Malan fructan and preparation method and application thereof

Abstract

The application discloses Malania oleifera polysaccharide as well as a preparation method and application thereof. Experimental research proves that the Malania oleifera polysaccharide can promote macrophages to polarize into M2 phenotype, reduce the expression of proinflammatory factors, increase the expression of anti-inflammatory factors, promote epithelialization, collagen deposition and accelerate angiogenesis on a wound surface, has a significant healing-promoting effect on the wound surface, can be prepared into various clinical application dosage forms such as gels and dressings, has excellent biocompatibility and high safety, is suitable for a wide range of application scenarios, can be used for repairing and intervention of various acute and chronic wounds, has important clinical value and market prospect, and provides a safe and efficient new option for clinical wound treatment.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine chemistry and wound repair technology, specifically relating to a koran polysaccharide, its preparation method, and its application. Background Technology

[0002] The skin is the body's first physiological barrier against external aggressors, and wound healing is a complex cascade of physiological processes involving inflammation regulation, cell proliferation, angiogenesis, and collagen remodeling. Intractable wounds, a common and difficult-to-treat clinical condition, are often induced by factors such as trauma, infection, diabetes, and aging. Conventional interventions result in long healing cycles and poor repair outcomes, making them highly susceptible to secondary infections and tissue necrosis. This severely reduces patients' quality of life and increases the burden on healthcare, making it a core challenge that urgently needs to be addressed in the field of wound repair.

[0003] Current clinical wound treatments have many limitations. Chemically synthesized anti-inflammatory drugs are prone to drug resistance and adverse reactions, while biological growth factor preparations suffer from poor stability, high costs, and difficulty in synergistically regulating multiple pathological processes. Although traditional Chinese medicine compound formulas or crude extracts have certain repair effects, their complex composition, unclear active ingredients, and poor quality control make them unsuitable for the research and application requirements of modern precision pharmacy, greatly limiting their clinical translation and large-scale application.

[0004] As a traditional medicinal and edible plant, *Kalimeris indica* has a long history of medicinal use. The ancient medical text *Ben Cao Zheng Yi* clearly records: "Kalimeris indica is most effective in clearing heat and toxins, specifically targeting the blood to stop bleeding and cool the blood, with particular strengths." Its traditional effects of clearing heat and toxins, cooling the blood and stopping bleeding, reducing swelling and promoting tissue regeneration suggest that *Kalimeris indica* has potential development value in the field of wound repair. However, current research on *Kalimeris indica* mainly focuses on basic pharmacological investigations of crude extracts, with a lack of research on the isolation, purification, structural identification, and mechanisms of its single active components; in particular, the regulatory role and molecular mechanism of *Kalimeris indica* polysaccharides in wound healing have not yet been reported in the literature. Summary of the Invention

[0005] To address the problems of the prior art, the present invention aims to provide a malachite polysaccharide, its preparation method, and its application. This malachite polysaccharide has a significant wound-healing effect and can be formulated into various clinically applicable dosage forms such as gels and dressings. It has both excellent biocompatibility and high safety, and has good clinical application prospects.

[0006] This invention is achieved through the following technical solution:

[0007] A kilane fucose, characterized in that the kilane fucose has the structure shown in formula (I):

[0008]

[0009] (I)

[0010] Wherein, β-D-Fruf is β-D-fructofuranose, α-D-Glcp is α-D-glucopyranose, and n is 17.

[0011] The weight-average molecular weight of the malachite polysaccharide provided by this invention is 3078 Da, and the monosaccharides are mainly composed of fructose and glucose, with molar percentages of 95.71% and 4.07%, respectively.

[0012] The present invention also provides a method for preparing the above-mentioned kilan kojisan, comprising the following steps:

[0013] (1) Soak the powdered Malan medicinal material in 95% ethanol to remove grease, filter, take the residue, and evaporate the ethanol;

[0014] (2) The residue was extracted with water and precipitated with alcohol to obtain a crude extract;

[0015] (3) The crude extract was subjected to protein removal by sevage method, decolorization by activated carbon, dialysis and drying to obtain crude polysaccharide of Malan;

[0016] (4) The crude polysaccharide of Malan was purified by DEAE agarose gel FF column, eluted with distilled water, and the elution peak was tracked and detected by phenol-sulfuric acid method. The eluent was collected, concentrated under reduced pressure, dialyzed and dried to obtain Malan fructan.

[0017] As a preferred technical solution of the present invention, in step (2), the material-to-liquid ratio of the water extraction is 1:20-30g / ml, the extraction time is 1-2h, and the number of extractions is 2-3 times; the alcohol precipitation is to add 3-4 times the volume of anhydrous ethanol and let it stand overnight at 4 ℃.

[0018] As a preferred technical solution of the present invention, in step (3), the activated carbon decolorization is to add 1-2% of activated carbon in the solution volume and decolorize at 35-45℃ for 20-40 min.

[0019] As a preferred technical solution of the present invention, in step (3) or step (4), the dialysis is performed using a dialysis bag with a molecular weight cutoff of 3500 Da, with distilled water for 48 h, and the water is changed every 12 h.

[0020] This invention also provides the application of the above-mentioned kilanin in the preparation of drugs that promote wound healing. Experimental studies have confirmed that the kilanin provided by this invention can promote macrophage polarization to the M2 phenotype, reduce the expression of pro-inflammatory factors, and increase the expression of anti-inflammatory factors. It promotes wound epithelialization, collagen deposition, and accelerates angiogenesis, thus having a significant wound-healing effect.

[0021] The wounds described in this invention include, but are not limited to, acute wounds, diabetic wounds, burns, pressure injuries, and chronic ulcers.

[0022] The present invention also provides a medicament for promoting wound healing, comprising an effective amount of the above-mentioned kilan glycosides and a pharmaceutically acceptable carrier.

[0023] The pharmaceutically acceptable carriers described in this invention include, but are not limited to, lubricants, fillers, binders, disintegrants, pH adjusters, surfactants, antioxidants, etc., in amounts that are conventional in the art.

[0024] The drug dosage forms of the present invention can be prepared according to conventional methods in the art, and the drug dosage forms include gels, medical dressings, patches, ointments, creams, powders or sprays, etc.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] The malachite polysaccharide provided by this invention can regulate the wound repair process through multiple synergistic pathways, and has a significant effect on promoting wound healing. Specifically, it significantly inhibits the release of pro-inflammatory factors such as TNF-α and IL-6, effectively reducing excessive inflammatory response in the wound; induces macrophages to polarize into the M2 (repair-oriented) type, upregulates the expression of M2 markers such as CD206, Arg-1, and IL-10, and accelerates inflammation resolution; promotes the proliferation and migration of vascular endothelial cells, and accelerates granulation tissue formation; upregulates the expression of angiogenic factors such as VEGF, increases the number of new blood vessels in the wound, and improves local blood supply to the wound; promotes the formation and orderly arrangement of collagen fibers, promotes wound re-epithelialization, and accelerates wound healing.

[0027] The koran polysaccharide provided by this invention is a single purified active ingredient with clearly defined pharmacological effects and easily controllable quality standards. It can be formulated into various clinically applicable dosage forms such as gels and dressings, and has both excellent biocompatibility and high safety. It is applicable to a wide range of scenarios and can be used for the repair and intervention of various acute and chronic wounds. It has important clinical value and market prospects, and provides a safe and efficient new option for clinical wound treatment. Attached Figure Description

[0028] Figure 1 (a) is the high-performance gel permeation chromatogram of the KIP-W of the present invention; (b) is the infrared spectrum; (cd) is the high-performance anion exchange chromatogram.

[0029] Figure 2 The diagram shows the in vitro anti-inflammatory effect of KIP-W in this invention (b represents the ability of KIP-W to promote macrophage proliferation, a and ce represent the expression of M1 / M2 protein as determined by Western blot, and fj represents the expression of M1 / M2 gene as determined by ELISA).

[0030] Figure 3 This is a diagram showing the migration of HUVECs promoted by KIP-W according to the present invention;

[0031] Figure 4 The images show the appearance of mice treated with KIP-W to promote wound healing and the statistical chart of the healing rate.

[0032] Figure 5 These are staining and semi-quantitative images of HE (a, c) and Masson (b, d) wounds treated with KIP-W according to the present invention. Detailed Implementation

[0033] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Example 1: Preparation of Malan fructan

[0035] (1) Raw material pretreatment: Fresh Malan medicinal material is washed to remove mud and sand, crushed into powder, and passed through No. 2 sieve for later use; it is dried at 60℃ to constant weight, and soaked overnight in 95% ethanol at a material-to-liquid ratio of 1:40 to remove lipids; the soaked Malan is filtered, the residue is retained, and the ethanol is recovered.

[0036] (2) Water extraction and alcohol precipitation: Add distilled water and decoct at a ratio of 1:25 for 1.5 hours each time. Extract 3 times in total, filter, combine the filtrates, concentrate the filtrate to 1 / 5 of the original volume using a rotary evaporator, add 3 times the volume of pre-cooled pure ethanol, precipitate overnight at 4 °C, filter, and take the precipitate.

[0037] (3) Protein removal, decolorization, and dialysis: The precipitate was washed with anhydrous ethanol and dissolved in an appropriate amount of distilled water. Then, it was reacted with Sevage reagent (chloroform: n-butanol = 5:1) with shaking for 30 min, centrifuged at 3000 r / min for 15 min, and the supernatant was collected to remove the precipitate. This operation was repeated until there was no separation between Sevage reagent and distilled water. The obtained supernatant was slowly added with 3 times the volume of pre-cooled pure ethanol and precipitated overnight at 4 ℃. After centrifugation, the precipitate was dissolved in an appropriate amount of distilled water and activated carbon was added at a ratio of 1.5%. The precipitate was decolorized at 40 ℃ for 30 min, centrifuged, and the supernatant was collected. The aqueous phase after protein removal was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with distilled water for 48 h, with the water changed every 12 h to remove small molecule impurities and salts. After dialysis, the precipitate was freeze-dried to obtain crude polysaccharide (KIP).

[0038] (4) Purification: After equilibration overnight on a DEAE agarose gel FF column, weigh approximately 5g of crude kohlii polysaccharide and dissolve it in 10 mL of distilled water. Centrifuge at 12000 r / min for 10 min and then aspirate the solution. When loading the solution, guide it slowly along the column arm with a glass rod. Elute with distilled water and use the phenol-sulfuric acid method to track and detect the elution peak. Collect the eluent, concentrate it under reduced pressure, and dialyze it in distilled water for 2 days using a 3500 Da dialysis tape, changing the distilled water regularly during the process. Concentrate under reduced pressure and freeze-dry to obtain kohlii fructan (KIP-W).

[0039] Example 2: Structural Identification of Malan Fructan

[0040] (1) Molecular weight

[0041] High-performance gel permeation chromatography (HPGPC) was used to analyze kilan grosvenorii (KIP-W). The results showed a single symmetrical peak (Figure 1a), indicating that the molecular weight distribution of KIP-W was uniform and its molecular weight was determined to be 3078 Da.

[0042] (2) Monosaccharide composition analysis

[0043] High-performance anion exchange chromatography (HPAEC) was used to determine the monosaccharide composition of KIP-W. The chromatogram is shown in Figure 1 (cd). By comparison with monosaccharide standards, fructose (Fru) was identified as the major component, with a small amount of glucose (Glc), at molar percentages of 95.71% and 4.07%, respectively. These results indicate that KIP-W is mainly composed of fructose.

[0044] (3) Infrared spectroscopy detection

[0045] Depend on Figure 1 The infrared spectral results of medium b show that 3377 cm⁻¹ -1 The broad peak at 2832 cm⁻¹ is attributed to the O–H stretching vibration associated with hydrogen bonding. -1 The weak absorption peak at 1453 cm⁻¹ corresponds to the C–H stretching vibrations of the –CH₂ and –CH₃ groups. -1 The absorption peak at 1597 cm⁻¹ corresponds to the C–O stretching vibration. -1 The absorption peak at 1033 cm⁻¹ is related to the O–H bending vibration of KIP-W in aqueous solution. -1 The peak at 1740 cm⁻¹ is a characteristic absorption peak for the C–O–C glycosidic bond vibration. Notably, this peak is also observed at 1740 cm⁻¹. -1 The absence of characteristic absorption peaks for the C=O stretching vibration of carboxylic acid groups in the vicinity indicates that the purified polysaccharide is mainly composed of neutral monosaccharides.

[0046] (4) Methylation analysis

[0047] Methylation analysis was used to elucidate the glycosidic bond linkage mode of kohlii polysaccharide. The specific method was as follows: 1 mg of KIP-W sample was dissolved in 1 mL of DMSO. 30 mg of NaOH was added, and the mixture was incubated for 30 min. 250 μL of iodomethane solution was added, nitrogen was introduced, and the mixture was reacted in the dark for 1 h. Another 250 μL of iodomethane solution was added, and the reaction was continued for 1 h. 1 mL of water and 2 mL of dichloromethane were added, vortexed, centrifuged, and the aqueous phase was discarded. The washing was repeated three times with water. The lower dichloromethane phase was collected and dried under nitrogen. 1 mL of 0.5 M TFA was added, and the mixture was reacted at 70 °C for 180 min. The mixture was dried under nitrogen at 30 °C. 1 mL of freshly prepared 1 M NaBD4 (ammonia solution) was added. The mixture was incubated with magnetic stirring at room temperature for 2.5 h. 300 μL of acetic acid was added to terminate the reaction, and the mixture was dried under nitrogen. The mixture was dried twice under nitrogen at 40 °C in 2 mL of 5% acetic acid in methanol, and then twice again under nitrogen at 40 °C in 2 mL of methanol. Add 1.5 mL of acetic anhydride, vortex to mix, and react at 100 °C for 2.5 h. Add 2 mL of water and let stand for 10 min. Add 1 mL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the washing with water 3 times. Take the lower dichloromethane phase and analyze it by GC-MS.

[0048] The results are summarized in Table 1, identifying four glycosidic bond linkage types: t-Fruf, t-Glcp, 1,2-Fruf, and 1,2,6-Fruf. Among these, 1,2-Fruf was the predominant linkage, indicating that the backbone is mainly composed of 1,2-Fruf residues. The terminal residues are primarily composed of t-Fruf and t-Glcp.

[0049] Table 1. Methylation analysis results of KIP-W

[0050] glycosidic bond Derivative Name Major mass spectrometry fragments (m / z) Retention time (min) molar ratio (%) t-Fruf 2,5-di-O-acetyl-1,3,4,6-tetra-O-methylmannitol 86.9, 100.9, 112.9, 128.9, 144.9,161.9 15.395 1.962 t-Fruf 2,5-di-O-acetyl-1,3,4,6-tetra-O-methylglucitol 86.9, 100.9，112.9, 128.9, 144.9,161.9 15.611 0.760 t-Glcp 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylglucitol 86.9, 101.9, 117.9, 128.9, 144.9,160.9, 205.0 16.773 2.219 1,2-Fruf 1,2,5-tri-O-acetyl-3,4,6-tri-O-methylmannitol 86.9, 99.9, 113.0, 128.9, 144.9,160.9, 189.9，205.0 19.479 58.460 1,2-Fruf 1,2,5-tri-O-acetyl-3,4,6-tri-O-methylglucitol 86.9, 100.9, 112.9, 128.9, 144.9,160.9, 189.9 19.584 34.330 1,2,6-Fruf 1,2,5,6-tetra-O-acetyl-3,4-di-O-methyl-hexitol (mannitol, glucitol) 86.9,98.9, 112.9,124.7, 128.9,189.9 23.391 2.268

[0051] Example 3: Cell viability verification of KIP-W

[0052] (1) Effects on macrophage polarization

[0053] To investigate the effect of KIP-W on the proliferation of RAW264.7 macrophages (ATCC), logarithmic growth phase RAW264.7 macrophages were cultured at a density of 1 × 10⁶ cells per well. 4 Cells were seeded at a density of 1 / mL in 96-well plates and cultured for 12 h until adherence. The culture medium was then discarded, and 100 μL of pre-prepared KIP-W solutions (3.906-1000 μg / mL) at different concentrations were added to each well. The plates were cultured for another 24 h. After 24 h of incubation, CCK-8 was added to the culture medium, and the plates were incubated for another 1 h. The absorbance of each well was recorded using a microplate reader. Figure 2The results of the study showed that KIP-W had no significant cytotoxicity on RAW 264.7 cells within the concentration range of 3.906-1000 μg / mL. Furthermore, concentrations of 31.25-250 μg / mL promoted the proliferation of RAW 264.7 cells. Therefore, 31.25 μg / mL, 62.5 μg / mL, and 125 μg / mL were selected as the low, medium, and high concentration groups of KIP-W.

[0054] To investigate the effect of KIP-W on macrophage polarization, RAW264.7 macrophages were cultured in lipopolysaccharide (LPS) medium for 24 h to induce M1 polarization. The medium was then discarded, and the cells were washed twice with PBS. LPS was added to induce the M1 phenotype in macrophages, serving as the positive control group (LPS group). Similarly, interleukin-4 (IL-4) was added to induce the M2 phenotype in macrophages, serving as the positive control group (IL-4 group). On the second day, macrophages polarized to the M1 phenotype were added to low, medium, and high concentrations of KIP-W (31.25, 62.5, and 125 μg / mL) as experimental groups (LSP+L-KIP-W group, LSP+M-KIP-W group, and LSP+H-KIP-W group) and cultured for another 24 h for subsequent measurements. The expression of macrophage-related inflammatory factors was strictly measured according to the instructions. The indicators and statistical results of M1 / M2 macrophage-related inflammatory proteins detected by Western blotting are shown in Figures 2a and 2ce. The expression of M1 protein (IL-6) was decreased, while the expression of M2 protein (Arg-1 and IL-10) was increased. Figure 2 ELISA results showed that the expression of inflammatory factors M1 (TNF-α, IL-6, IL-1β) was decreased, while the expression of M2 (IL-1RA, IL-10) was increased. Therefore, the KIP-W of this invention can significantly reduce the level of M1 markers and significantly increase the level of M2 markers, indicating that it has a significant ability to induce M2 polarization in macrophages.

[0055] (2) Experiment on migration of human umbilical vein endothelial cells

[0056] Transwell assay was used to detect cell migration ability. Supernatant from different macrophage groups was collected under (1). Human umbilical vein endothelial cells (HUVECs) were seeded in the upper chamber, and 200 μL of low serum culture medium was added to each well. Approximately 600 μL of culture supernatant from different macrophage groups (LPS group, LPS+L-KIP-W, LPS+M-KIP-W, LPS+H-KIP-W group) was added to the lower chamber. After incubation, the migrating cells were fixed, stained with crystal violet, and quantitatively analyzed. Figure 3 KIP-W significantly promoted scratch closure. It also increased the number of HUVECs.

[0057] Example 4: In vivo study of KIP-W's effect on mouse wound healing

[0058] (1) Preparation of KIP-W wound repair hydrogel

[0059] Carbomer 940 (CBM) was added to an appropriate amount of purified water and allowed to swell overnight. A sodium carboxymethyl cellulose (CMC) aqueous solution was prepared separately, and the two solutions were mixed thoroughly to form a homogeneous blank gel matrix. KIP-W aqueous solutions with concentrations of 0.05 mg / mL and 0.1 mg / mL were prepared separately, and added to the blank gel matrix CBM / CMC, stirring thoroughly. Triethanolamine was added dropwise to adjust the pH to 7.0, making the gel transparent and with suitable viscosity, thus obtaining KIP-W wound repair hydrogels of different concentrations, denoted as KIP-W... 0.05 / CBM / CMC (L-KIP-W) and KIP-W 0.1 / CBM / CMC (H-KIP-W).

[0060] (2) Model building

[0061] Six-week-old SPF-grade male BALB / c mice were housed at the Experimental Animal Center of Anhui University of Traditional Chinese Medicine. After one week of acclimatization, the mice were randomly divided into four groups of 24 mice each. Group I mice were treated with CBM / CMC as the Normal group (CBM / CMC) and KFX as the Positive group. The other two groups were treated with hydrogels composed of different concentrations of KIP-W, namely the low-dose group (L-KIP-W) and the high-dose group (H-KIP-W), with 16 mice in each group. Mice were anesthetized with 10% urethane. Hair on the back of the mice was removed using a shaving tool and depilatory cream. A circular wound with a diameter of 1 cm was made on the back of each mouse using a sterile punch to establish a wound model. CBM / CMC, KEX, L-KIP-W, and H-KIP-W were applied to the wound surface of the mice once a day. Wound healing was observed and photographed on days 0, 3, 7, 10, and 14, and the wound size was recorded. The wound area was measured using ImageJ software, and the wound healing status was calculated.

[0062] (3) Experimental results

[0063] Wound healing rate: Photos were taken and the healing rate was calculated on postoperative days 3, 7, 10 and 14. Figure 4The results showed that on day 3, the wound healing rate was 22.11 ± 1.73% in the normal group, 23.94 ± 1.22% in the L-KIP-W group, and 23.57 ± 1.21% in the H-KIP-W group. There was no statistically significant difference between the normal group and the KIP-W group (p > 0.05). By day 7, the wound healing rate in the normal group reached 33.02 ± 1.74%, while it increased to 47.67 ± 3.82% and 56.94 ± 1.16% in the L-KIP-W group and H-KIP-W group, respectively (p < 0.001 compared with the normal group). On day 10, the wound healing rates in the L-KIP-W group (77.56 ± 8.04%) and the H-KIP-W group (83.42 ± 2.39%) were significantly higher than those in the normal group (55.02 ± 3.10%) (both p < 0.001). By day 14, the wounds in the L-KIP-W group (97.02 ± 2.69%) and the H-KIP-W group (99.10 ± 0.54%) had essentially healed; however, residual scabs were still visible in the normal group, indicating relatively delayed healing. This suggests that KIP-W can significantly promote wound healing in vivo.

[0064] Histopathological observation: H&E staining results of wound tissue on postoperative days 7, 10 and 14 showed ( Figure 5 Compared with the control group, the L-KIP-W and H-KIP-W groups showed significantly reduced inflammatory cell infiltration. Re-epithelialization before dermal repair is a crucial step in skin wound healing, enabling the restoration of the epidermal barrier and preventing excessive transepidermal water loss and wound infection. Wounds treated with KIP-W formed a complete epidermal structure with a clear boundary between the epidermis and dermis, a phenomenon not observed in the normal group. Fourteen days post-surgery, the scabs in all KIP-W-treated groups had completely fallen off, revealing intact epidermis and orderly arranged hair follicle structures, similar to normal skin tissue. Granulation tissue plays a vital role in wound regeneration and repair during the inflammatory phase. Ten days after KIP-W treatment, the granulation tissue thickness in the L-KIP-W group (375.50 ± 20.82 μm) and the H-KIP-W group (442.80 ± 75.44 μm) was significantly greater than that in the normal group (214.10 ± 5.49 μm), further confirming the wound-healing effect of KIP-W. Additionally, KIP-W significantly increased collagen fibers, which were more tightly packed.

Claims

1. A kind of kilan kojisan, characterized in that, The malachite has the structure shown in formula (I): （I） Wherein, β-D-Fruf is β-D-fructofuranose, α-D-Glcp is α-D-glucopyranose, and n is 17.

2. The kilan fructan according to claim 1, characterized in that, The weight-average molecular weight of the malachite polysaccharide is 3078 Da, and the monosaccharides are mainly composed of fructose and glucose, with molar percentages of 95.71% and 4.07%, respectively.

3. The method for preparing malachite polysaccharide according to claim 1, characterized in that, Includes the following steps: (1) Soak the powdered Malan medicinal material in 95% ethanol to remove grease, filter, take the residue, and evaporate the ethanol; (2) The residue was extracted with water and precipitated with alcohol to obtain a crude extract; (3) The crude extract was subjected to protein removal by sevage method, decolorization by activated carbon, dialysis and drying to obtain crude polysaccharide of Malan; (4) The crude polysaccharide of Malan was purified by DEAE agarose gel FF column, eluted with distilled water, and the elution peak was tracked and detected by phenol-sulfuric acid method. The eluent was collected, concentrated under reduced pressure, dialyzed and dried to obtain Malan fructan.

4. The method for preparing malachite polysaccharide according to claim 3, characterized in that: In step (2), the material-to-liquid ratio of the water extraction is 1:20-30 g / mL, the extraction time is 1-2 h, and the extraction is performed 2-3 times; the alcohol precipitation is performed by adding 3-4 times the volume of anhydrous ethanol and letting it stand overnight at 4 ℃.

5. The method for preparing malachite polysaccharide according to claim 3, characterized in that: In step (3), the activated carbon decolorization is achieved by adding 1-2% activated carbon by volume of the solution and decolorizing at 35-45℃ for 20-40 min.

6. The method for preparing kilan fructan according to claim 3, characterized in that: In step (3) or step (4), the dialysis is performed using a dialysis bag with a molecular weight cutoff of 3500 Da, with distilled water for 48 hours and the water changed every 12 hours.

7. The use of the argan polysaccharide as described in claim 1 or 2 in the preparation of a wound-healing agent.

8. The application according to claim 7, characterized in that, The wounds include acute wounds, diabetic wounds, burns, pressure injuries, and chronic ulcers.

9. A drug for promoting wound healing, characterized in that, It contains an effective amount of the kilanin as described in claim 1 or 2 and a pharmaceutically acceptable carrier.

10. The wound-healing agent according to claim 9, characterized in that, The dosage form of the drug is a gel, medical dressing, patch, ointment, cream, powder, or spray.

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