Preparation and application of fucoidan for promoting healing of diabetic ulcers

Abstract

The application discloses a novel brown algae polysaccharide SARP for promoting healing of diabetic ulcers and a preparation and application thereof. A novel SARP is obtained through Sephacryl S-300 / HR gel column separation and purification, and the molecular weight of the SARP is about 45.4 kDa, which is composed of mannuronic acid (76.56%), guluronic acid (18.89%) and a small amount of glucuronic acid (4.55%). Compared with alginate separated from other brown algae, the SARP has a higher content of mannuronic acid. The SARP shows a better in-vitro antioxidant capacity, and can obviously reverse the cell damage of HUVECs caused by H2O2 and high sugar in the HUVECs. In addition, the SARP can promote the migration and tubular formation of the HUVECs. In-vivo experiments further show that the SARP can promote the healing of diabetic ulcer in rats, and can reduce the oxidative stress, inflammation level and lipid disorder in the rats. The SARP has a simple purification route, can be prepared in a large amount, and is a very promising novel drug for treating diabetic foot ulcers.

Description

I. Technical Field

[0001] This invention relates to the application of a novel brown algae polysaccharide (SARP) derived from Sargassum kjellmanianum in the treatment of diabetic ulcers, and belongs to the field of traditional Chinese medicine. II. Background Technology

[0002] Diabetic foot ulcers are one of the most serious complications of diabetes, a major cause of disability and even death among diabetic patients, causing not only pain but also a significant financial burden. Statistics show that 15% of diabetic patients will develop foot ulcers in their lifetime, and globally, a leg is amputated every 30 seconds due to diabetic foot. The causes of diabetic foot ulcers are complex, and currently there is no effective treatment. Existing treatments have low clinical cure rates and long treatment cycles. More seriously, the high 5-year recurrence rate of diabetic foot ulcers significantly reduces the effectiveness of existing treatments, making the search for new drugs to treat diabetic foot ulcers an urgent priority.

[0003] Brown algal polysaccharides, primarily derived from the cell walls of brown algae, are mainly found in seaweeds such as kelp, *Sargassum jellmanianum*, giant kelp, and *Fucus vesiculosus*. They possess unique advantages such as water solubility, non-cytotoxicity, and non-immunogenicity. Reportedly, brown algal polysaccharides exhibit excellent anti-inflammatory and antioxidant activities and have been widely used in food and pharmaceutical fields. In this invention, we isolated a novel type of brown algal polysaccharide from *Sargassum jellmanianum* (SARP), which has a high mannulic acid content. We evaluated its therapeutic effect on diabetic foot ulcers and elucidated its mechanism of promoting diabetic ulcer healing.

[0004] This invention does not previously demonstrate the application of SARP in diabetic foot ulcers. Furthermore, SARP has a simple purification route, can be prepared in large quantities, and can promote the healing of ulcers in diabetic rats, making it a very promising novel drug for treating diabetic foot ulcers. III. Summary of the Invention

[0005] One of the objectives of this invention is to elucidate the purification and preparation process of SARP.

[0006] The second objective of this invention is to evaluate the physicochemical properties and structural characteristics of SARP.

[0007] The third objective of this invention is to evaluate the effect of SARP in promoting the healing of diabetic ulcers.

[0008] The fourth objective of this invention is to elucidate the mechanism by which SARP promotes the healing of diabetic ulcers.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] High-purity SARP was purified and prepared using modern chromatographic separation techniques.

[0011] The molecular weight and purity of SARP were determined using high-performance gel permeation liquid chromatography.

[0012] The monosaccharide composition of SARP was determined using PMP pre-column derivatization technique.

[0013] The infrared spectral characteristics of SARP were detected using KBr pellet compression technology.

[0014] SEM technology was used to detect the morphological characteristics of SARP.

[0015] NMR technology was used to detect SARP. 1 H, 13 C characteristic.

[0016] The SRB technique was used to detect the in vitro reversal effect of SARP on the oxidative stress in H2O2 and high-sugar HUVECs.

[0017] The effects of SARP on HUVECs cell migration were detected using the scratch and transwell assays.

[0018] We used tubule generation technology to investigate the effect of SARP on angiogenesis in HUVECs.

[0019] Using a diabetic rat wound model, the effect of SARP on ulcer healing in diabetic rats was investigated.

[0020] Immunohistochemistry was used to detect the effect of SARP on wound angiogenesis in diabetic rats.

[0021] The effects of SARP on blood biochemistry in diabetic rats were detected using a fully automated blood biochemistry analyzer.

[0022] Compared with the prior art, the advantages and technical effects of the present invention are as follows: The present invention purifies and prepares a novel alginate SARP from Sargassum kjellmanianum, demonstrating for the first time the role of SARP in promoting the healing of diabetic ulcers, and systematically elucidating its mechanism of promoting the healing of diabetic ulcers.

[0023] SARP has a molecular weight of approximately 45.4 kDa and is composed of mannuronic acid (76.56%), guluronic acid (18.89%), and a small amount of glucuronic acid (4.55%). Compared with alginate isolated from other brown algae, SARP has a higher mannuronic acid content. SARP exhibits significant antioxidant activity in vitro, inhibiting HUVEC cell damage induced by H2O2 and high glucose. SARP also promotes HUVEC cell migration and angiogenesis in vitro. We further evaluated the effect of SARP on ulcer healing in diabetic rats, finding that SARP significantly promoted ulcer healing in diabetic rats. Further experiments demonstrated that SARP reduced oxidative stress, inflammation levels, and lipid disorders in diabetic rats. These findings indicate that SARP has a good therapeutic effect on diabetic foot ulcers and has broad application prospects. IV. Description of the attached drawings

[0024] Figure 1 This indicates that SARP in this invention is separated and purified using a Sephacryl S-300 / HR gel column.

[0025] Figure 2 This indicates that the SARP in this invention has high purity and a molecular weight of approximately 45.4 kDa.

[0026] Figure 3 This indicates that the SARP in this invention is composed of mannuronic acid (76.56%), guluronic acid (18.89%), and a small amount of glucuronic acid (4.55%).

[0027] Figure 4 This indicates that mannuronic acid and guluronic acid in SARP in this invention... 1 H, 13 Chemical shift value in C.

[0028] Figure 5 This indicates that SARP in this invention can scavenge free radicals in vitro and reverse the damage to HUVECs cells caused by H2O2 and high glucose.

[0029] Figure 6 This indicates that SARP in this invention can promote the migration of HUVECs.

[0030] Figure 7 This indicates that SARP in this invention can promote angiogenesis in HUVECs.

[0031] Figure 8 This indicates that SARP in this invention can promote the healing of skin ulcers in diabetic rats.

[0032] Figure 9This indicates that SARP in this invention can reduce oxidative stress in diabetic rats.

[0033] Figure 10 This indicates that SARP in this invention can promote angiogenesis at the wound site in diabetic rats.

[0034] Figure 11 This indicates that SARP in this invention can reduce inflammation levels in diabetic rats.

[0035] Figure 12 This indicates that the SARP of the present invention can alleviate lipid metabolism disorders in diabetic rats. V. Detailed Implementation Methods

[0036] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and test examples.

[0037] Experimental Example 1

[0038] The elution curve of SARP on a Sephacryl S-300 gel chromatography column was determined.

[0039] Reagents: Ammonium bicarbonate, sulfuric acid, phenol (Sinopharm Group, China)

[0040] Instrumentation: Sephacryl S-300 gel chromatography column (2.6 × 100 cm), automated collector (Bio-Rad Model 2110, USA)

[0041] Methods: The crude polysaccharide powder was dissolved in distilled water (20 mg / mL) and further purified using a Sephacryl S-400 gel column. The eluent was 0.2 mol / L NH4HCO3 at a flow rate of 0.3 mL / min. The eluent was automatically collected, and the sugar content was determined by the sulfuric acid-phenol method. Elution curves were plotted, and the peaks of the curves were collected. The polysaccharide was concentrated, deammoniated under reduced pressure, and lyophilized to obtain a homogeneous polysaccharide, which was named SARP.

[0042] Test results Figure 1 This indicates that SARP was prepared by collecting the peaks with high absorbance, concentrating, dialyzing, and lyophilizing.

[0043] Experimental Example 2

[0044] The molecular weight and purity of SARP were determined.

[0045] Reagents: Anhydrous Na2SO4; Dextran standards Mw: 5.9, 9.6, 21.1, 47.7, 107, 200, 344, 708 kDa (Showa Denko KK Corporation, Japan).

[0046] Instruments: Agilent 1290 high performance liquid chromatograph; RID-10A differential detector; Shodex Ohpak SB-804HQ column.

[0047] Experimental Methods: A standard curve was plotted with the logarithm of the weight-average molecular weight (logMw) of the standard as the ordinate and retention time (RT) as the abscissa to obtain a linear regression equation. 2 mg of polysaccharide was accurately weighed and added to 400 μL of Na2SO4 solution to obtain a 5 mg / mL polysaccharide sample. The sample was filtered through a 0.22 μm filter, and the retention time was measured by HPGPC. The weight-average molecular weight of the sample was calculated based on the linear regression equation.

[0048] Test results Figure 2 This indicates that the SARP has high purity and a molecular weight of 45.4 kDa.

[0049] Experimental Example 3

[0050] The monosaccharide composition of SARP was determined.

[0051] Reagents: 1-Phenyl-3-methyl-5-pyrazolone (PMP); trifluoroacetic acid, methanol (Sinopharm Group, China); monosaccharide standards: mannose, glucosamine, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, fucose (Sigma-Aldrich, USA); mannuronic acid and guluronic acid (Yuanye Biotechnology Co., Ltd., China); chromatographic grade acetonitrile (Sinopharm Group, China).

[0052] Instruments: Agilent 1290 high performance liquid chromatograph; DAD ultraviolet detector; Eclipse XDB-C18 (5μm, 4.6μm×25.0cm) column.

[0053] Experimental Methods: Polysaccharide degradation products and isomonosaccharide standards were thoroughly dissolved in 100 μL of distilled water, followed by the addition of 100 μL of 0.3 mol / L NaOH and 120 μL of 0.5 mol / L PMP methanol solution. The mixture was reacted in a water bath at 70 °C for 60 min. After the reaction was completed and cooled to room temperature, 100 μL of 0.3 mol / L HCl solution was added for neutralization. The mixture was extracted three times with dichloromethane to remove unreacted PMP. The supernatant was filtered through a 0.22 μm microporous membrane for later use. High-performance liquid chromatography (HPLC) conditions: Mobile phase: acetonitrile:phosphate buffer (pH 6.7) = 17:83 (v / v volume ratio); injection volume: 10 μL; column temperature: 35 °C; detector: UV detector (254 nm); flow rate: 1.0 mL / min. The monosaccharide composition and proportion of SARP were determined by comparing the peak time and peak area with those of the standards.

[0054] Test results Figure 3The results indicate that it is composed of mannuronic acid, guluronic acid and a small amount of glucuronic acid, with contents of 76.56%, 18.89% and 4.55%, respectively.

[0055] Test Example 4

[0056] SARP was measured 1 H, 13 C characteristics

[0057] Reagents: Deuterated heavy water, deuterated acetone (Sigma, USA)

[0058] Instrument: Agilent DDZ 500MHz nuclear magnetic resonance spectrometer

[0059] Experimental method: 60 mg of polysaccharide sample was dissolved in 0.5 mL L₂O, lyophilized, and this operation was repeated three times. After centrifugation, the sample was placed in an NMR tube, and an appropriate amount of deuterated acetone was added as an internal standard. One-dimensional NMR spectroscopy was performed using an Agilent DDZ 500 MHz NMR spectrometer. 1 H-NMR, 13 C-NMR (nuclear magnetic resonance) spectroscopy.

[0060] Test results Figure 4 This indicates that SARP's 1 The H-NMR spectrum showed two anomeric hydrogens at 5.09 ppm and 4.67 ppm, with the 5.09 ppm signal attributed to H-1 of α-L-guluronic acid and the strong signal at 4.67 ppm attributed to H-1 of β-D-mannuronic acid. The resonance at 4.54 ppm was caused by H-5 of α-L-guluronic acid. Furthermore, according to… 1 The 1H NMR data allowed for the derivation of the M:G ratio. The result was consistent with the HPLC monosaccharide composition analysis, showing a ratio of 4.02:1.00. SARP's... 13 The C2-C6 NMR spectrum includes three regions: the anomeric carbon region is 100.2-101.9 ppm, the C2-C6 region is 65.3-80.7 ppm, and the carbon signal region of the carboxyl group is at 176.6 ppm.

[0061] Experimental Example 5

[0062] The study investigated the free radical scavenging ability of SARP and the protective effect of different concentrations of SARP on cells under oxidative stress.

[0063] Cells: Human umbilical vein endothelial cells (HUVECs), purchased from the China Center for Type Culture Collection.

[0064] Reagents: DMEM dry powder culture medium (GIBCO, USA); PBS; SRB (Shanghai Solarbio Biotechnology Co., Ltd.); H2O2, glucose (Beyotime Biotechnology Co., Ltd.).

[0065] Instruments: CO2 incubator (Heraeus, Germany); inverted microscope (Olympus, Japan); clean bench (Heraeus, Germany); microplate reader (Bio-Tek, USA).

[0066] Experimental Methods: In vitro total antioxidant capacity assay: Dilute the standard with distilled water or sample preparation solution. Add 20 μL of peroxidase working solution to each well of a 96-well plate. Add 10 μL of distilled water to the blank control wells; add 10 μL of Trolox standard solutions of various concentrations to the standard curve wells; add 10 μL of various samples to the sample wells. Mix gently. Add 170 μL of ABTS working solution to each well and mix gently. After incubation at room temperature for 6 minutes, measure the OD value at 414 nm. Determine the antioxidant capacity of SARP according to the standard curve.

[0067] Experimental Methods: SRB Method: HUVECs in the logarithmic growth phase were seeded into 96-well plates. After cell adhesion, different concentrations of SARP were added to the experimental groups, while DMSO was added to the control group at a proportionally diluted level. Doxorubicin was used as a positive control (concentration of 1 μM). The 96-well plates were incubated at 37°C with 5% CO2 for 24 h. After incubation, 500 μM H2O2 was added to each well for 3 h. After incubation, the supernatant was discarded, and 100 μL of pre-chilled 10% TCA was added to fix the cells. The cells were allowed to stand for 5 min, then fixed at 4°C for at least 1 h. The cells were rinsed 5–6 times with tap water and allowed to air dry or dried in a 37°C incubator. 100 μL of prepared SRB dye was added to each well and stained at room temperature for 15 min. The cells were washed 5–6 times with 1% glacial acetic acid to remove excess dye and allowed to air dry or dried in a 37°C incubator. Finally, add 150 μL / well of 10 mM Tris-HCl solution and incubate at 37 °C for 10 min. Shake with a microplate reader for 3–5 min and measure the absorbance (OD) at 515 nm or 540 nm using a microplate reader.

[0068] Experimental methods: Establishment of high glucose cell model: HUVECs in the logarithmic growth phase were seeded into 96-well plates. After cell adhesion, the culture medium was removed and the cells were washed twice with PBS. The culture medium for the high glucose group was replaced with glucose containing 300 mM, and different concentrations of SARP were added and cultured for 24 h. After culture, cell viability was detected by SRB method.

[0069] according to Figure 5Experimental results showed that SARP can significantly scavenge free radicals in vitro. In H2O2 and high glucose-induced oxidative stress cell models, SARP can significantly reverse cell damage caused by H2O2 and high glucose, indicating that SARP can protect cells from oxidative stress damage.

[0070] Experimental Example 6

[0071] To investigate the effects of SARP on HUVEC cell migration.

[0072] Cells: Human umbilical vein endothelial cells (HUVECs), purchased from the China Center for Type Culture Collection.

[0073] Reagents and consumables: DMEM dry powder culture medium (GIBCO, USA); PBS; ROS reagent kit (Beyotime Biotechnology Co., Ltd.); Transwell chambers (Corning, USA).

[0074] Instruments: CO2 incubator (Heraeus, Germany); inverted microscope (Olympus, Japan); clean bench (Heraeus, Germany).

[0075] Experimental method: Scratch assay: Cells in the logarithmic growth phase were taken and their density adjusted to 3–4 × 10⁻⁶. 4 ×10 5 Cells were seeded at a rate of 100 μL / well in 96-well plates and incubated at 37°C for 24 h. When the cell adhesion density reached 80-90%, a 10 μL pipette tip was used to make a central scratch in each well. After washing twice with PBS, the medium was replaced with fresh serum-free medium, and different concentrations of SARP were added for treatment. The central scratched area was photographed under a microscope, and then photographed every 12 hours thereafter. Cell migration rate was calculated as follows: Cell migration rate % = (edge ​​distance 0h - edge distance th) / edge distance 0h × 100.

[0076] Experimental method: Transwell assay: Cells in logarithmic growth phase were seeded into transwell chambers at a density of 8000 cells / well. Different concentrations of SARP were added to the chambers, with a serum concentration of 1% in the upper chamber and a corresponding concentration of SARP (10% serum concentration) in the lower chamber. The transwell chambers were incubated for 24 hours. After incubation, the culture medium was discarded, and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde for 15 minutes, washed three times with PBS after fixation, and stained with 0.1% crystal violet at room temperature for 15 minutes. After wiping the cells from the upper chamber with a cotton swab, the cells that had migrated to the lower chamber were photographed under a microscope and destained with 33% acetic acid. The OD value was measured at 570 nm.

[0077] according to Figure 6 The experimental results showed that, compared with the control group, the migration rate of scratched cells towards the center was significantly increased after 24 hours of SARP treatment, indicating that SARP can promote cell migration. To further demonstrate the effect of SARP on HUVECs cell migration, cells were placed in a transwell, and it was found that after the addition of SARP, the number of cells migrating to the lower chamber was significantly increased. These experiments demonstrate that SARP can promote HUVECs migration.

[0078] Experimental Example 7

[0079] The effect of SARP on K562 on tubule formation was investigated.

[0080] Cells: Human umbilical vein endothelial cells (HUVECs), purchased from the China Center for Type Culture Collection.

[0081] Reagents and consumables: DMEM dry powder culture medium (GIBCO, USA); PBS; trypsin (Beyotime Biotechnology Co., Ltd.); Matrigel (Corning, USA)

[0082] Instruments: CO2 incubator (Heraeus, Germany); inverted microscope (Olympus, Japan); clean bench (Heraeus, Germany).

[0083] Experimental Method: Tube Formation Method: Matrigel was melted to liquid state at 4℃. 50 μL was spread evenly in a 96-well plate and incubated at 37℃ for solidification. Cells in the logarithmic growth phase were taken and mixed with different concentrations of SARP at a density of 10,000 cells / well. The cells were then seeded onto Matrigel. After 3 hours, tube formation was observed under a microscope.

[0084] according to Figure 7 The experimental results showed that, compared with the control group, HUVECs formed a distinct network structure after the addition of SARP, while the control group had not yet formed a complete luminal structure. At the same time, we counted the nodes, and our results indicate that SARP can promote the formation of tubules in HUVECs, suggesting that SARP can promote angiogenesis.

[0085] Experimental Example 8

[0086] The in vivo effect of SARP on the healing of skin ulcers in diabetic rats was investigated.

[0087] Animals: SD rats (male, 200–220g), purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.

[0088] Reagents: streptozotocin (STZ, Sigma, USA); sodium citrate (Solepro Technology Co., Ltd.).

[0089] Instruments and consumables: Clean bench (Heraeus GmbH, Germany); Syringe (Henan Shuguang Jianshi Medical Instrument Group Co., Ltd.).

[0090] Experimental Methods: Diabetic Model Establishment: SD rats were fasted overnight. STZ was dissolved in a pH 4.5 sodium citrate solution, protected from light, and placed on ice. SD rats were intraperitoneally injected with 50 mg / kg over 5 minutes. Food was given 3 hours after injection. Diabetic symptoms were observed during the period, and blood glucose was measured on the third day after STZ injection. Only rats with blood glucose ≥16.67 mmol / L were used for subsequent experiments.

[0091] Experimental method: Diabetic wound ulceration method: The back hair of diabetic rats was shaved and anesthetized. In a sterile operating table, a wound of the same diameter was made on each side of the back using a biopsy punch. Each rat was housed alone to prevent bacterial infection.

[0092] Experimental Methods: Animal grouping and administration method: Rats were divided into 3 groups of 6 rats each: (1) control group; (2) STZ group; (3) STZ+SARP group. SARP powder was dissolved in PBS and filtered through a 0.22 μM sterile filter membrane. SARP (200 mg / kg) was injected intraperitoneally and administered continuously for 10 days. The wound diameter was measured and photographed regularly.

[0093] according to Figure 8 The experimental results showed that, compared with the control group, the wound healing of rats in the STZ group was significantly slower, with deeper and larger wounds, indicating a slower healing of diabetic ulcers; while after administration of SARP, compared with the STZ group, the wound depth and diameter of the wounds were smaller after SARP treatment, indicating that SARP can promote the healing of diabetic ulcers.

[0094] Experimental Example 9

[0095] The effects of SARP on oxidative stress in diabetic rats were investigated.

[0096] Animals: SD rats (male, 200–220g), purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.

[0097] Instruments and consumables: Inverted microscope (Olympus Corporation, Japan); capillary tubes, blood collection needles, and vacuum blood collection anticoagulant tubes (Jiangxi Saihua Technology Co., Ltd.); syringes (Henan Shuguang Jianshi Medical Instrument Group Co., Ltd.); lipid oxidation (MDA) detection kit, glutathione detection kit, and SOD detection kit (Beyotime Biotechnology Co., Ltd.).

[0098] Experimental method: Rat ocular venous blood was collected, mixed by inversion, and allowed to stand at room temperature for 20 min. After centrifugation at 4℃ and 600g for 10 min, the supernatant was transferred to another new 1 mL centrifuge tube. MDA, glutathione and SOD were measured according to the kit instructions.

[0099] according to Figure 9 The experimental results showed that, compared with the control group, the STZ group rats had increased oxidative stress levels, manifested as increased MDA and GSSG levels and decreased SOD levels. After SARP treatment, the oxidative stress levels of diabetic rats decreased, manifested as decreased MDA and GSSG levels and increased SOD levels. This indicates that SARP can improve oxidative stress in diabetic rats and thus promote the healing of diabetic ulcers.

[0100] Experimental Example 10

[0101] The effects of SARP on wound angiogenesis in diabetic rats were investigated.

[0102] Animals: SD rats (male, 200–220g), purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.

[0103] Reagents: Hematoxylin, CD31 antibody (Wuhan Saiweier Biotechnology Co., Ltd.)

[0104] Instruments and consumables: scissors; inverted microscope (Olympus Corporation, Japan).

[0105] Experimental Methods: Each group of wounds and surrounding normal skin were cut with scissors to prepare 5μm sections. The sections were dewaxed and hydrated. After antigen retrieval, 5% normal serum (of the same or similar origin as the secondary antibody) was added to the slides, and the slides were blocked at 37℃ for 30 min. Then, diluted primary antibody was added and incubated overnight at 4℃. The slides were washed three times with PBS for 3 min each time. After drying with absorbent paper, horseradish peroxidase-labeled secondary antibody was added, and the slides were incubated at 37℃ for 30 min. The slides were washed four times with PBS for 3 min each time. After discarding the PBS, the slides were dried with absorbent paper. Freshly prepared DAB chromogenic solution was added to each slide, and the slides were observed under a microscope. A positive signal was brownish-yellow or brownish-red. The slides were rinsed with tap water to stop the chromogenic process. Hematoxylin counterstaining was performed. Finally, the slides were dehydrated, fixed, mounted, and observed under a microscope.

[0106] according to Figure 10 The experimental results showed that, compared with the control group, the STZ group had significantly reduced angiogenesis, which was manifested by less brownish-yellow signal, while SARP can promote angiogenesis.

[0107] Experimental Example 11

[0108] The effects of SARP on inflammation in diabetic rats were investigated in vivo.

[0109] Animals: SD rats (male, 200–220g), purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.

[0110] Instruments and consumables: capillary tubes, blood collection needles, vacuum blood collection anticoagulant tubes (Jiangxi Saihua Technology Co., Ltd.), syringes (Henan Shuguang Jianshi Medical Instrument Group Co., Ltd.).

[0111] Experimental method: Blood was collected by a lancet and placed in a vacuum blood collection anticoagulant tube. After being slowly mixed, the changes in the number of each cell were detected by a fully automated blood biochemistry analyzer.

[0112] according to Figure 11 The experimental results showed that, compared with the control group, the STZ group had a significantly higher level of inflammation, manifested by an increase in white blood cells, lymphocytes, and monocytes. After SARP treatment, the inflammatory symptoms were significantly reduced and the number of inflammatory cells decreased. The above experiments indicate that SARP can reduce the level of inflammation in diabetic rats.

[0113] Experimental Example 12

[0114] The effects of SARP on lipid metabolism in diabetic rats were investigated.

[0115] Animals: SD rats (male, 200–220g), purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.

[0116] Instruments and consumables: capillary tubes, blood collection needles, vacuum blood collection anticoagulant tubes (Jiangxi Saihua Technology Co., Ltd.), syringes (Henan Shuguang Jianshi Medical Instrument Group Co., Ltd.).

[0117] Experimental method: Blood was collected by a lancet and placed in a vacuum blood collection anticoagulant tube. After being slowly mixed, the changes in the number of each cell were detected by a fully automated blood biochemistry analyzer.

[0118] according to Figure 12 The experimental results showed that the STZ group had elevated blood lipids, manifested as elevated serum total cholesterol, triglycerides, and low-density lipoprotein. After SARP treatment, serum total cholesterol, triglycerides, and low-density lipoprotein decreased overall, indicating that SARP can alleviate lipid metabolism disorders in diabetic rats.

Claims

1. Sargassum kjellmanianum The application of SARP, a polysaccharide derived from brown algae, in the preparation of formulations that promote the healing of diabetic ulcers, is characterized by, The SARP has a molecular weight of 45.4 kDa and is composed of 76.56% mannuronic acid, 18.89% guluronic acid and 4.55% glucuronic acid.

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