanol and chloroform, wherein V 正丁醇 V 三氯甲烷 =1:4; the volume ratio of CSSBP solution to Sevage reagent is 1:5.

[0029] Preferably, in step 4), the dialysis bag is 3500 Da, the running water treatment is 72 h, and the static water treatment conditions are 4℃ for 24 h.

[0030] Preferably, in step 5), the purification column is DEAE seplife FF, the flow rate of the column is 4 ml / min, and the column is eluted sequentially with pure water, 0.1 M, 0.2 M and 0.3 M NaCl solutions, and all eluents are collected.

[0031] The application of Sedum sarmentosum polysaccharide SSBP in the preparation of anti-inflammatory drugs or functional foods, wherein the SSBP can inhibit LPS-induced NO release from RAW264.7 macrophages and the secretion of inflammatory factors IL-6, IL-1β and TNF-α.

[0032] The beneficial effects of this invention are:

[0033] 1) First, in terms of preparation method, the present invention uses a DEAE seplife FF ion exchange column combined with gradient NaCl solution elution, which can obtain salt-eluted polysaccharide components that are different from the water-eluted components of the prior art, thus enriching the structural diversity of Sedum sarmentosum polysaccharides; at the same time, the extraction process parameters are systematically optimized by response surface methodology, so that the crude polysaccharide yield reaches 19.06%, providing a reliable process basis for industrial production.

[0034] 2) Secondly, regarding bioactivity, this invention is the first to systematically verify the anti-inflammatory activity of Sedum sarmentosum polysaccharide SSBP, confirming that it can dose-dependently inhibit LPS-induced NO release from RAW264.7 macrophages and the secretion of inflammatory factors IL-6, IL-1β, and TNF-α, with inhibition rates of 74.41%, 92.53%, 95.27%, and 95.73%, respectively, at a concentration of 1.2 mg / mL, filling the gap in existing research on the anti-inflammatory activity of Sedum sarmentosum polysaccharides; simultaneously, SSBP also exhibits good antioxidant activity against DPPH and ABTS. + The free radical scavenging rates reached 77.31% and 99.28%, respectively;

[0035] 3) Third, the present invention uses the Sevage method to specifically remove proteins, which improves the purity and activity stability of polysaccharides, laying a quality foundation for subsequent structural analysis and activity studies.

[0036] In summary, this invention provides new technical solutions and scientific basis for in-depth research on Sedum sarmentosum polysaccharides and the development of anti-inflammatory and antioxidant products. Attached Figure Description

[0037] Figure 1 This is a schematic diagram illustrating the effect of liquid-to-solid ratio on CSSBP yield;

[0038] Figure 2 To investigate the effect of temperature on CSSBP yield;

[0039] Figure 3 The impact of extraction time on CSSBP yield;

[0040] Figure 4 The response surface plot and corresponding contour plot were constructed for the interaction of three factors A, B, and C (where A is the extraction temperature, B is the extraction time, and C is the solid-liquid ratio).

[0041] Figure 5 Separation and purification of crude polysaccharides (A); HPLC chromatogram of CSSBP (B); elution curve of ion purification (C); elution curve of gel purification (D); HPLC chromatogram of SSBP.

[0042] Figure 6 This is a schematic diagram of the DPPH radical scavenging rate of SSBP.

[0043] Figure 7 ABTS for SSBP + A schematic diagram illustrating the free radical scavenging rate;

[0044] Figure 8 OH of SSBP - A schematic diagram illustrating the free radical scavenging rate;

[0045] Figure 9 A schematic diagram illustrating the effects of LPS and SSBP on the viability of RAW264.7 cells;

[0046] Figure 10 A schematic diagram showing the effect of SSBP on NO content in RAW264.7 cells (A: NaNO2 standard curve; B: cell NO content).

[0047] Figure 11 A schematic diagram showing the effect of SSBP on the levels of inflammatory factors in RAW264.7 cells (A: IL-6 concentration; B: IL-1β concentration; C: TNF-α concentration).

[0048] Figure 12 This is a chromatogram showing the absolute molecular weight analysis of SSBP;

[0049] Figure 13 This is a full-wavelength scan of iodine-potassium iodide.

[0050] Figure 14 Ion chromatograms of monosaccharide standards and SSBP;

[0051] Figure 15 This is the infrared spectrum of SSBP. Detailed Implementation

[0052] The present invention will be further described below through specific embodiments. To make the inventive objectives, technical solutions, and beneficial technical effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the embodiments described in this specification are merely for explaining the present invention and are not intended to limit the present invention.

[0053] Unless otherwise stated, all instruments and reagents used in the examples are commercially available or synthesized using conventional methods and can be used directly without further processing, and all instruments used in the examples are commercially available.

[0054] The reagents are as follows:

[0055]

[0056] The experimental instruments and equipment are listed in the table below:

[0057]

[0058] Example 1: Extraction of crude polysaccharides from Sedum sarmentosum

[0059] Sedum sarmentosum samples (whole plant) were collected uniformly in the Dabie Mountains of Anhui Province, China in December 2022. The whole plant was washed, dried in an oven at 37℃, and ground into powder using a pulverizer. The powder was then passed through a 50-mesh sieve for later use. The powder was then mixed with distilled water at a material-to-liquid ratio of 1:40 and extracted at 100℃ for 3 hours. After centrifugation, the supernatant was collected and concentrated to 1 / 10 of its original volume by rotary evaporation at 65℃. Anhydrous ethanol was added at a ratio of 1:4, and the mixture was allowed to stand overnight at 4℃. After centrifugation, the precipitate was collected, redissolved, and freeze-dried to obtain crude polysaccharide of Sedum sarmentosum (CSSBP).

[0060] The yield of crude polysaccharides is calculated according to formula 1-1.

[0061]

[0062] In the formula: M1: Mass of crude polysaccharide (m)

[0063] M2: Raw material mass (m)

[0064] 1.1 Single-factor experiment

[0065] Weigh 20g of Sedum sarmentosum powder and, according to the extraction process described above, set the following conditions: ① extraction temperature 90℃, extraction time 3h; ② extraction temperature 90℃, material-liquid ratio 1:40; ③ material-liquid ratio 1:40, extraction time 3h. Single-factor experiments were conducted with different material-liquid ratios (1:20, 1:30, 1:40, 1:50, 1:60 (g / mL)), extraction times (1, 2, 3, 4, 5 (h)), and extraction temperatures (60, 70, 80, 90, 100 (°C)). Each experiment was repeated 3 times to explore the effect of different extraction conditions on the yield of crude polysaccharides from Sedum sarmentosum.

[0066] 1.2 Results of Single-Factor Experiments

[0067] 1.2.1 Effect of liquid-to-solid ratio on CSSBP yield

[0068] Depend on Figure 1 It was observed that within the extraction solvent ratio range of 1:20 to 1:60 (g / mL), the crude polysaccharide yield increased rapidly with the increase of the extraction solvent-solid ratio. The CSSBP yield reached its maximum value of (17.26±0.18)% at a solvent-solid ratio of 1:40 g / mL, after which the yield gradually decreased. This may be due to excessive extraction solvent, leading to over-dilutement of the polysaccharide in the system, reducing the concentration difference between the inside and outside, weakening the diffusion driving force, and thus decreasing the rate and efficiency of polysaccharide transfer from the raw material to the solvent; or it may be due to increased system heat capacity and low mass transfer efficiency.

[0069] 1.2.2 Effect of extraction temperature on CSSBP yield

[0070] Depend on Figure 2It can be seen that within the range of 60~90℃, the yield of crude polysaccharides increases rapidly with increasing extraction temperature, reaching a maximum of 17.56±0.20% at 90℃. When the temperature reaches 100℃, the yield of crude polysaccharides decreases slightly. This may be because excessively high extraction temperatures cause the polysaccharide chains to hydrolyze and break down, transforming them into oligosaccharides or monosaccharides with smaller molecular weights. These components may not be effectively precipitated during subsequent alcohol precipitation.

[0071] 1.2.3 The impact of extraction time on CSSBP yield

[0072] Depend on Figure 3 It can be seen that the crude polysaccharide yield increases rapidly within the range of 1 to 4 hours; when the extraction time is 4 hours, the crude polysaccharide yield reaches its maximum value of 18.86±0.26%, and then begins to decline. This may be because all the polysaccharides have been precipitated, while impurities such as proteins and inorganic salts in the raw materials continue to dissolve, affecting the crude polysaccharide yield.

[0073] 1.3 Optimization of Response Surface Experiment

[0074] Based on the results of the single-factor experiment, a three-factor, three-level response surface experiment was conducted with (A) material-liquid ratio, (B) extraction time, and (C) extraction temperature as variables (Table 1), and the results were analyzed using Design Expert 13.0 software.

[0075] Table 1. Levels of Experimental Factors

[0076]

[0077] 1.3.1 Optimization of the extraction process of crude polysaccharide from Sedum sarmentosum using response surface methodology

[0078] Data was analyzed using Design-Expert 13 software, and a fitted quadratic polynomial regression model was constructed: Y=18.70-0.5585A+0.9720B+0.6312C+0.6528AB+0.7747AC-0.7557BC-1.47A²-0.8422B²-1.55C².

[0079] Table 2 Experimental Design and Results

[0080]

[0081] Table 3. Analysis of Variance of the Regression Model

[0082]

[0083] The analysis of variance results show that the regression model has F > 0.05, P < 0.01, and the p-value of the lack-of-fit term is not significant (0.8579 > 0.05), indicating that the model is highly significant and the lack-of-fit term is not significant, thus it fits the data well. The model's R² is 0.9859, and the adjusted R² is 0.9677, indicating that the model can explain 98.59% of the experimental results, and the predicted values ​​can explain 96.77% of the problems. Furthermore, the close proximity of R² and adjusted R² indicates a high correlation between the predicted and actual values.

[0084] The order of influence of the linear terms A (extraction temperature), B (extraction time), and C (solid-liquid ratio) on the extraction rate in the regression equation is B>C>A. Furthermore, the linear terms A, B, and C, the interaction terms AB, AC, and BC, and the quadratic terms A², B², and C² all have a significant impact on the extraction rate (P<0.05), indicating that the change in the response value exhibits a quadratic relationship.

[0085] For further details, please refer to Figure 4 From the three-dimensional response surface plots, the surfaces corresponding to the pairwise interactions between AB, AC, and BC all exhibit significant slope changes, indicating a synergistic or antagonistic interaction effect among the three factors: extraction temperature, extraction time, and material-to-liquid ratio. The contour lines are densely distributed in sloping elliptical shapes, and the slope of the response surface changes dramatically, indicating that the interactions between extraction temperature (A) and extraction time (B), extraction temperature (A) and material-to-liquid ratio (C), and extraction time (B) and material-to-liquid ratio (C) have a significant impact on the extraction rate (P<0.05). At the same time, the slope of the response surface between groups AB and BC is more pronounced in the direction of extraction time (B), indicating that the extraction time has a stronger effect on the extraction rate and is the dominant factor affecting the extraction rate.

[0086] Analysis of the equations revealed the extreme points of the regression model as: A = -0.0562438, B = 0.52807, and C = 0.0611224, corresponding to a theoretical extraction rate of 18.99%. Based on actual operating conditions, the coded values ​​were restored and optimized to actual process parameters: extraction temperature 89°C, extraction time 4.5 h, and material-to-liquid ratio 41 g / ml. To verify the model's reliability, three parallel validation experiments were conducted. The results showed an actual extraction rate of 19.06 ± 0.06%, with relative errors from the model's predictions all less than 5%, indicating high prediction accuracy of the regression model and good stability of the optimized process parameters.

[0087] Example 2: Isolation and purification of Sedum sarmentosum polysaccharides

[0088] The crude polysaccharide was reconstituted and mixed with Sevage reagent (butanol to chloroform, volume ratio 1:4) at a volume ratio of 1:5 to remove proteins. After removing other impurities, the sample was treated with running water through a 3500 Da dialysis bag for 72 h, followed by static water treatment at 4 °C for 24 h. The dialysate was collected again and lyophilized. The lyophilized sample was dissolved in distilled water, centrifuged, and the supernatant was purified by passing it through a DEAE Seplife FF column at a flow rate of 4 ml / min. A gradient elution was performed sequentially with pure water, 0.1 M, 0.2 M, and 0.3 M NaCl solutions, and all eluents were collected. The absorption curve was then determined using the phenol-sulfuric acid method. The highest peak in the distilled water eluent was collected and lyophilized. The lyophilized sample was purified by Sephadex G-200 column, and the major fraction was collected. The purified Sedum sarmentosum Bunge polysaccharides (SSBP) was obtained by high-performance liquid chromatography (HPLC) and then lyophilized.

[0089] The crude polysaccharide from *Sedum sarmentosum* was purified using a DEAE Seplife FF column. Elution with different concentrations of NaCl solution yielded three fractions: SSBP-0.1M, SSBP-0.2M, and SSBP-0.3M, with purities of 79.6%, 81.4%, and 73.8%, respectively. The fraction with the highest purity, SSBP-0.2M, was selected for further purification. The purification results using a Sephadex G-200 column are shown below. Figure 5 As shown, two components were obtained. After being collected and mixed separately, they exhibited a single peak shape, with purities of 84.0% and 85.8%, respectively. The second component was selected, and the HPLC results showed that it had a single symmetrical peak shape (Figure), indicating that a relatively pure polysaccharide was obtained, which was named SSBP.

[0090] Example 3: Chemical Composition Analysis

[0091] 3.1 The total sugar content was determined using the phenol-sulfuric acid method.

[0092] Prepare 0.1 mg / mL glucose standard solution and 0.05 mg / mL and 0.08 mg / mL SSBP solutions. Set up three replicates for each group, placing 0, 0.2, 0.4, 0.6, 0.8, and 1 mL of the standard solution into test tubes, respectively, and adding distilled water to bring the volume to 1 mL. Add 1 mL of 6% phenol solution and 5 mL of concentrated sulfuric acid sequentially, mix thoroughly, react at room temperature for 5 min, then continue the reaction under boiling water for 15 min. After cooling for 10 min, measure the absorbance at 490 nm and plot the glucose standard curve. Determine the SSBP content using the above method, and calculate the total sugar content based on the standard curve.

[0093] 3.2 Determination of uronic acid content using the m-hydroxybiphenyl method

[0094] Prepare a 0.1 mg / mL galacturonic acid standard solution and 0.05 mg / mL and 0.08 mg / mL SSBP solutions. Set up three replicates for each group, placing 0, 0.2, 0.4, 0.6, 0.8, and 1 mL of the standard solution into test tubes, respectively, and adding distilled water to bring the volume to 1 mL. After mixing, add 5 mL of sodium tetraborate solution, incubate in a boiling water bath for 5 min, and then add 0.1 mL of m-hydroxybiphenyl solution. After mixing, let stand at room temperature for 5 min, then measure the absorbance at 520 nm and plot a uronic acid standard curve. Repeat the above method for SSBP determination, and calculate the uronic acid content based on the standard curve.

[0095] 3.3 Coomassie Brilliant Blue Method for Determining Protein Content

[0096] Prepare 0.1 mg / mL BSA standard solution and 0.05 mg / mL and 0.08 mg / mL SSBP solutions. Set up three replicates for each group, placing 0, 0.2, 0.4, 0.6, 0.8, and 1 mL of the standard solution into test tubes, and adding distilled water to bring the volume to 1 mL. Then, add 5 mL of Coomassie Brilliant Blue solution to each test tube, let stand for 30 min, and measure the absorbance at 595 nm to plot a standard curve. Measure SSBP using the above method, and calculate the protein content based on the standard curve.

[0097] Standard curves were plotted using glucose, galacturonic acid, and BSA as standards. Based on the standard curves for total sugar (y = 348.63x -21.257, R² = 0.9991), protein (y = 0.3679x + 0.5958, R² = 0.9804), and uronic acid (y = 2.5881x + 0.097, R² = 0.994), the total sugar content in SSBP was found to be 85.8±0.23%, the protein content was 1.65±0.07%, and the uronic acid content was 36.12±0.34%.

[0098] Example 4: Structural Characterization of SSBP

[0099] 4.1 The absolute molecular weight of SSBP was determined using the SEC-MALLS-RI technique.

[0100] SSBP was dissolved in a 0.1M NaNO3 aqueous solution containing 0.02% NaN3, at a concentration of 1 mg / mL. The solution was filtered through a 0.45 μm filter before being analyzed. The homogeneity and molecular weight of each fraction were determined using SEC-MALLS-RI technology. The results showed that the relative molecular mass of SSBP was 7.484 kDa (see...). Figure 12 ).

[0101] 4.2 Iodine-potassium iodide reaction

[0102] Prepare a 0.01 mg / mL SSBP solution and iodine reagent (0.02% I₂ and 0.2% KI). Add 1.2 mL of iodine reagent to 1 mL of SSBP solution and observe for a blue-violet reaction, and check for absorption peaks at 350 nm and 565 nm. The results showed that SSBP did not react with iodine-potassium iodide reagent to produce a blue-violet reaction, and there were no characteristic absorption peaks in the 540-650 nm range (see [link to study]). Figure 13 This indicates that SSBP does not contain starchy substances.

[0103] 4.3 Fourier Transform Infrared Spectroscopy Scan

[0104] Mix SSBP and potassium bromide at a ratio of 1:150 and compress into tablets. [The solution is then stored at 4000-400 cm⁻¹.] -1 Infrared spectral scanning was performed within the range. Results are shown (see...). Figure 15 ): 3415.8 cm -1 The point is the OH stretching vibration; 2936.57 cm. -1 The point is a CH stretching vibration; 1743.82 cm. -1 and 1611.23 cm -1 The positions correspond to esterified carboxyl groups (C=O) and free carboxyl groups (COO), respectively. - The asymmetric stretching vibrations of SSBP confirm the presence of uronic acid units; 1416.46 cm -1 The vibrations at these locations are CH bending vibrations; 1146.47, 1102.12, and 1024.50 cm. -1 The absorption peak at 833.10 cm⁻¹ is characteristic of the pyranose ring; -1 The characteristic absorption peak at the point indicates that SSBP contains α-glycosidic bonds.

[0105] 4.4 Monosaccharide Composition Analysis

[0106] Take about 5 mg of Sedum sarmentosum polysaccharide and place it in a stoppered test tube. Add 1 ml of 2M trifluoroacetic acid and dissolve it completely. Purge with nitrogen, seal with sealing film, and incubate in an oil bath at 121°C for 2 hours. Then cool. Dry the contents of the test tube with a nitrogen evaporator, add 2 ml of methanol to dissolve again and dry. Repeat the methanol washing 3-4 times, dry, and store in a desiccator.

[0107] Thirteen monosaccharide samples (fucose (Fuc), rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), fructose (Fru), ribose (Rib), galacturonic acid (Gal-UA), glucuronic acid (Glc-UA), mannuronic acid (Man-UA), and guluronic acid (Gul-UA)) were accurately weighed and prepared into a 0.1 mg / mL mixed standard stock solution using ultrapure water. Before instrumental analysis, the mixed standard was diluted to 50 μg / mL with ultrapure water. A 1 mg / mL *Sedum sarmentosum* polysaccharide solution was prepared and then diluted to 50 mg / L. 1 mL of the mixed standard dilution and the *Sedum sarmentosum* polysaccharide solution were separately drawn using a syringe and filtered sequentially through a 0.22 μm aqueous filter before injection. The peak times of the polysaccharide samples were compared with those of the mixed standard to determine the monosaccharide composition.

[0108] Chromatographic conditions: The liquid chromatography column was a Dionex™ CarboPac™ PA20 (150 mm × 3.0 mm, 10 μm); the injection volume was 5 μL; mobile phase A was ultrapure water, mobile phase B was 0.1 mol / L NaOH solution, and mobile phase C was a mixed solution of 0.1 mol / L NaOH and 0.2 mol / L CH3COONa; the flow rate was 0.5 mL / min; and the column temperature was 30 °C. Elution gradient: 0 min A / B / C phase (95:5:0, V / V), 26 min A / B / C phase (85:5:10, V / V), 42 min A / B / C phase (85:5:10, V / V), 42.1 min A / B / C phase (60:0:40, V / V), 52 min A / B / C phase (60:40:0, V / V), 52.1 min A / B / C phase (95:5:0, V / V), 60 min A / B / C phase (95:5:0, V / V).

[0109] The results show (see) Figure 14 SSBP is composed of five monosaccharides: rhamnose (Rha), arabinose (Ara), galactose (Gal), galacturonic acid (Gal-UA), and glucuronic acid (Glc-UA), with a molar ratio of 12.85:19.13:18.98:35.73:3.35.

[0110] Example 5: In vitro antioxidant activity of Sedum sarmentosum polysaccharides

[0111] 5.1 Scavenging of DPPH free radicals

[0112] Prepare a 2 mg / mL SSBP solution. Set up three replicates for each group, taking 10, 20, 40, 60, 80, and 100 μL of SSBP solution respectively, and bringing the volume to 100 μL with distilled water. Then add 100 μL of 0.1 mmol / L DPPH-ethanol solution. After adding the sample, remove air bubbles, incubate in the dark for 30 min, and measure the absorbance (A1) at 517 nm. The blank control group (A0) and background group (A2) were prepared using anhydrous ethanol instead of the sample solution and DPPH-ethanol solution, respectively. VC was used as a positive control, and the procedure was the same. Calculate the scavenging rate of SSBP against DPPH free radicals using the following formula.

[0113]

[0114] DPPH is a stable free radical and is often used to evaluate the antioxidant properties of substances. For example... Figure 6 As shown, within the range of 0.2-2.0 mg / ml, the free radical scavenging rate of SSBP increased with increasing concentration, rising from 33.68% to 77.31%, with an IC50 value of 0.69, indicating that SSBP has good DPPH free radical scavenging ability.

[0115] 5.2 Scavenging of ABTS+ free radicals

[0116] Mix an equal volume of 7 mmol / L ABTS solution with 2.45 mmol / L potassium persulfate solution and react in the dark for 12 h. Then dilute with distilled water until the absorbance at 734 nm is 0.70 ± 0.02. Prepare a 2 mg / mL SSBP solution. Set up three replicates for each group, taking 4, 8, 16, 24, 32, and 40 μL of SSBP solution respectively, and bringing the volume to 40 μL with distilled water. Add 160 μL of ABTS solution, mix well, and react in the dark for 30 min. Measure the absorbance at 734 nm. For the blank control group A0 and background group A2, use distilled water instead of the sample solution and ABTS solution, respectively. VC serves as a positive control, and the procedure is the same as above. Calculate the scavenging rate of ABTS free radicals by SSBP using the following formula.

[0117]

[0118] ABTS + Determining free radical scavenging capacity is a commonly used method for evaluating the activity of antioxidants. When a substance with antioxidant activity reacts with ABTS... + During the reaction, electrons or hydrogen atoms are donated to reduce it, resulting in the solution becoming lighter or fading in color.

[0119] like Figure 7As shown, the scavenging ability of SSBP against ABTS free radicals increases with increasing concentration, with an IC50 value of 0.58. Within the range of 0.2-1.6 mg / ml, the scavenging rate of SSBP against ABTS free radicals rapidly increases from 23.19% to 96.65%, and then gradually stabilizes. At a concentration of 2.0 mg / ml, the scavenging rate of SSBP against ABTS free radicals reaches 99.28%, approaching the scavenging rate of VC, indicating that SSBP has good antioxidant capacity.

[0120] 5.3 Scavenging of hydroxyl radicals

[0121] Prepare a 2 mg / mL SSBP solution. Set up three replicates per group, taking 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of SSBP solution into test tubes, respectively, and bringing the volume to 1 mL with distilled water. Add 1 mL of 0.01 mol / L ferrous sulfate solution and 0.01 mol / L salicylic acid-ethanol solution to the test tubes sequentially, mix well, and then add 0.1 mL of 0.03% H₂O₂ solution to initiate the reaction. After shaking well, incubate at 37°C for 30 min, and measure the absorbance at 510 nm. The blank control group A0 and background group A2 use distilled water instead of H₂O₂ solution and sample solution, respectively. VC serves as a positive control, and the same procedure is followed. Calculate the scavenging rate of OH free radicals by SSBP using the following formula.

[0122]

[0123] Hydroxyl radicals are known as the most reactive and harmful free radicals, and are closely related to the occurrence of many diseases. For example... Figure 8 As shown, the scavenging ability of SSBP against OH- radicals is positively correlated with concentration. Within the range of 0.2-2 mg / ml, the scavenging rate of SSBP against ABTS radicals increased from 13.55% to 40.57%, but it still lags significantly behind that of VC.

[0124] Example 6: In vitro anti-inflammatory activity of Sedum sarmentosum polysaccharides

[0125] 6.1 Cell Culture

[0126] Thaw the cryopreserved cells rapidly in a 37°C water bath, then transfer them to centrifuge tubes, add 2 ml of complete culture medium, and centrifuge at 1000 rpm for 5 min. Discard the supernatant, add fresh culture medium, transfer to a sterile culture flask, and incubate in a cell culture incubator (37°C, 5% CO2). Passage the cells when they reach 80% confluence; continue culturing and observe cell status regularly. Cells are ready for subsequent experiments after passage 3.

[0127] 6.2 Investigating the effect of Sedum sarmentosum polysaccharide on the viability of RAW264.7 macrophages using the CCK8 assay

[0128] The experiment included a blank control group, a control group, and a drug-treated group, with three replicates for each group. RAW 264.7 cells in logarithmic growth phase were injected with 1×10⁻⁶ cells / cells. 4 Cells were evenly seeded into 96-well plates at a density of [number] cells / well and cultured for 24 h. The supernatant was discarded, and 200 µL of SSBP solution at different concentrations (0.4, 0.8, 1.2, 1.6, 2.0 mg / mL) and LPS solution at different concentrations (1, 5, 10, 20, 30, 40, 50 μg / mL) were added for pretreatment. After 24 h of culture, the drug-containing medium was removed, and 90 µL of complete medium and 10 µL of CCK-8 solution were added to each well. The plates were then incubated for 2 h. The absorbance (A) of each well was measured at 450 nm, and cell viability was calculated using the following formula: Blank group (A0) contained only medium and CCK-8; control group (A1) contained cells, medium, and CCK-8; and drug-treated group (A2) contained cells, drug, medium, and CCK-8.

[0129]

[0130] like Figure 9 As shown, the cell viability was highest when the LPS concentration was 30 µg / mL. Therefore, this concentration was selected for subsequent experiments.

[0131] As shown in the figure, cell viability increased after treatment with different concentrations of SSBP, stabilizing between 110-120%. This indicates that within this concentration range, SSBP promoted the proliferation of RAW 264.7 cells without cytotoxicity. The cell viability reached its maximum (119.54%) at an SSBP concentration of 1.2 mg / mL, subsequently decreasing slightly. This may be due to the inhibitory effect of high concentrations of polysaccharides on cell growth. Therefore, concentration gradients of 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mg / mL were selected for subsequent experiments.

[0132] 6.3 Effects of Sedum sarmentosum polysaccharide on NO release from RAW264.7 macrophages

[0133] Prepare a 1 mol / L sodium nitrite standard solution and dilute it to concentrations of 1, 2, 5, 10, 20, 40, 60, and 100 μmol / L. Add 50 μL of each of the 0, 1, 2, 5, 10, 20, 40, 60, and 100 μmol / L sodium nitrite standard solutions to a 96-well plate. Then, add 50 μL of Griess reagent I and Griess reagent II to each well sequentially, and measure the absorbance at 540 nm. Plot a standard curve with the sodium nitrite standard concentration on the x-axis and the absorbance value on the y-axis.

[0134] The experiment included a control group, a model group, and an SSBP group. RAW 264.7 cells were cultured at a rate of 2 × 10⁻⁶ cells / mL. 4 After incubation at a density of 100 cells / well in 96-well plates for 24 hours, drug treatment was administered. Different concentrations (0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mg / mL) of SSBP were added to the SS polysaccharide group, while the control and model groups were treated with an equal volume of complete culture medium for 24 hours. LPS was then added to each well to achieve the optimal concentration selected by CCK8 screening. After incubation at 37°C, 95% air, and 5% CO2 for 24 hours, 50 μL of the supernatant from each well was transferred to a new 96-well plate. Equal volumes of room temperature Griess reagent I and Griess reagent II were then added to each well, and the plates were incubated at 37°C for 1–4 hours. The absorbance was then measured at 540 nm using a microplate reader. NO production was calculated using the standard curve method.

[0135] The Griess process generates nitrite (NO) through a reaction. 2- SSBP can be used to indirectly detect NO release, as one of the metabolic products of NO in organisms is nitrite. Macrophages produce nitric oxide (NO) after being stimulated by cytokines, which can be used to indirectly determine the effect of SSBP on the macrophage's response to inflammation. As shown in the figure, in the RAW264.7 macrophage inflammation modeling at a concentration of 30 μg / ml LPS, the NO release in the LPS group was significantly higher than that in the control group, indicating successful modeling. Under the action of SSBP, the NO release from macrophages decreased in a dose-dependent manner, reaching the lowest level at 1.2 mg / ml, a decrease of 74.41%. ELISA results showed that within the concentration range of 0.2-1.2 mg / ml, SSBP significantly reduced the secretion of IL-6, IL-1β, and TNF-α. At a concentration of 1.2 mg / ml, the secretions were reduced by 92.53%, 95.27%, and 95.73%, respectively. Figure 10 The above results all indicate that SSBP has a significant anti-inflammatory effect.

[0136] 6.4 Effects of SSBP on LPS-induced inflammatory factor levels in RAW264.7 cells

[0137] Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria. LPS activation of macrophages and the resulting inflammatory cascade are core components of the host's defense against bacterial infection. In experimental studies and preclinical models, the degree of macrophage inflammation cannot usually be directly measured in vivo. Therefore, quantitative detection of specific inflammatory factors has become a core indirect method for evaluating their activation status and inflammation level. ELISA results are as follows... Figure 11 As shown, compared with the blank group, the LPS group cell supernatant contained significantly higher levels of TNF-α, IL-1β, and IL-6, indicating successful model establishment. Simultaneously, compared with the LPS group, the SSBP dosage group showed significantly lower levels of TNF-α, IL-1β, and IL-6 in a dose-dependent manner. This indicates that SSBP can reduce the levels of inflammatory factors in cells and alleviate damage.

[0138] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A polysaccharide from Sedum sarmentosum, characterized in that, The polysaccharide is an acidic polysaccharide containing uronic acid, with a total sugar content of 85%~87%, a uronic acid content of 35%~37%, a protein content of 1.5%~1.8%, and a molecular weight of 7~8 kDa.

2. The *Sedum sarmentosum* polysaccharide according to claim 1, characterized in that, The monosaccharide composition of the SSBP includes rhamnose (Rha), arabinose (Ara), galactose (Gal), galacturonic acid (Gal-UA), and glucuronic acid (Glc-UA), with a molar ratio of (12~13):(19~20):(18~19):(35~36):(3~4).

3. The *Sedum sarmentosum* polysaccharide according to claim 1, characterized in that, The infrared spectrum of the SSBP has characteristic absorption peaks at 3415 cm⁻¹, 2936 cm⁻¹, 1743 cm⁻¹, 1611 cm⁻¹, and 833 cm⁻¹. The absorption peaks at 1743 cm⁻¹ and 1611 cm⁻¹ correspond to esterified carboxyl groups and free carboxyl groups, respectively, indicating the presence of uronic acid units. The absorption peak at 833 cm⁻¹ indicates the presence of α-glycosidic bonds.

4. A process for extracting Sedum sarmentosum polysaccharide according to any one of claims 1-3, characterized in that, Includes the following steps: 1) Wash the whole plant of Sedum sarmentosum, dry it in an oven and then crush it for later use; 2) Mix Sedum sarmentosum powder with distilled water, heat to extract, then centrifuge, collect the supernatant and concentrate; 3) Add anhydrous ethanol in proportion, let stand overnight, centrifuge, collect the precipitate and redissolve, freeze dry to obtain Sedum sarmentosum crude polysaccharide CSSBP; 4) Reconstitute CSSBP, mix with Sevage reagent to remove impurities; then treat with running water through a dialysis bag and then with static water, collect the dialysate and freeze dry; 5) Dissolve the freeze-dried sample in water, centrifuge, and purify the supernatant by column chromatography; then elute with gradient and collect, freeze, and dry to obtain Sedum sarmentosum polysaccharide SSBP.

5. The extraction process of Sedum sarmentosum polysaccharides according to claim 4, characterized in that, The drying temperature in step 1) is 37°C, and the Sedum sarmentosum powder is passed through a 50-mesh sieve.

6. The extraction process of Sedum sarmentosum polysaccharides according to claim 4, characterized in that, In step 2), the ratio of the powder of *Sedum sarmentosum* to distilled water is 1:20-1:60 (g / mL); the extraction temperature is 60-100℃, the extraction time is 1-5h, and the mixture is concentrated to 1 / 10 of its original volume by rotary evaporation at 65℃.

7. The extraction process of Sedum sarmentosum polysaccharides according to claim 4, characterized in that, In step 3), the ratio of anhydrous ethanol added is 1:4 by volume, and the standing condition is 4°C.

8. The extraction process of Sedum sarmentosum polysaccharides according to claim 4, characterized in that, In step 4), the Sevage reagent is a mixture of n-butanol and chloroform, wherein V 正丁醇 V 三氯甲烷 =1:4; the volume ratio of CSSBP solution to Sevage reagent is 1:5; the dialysis bag is 3500 Da, and the water treatment is carried out for 72 hours. The conditions for static water treatment are 4℃ for 24 hours.

9. The extraction process of Sedum sarmentosum polysaccharides according to claim 4, characterized in that, In step 5), the purification column was DEAE seplife FF, the flow rate was 4 ml / min, and the column was eluted sequentially with pure water, 0.1 M, 0.2 M and 0.3 M NaCl solutions, and all eluents were collected.

10. The application of Sedum sarmentosum polysaccharide SSBP in the preparation of anti-inflammatory drugs or functional foods, characterized in that, The SSBP can inhibit LPS-induced NO release from RAW264.7 macrophages and the secretion of inflammatory factors IL-6, IL-1β, and TNF-α.

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