Eucommia ulmoides extract, preparation method thereof and application thereof in preparing products for promoting skin wound healing

Abstract

The invention discloses an eucommia extract and a preparation method thereof and its application in preparing a product that promotes skin wound healing, and relates to the field of plant extraction. The invention provides a method for preparing an eucommia extract, which includes extracting eucommia leaf crude polysaccharides from eucommia leaf water extract using a two-phase aqueous system formed by an ethanol aqueous solution and an ammonium sulfate aqueous solution, taking the polysaccharide solution phase, standing to allow ammonium sulfate crystallization to precipitate, and dialysis to obtain an eucommia leaf crude polysaccharide solution; alcohol precipitation is performed on the obtained eucommia leaf crude polysaccharide solution to obtain eucommia leaf crude polysaccharides. The whole preparation process has little environmental pollution and high yield, and the ethanol-ammonium sulfate two-phase aqueous system can also remove pigments therein while removing proteins in crude polysaccharides, thereby increasing the purity of polysaccharides; the scheme of the eucommia leaf polysaccharide provided by the invention is reasonable, the process is simple, and the prepared eucommia polysaccharide provides a theoretical basis and new possibilities for drug development of diabetic wound healing.

Description

Technical Field

[0001] The invention relates to the field of plant extraction, in particular to an eucommia extract and a preparation method thereof and application of the extract in preparing a product for promoting skin wound healing. Background Art

[0002] Skin wound healing is a complex biological process involving multiple cells and factors, mainly including inflammatory response, cell proliferation, collagen formation and tissue remodeling, and these periods are intertwined and infiltrated. Epidermal cells (HaCaT) and fibroblasts (HF) are important cells involved in the skin wound repair process. Their proliferation and migration are directly related to the wound healing rate. In the treatment of skin, cornea, gastrointestinal tract, burns and various chronic wounds, the proliferation, differentiation and migration of epidermal cells and fibroblasts can promote the formation of new granulation tissue and re-epithelialization of the wound, thereby healing.

[0003] The human body has limited wound healing ability. When the skin is damaged, if the wound is not treated in time or the wound healing factor is not given, it will cause wound ulceration, even amputation and death. Now, there is still a need to provide products that can effectively improve skin wound repair.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide an eucommia extract and a preparation method thereof and application of the eucommia extract in preparing a product for promoting skin wound healing.

[0006] The present invention is achieved in that:

[0007] In the first aspect, an embodiment of the present invention provides a method for preparing an eucommia leaf extract, which comprises: extracting crude eucommia leaf polysaccharides from an eucommia leaf water extract using a two-phase aqueous system formed by an ethanol aqueous solution and an ammonium sulfate aqueous solution, taking the polysaccharide solution phase, standing to allow ammonium sulfate to crystallize out, and then dialyzing to obtain a crude eucommia leaf polysaccharide solution; and subjecting the obtained crude eucommia leaf polysaccharide solution to alcohol precipitation to obtain crude eucommia leaf polysaccharides.

[0008] In a second aspect, an embodiment of the present invention provides an eucommia extract, which is prepared by the preparation method of the aforementioned embodiment.

[0009] In a third aspect, embodiments of the present invention provide use of the Eucommia ulmoides extract obtained by the preparation method described in the aforementioned embodiments or the Eucommia ulmoides extract described in the aforementioned embodiments in the preparation of a product for promoting skin wound healing.

[0010] In a fourth aspect, an embodiment of the present invention provides a product for promoting skin wound healing, the main ingredient of which includes: the Eucommia ulmoides extract described in the above embodiment.

[0011] It has the following beneficial effects:

[0012] The invention provides a method for preparing an eucommia extract. The whole preparation process has little environmental pollution and high yield. The ethanol-ammonium sulfate aqueous two-phase system can remove pigments in crude polysaccharides while removing proteins therein, thereby increasing the purity of the polysaccharides. The eucommia leaf polysaccharide provided by the invention has a reasonable scheme and a simple process. The prepared eucommia polysaccharide provides a theoretical basis and new possibilities for the research and development of drugs for diabetic wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0014] Figure 1 This is a diagram of the animal test results of dry film dressing;

[0015] Figure 2 The wound healing rate of mice within 2 weeks of the Eucommia polysaccharide prepared in Example 1-8. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0017] The embodiment of the present invention provides a method for preparing an eucommia extract, which comprises:

[0018] Extracting crude polysaccharides from eucommia leaf water extract by using a two-phase system formed by ethanol aqueous solution and ammonium sulfate aqueous solution, taking the upper phase, i.e. the polysaccharide solution layer, standing to allow ammonium sulfate crystals to precipitate, and dialyzing to obtain a crude polysaccharide solution from eucommia leaf;

[0019] The obtained crude polysaccharide solution of Eucommia ulmoides leaves is subjected to alcohol precipitation to obtain crude polysaccharide of Eucommia ulmoides leaves.

[0020] After a series of creative work, the inventors of the present application discovered that the use of an ethanol-ammonium sulfate aqueous two-phase system can not only remove proteins from crude polysaccharides, but also remove pigments therein, thereby increasing the purity of the polysaccharides; and that the entire extraction scheme is reasonable and the process is simple. The prepared Eucommia polysaccharide has the effect of promoting wound healing (including diabetic patients), providing a theoretical basis and new possibilities for the development of related drugs.

[0021] In some embodiments, in the aqueous two-phase system, the mass concentration of ethanol is 20-30%, and the mass concentration of ammonium sulfate is 10-20%. Suitable alcohol and ammonium sulfate concentrations are conducive to the formation of an aqueous two-phase system. Within this concentration range, the partition coefficient of sugar in the ammonium sulfate phase is high. Reducing or increasing the amount of alcohol and ammonium sulfate will cause the solubility of sugar to decrease. Specifically, the mass concentration of ethanol in the aqueous two-phase system can be any one of 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, or any two of the range. The mass concentration of ammonium sulfate in the aqueous two-phase system can be any one of 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, and 20%, or any two of the range.

[0022] In some embodiments, the dialysis includes: using a dialysis bag with a molecular weight of 3000-4000KD for interception, which can remove inorganic salts and small molecular compounds in the solution inside and improve the purity of the polysaccharide.

[0023] In some embodiments, the dialysis time is 1 to 3 days, specifically 1 day, 2 days or 3 days.

[0024] In some embodiments, the static temperature is 0-5°C, specifically any one of 0°C, 1°C, 2°C, 3°C, 4°C and 5°C, or a range between any two of them.

[0025] In some embodiments, the method for extracting the eucommia leaf water extract comprises: heating and extracting a mixture of eucommia leaf powder and water, and concentrating the supernatant obtained after the extraction to obtain the eucommia leaf water extract.

[0026] In some embodiments, the temperature of the heating extraction is 80-100°C, and the time is 1-2 hours. The temperature of the heating extraction can be any one of 80°C, 82°C, 84°C, 86°C, 88°C, 90°C, 92°C, 94°C, 96°C, 98°C, 100°C, or any range between two thereof. The time can be 1 hour, 1.5 hours, or 2 hours.

[0027] In some embodiments, the heating extraction is performed 1 to 3 times, and the number can be 1 time, 2 times or 3 times.

[0028] In some embodiments, in the mixed solution, the volume ratio of eucommia leaf powder to water is 1:(5-10), and the volume ratio can specifically be any one of 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or a range between any two of them.

[0029] In some embodiments, the mixture of Eucommia leaf powder and water is obtained by mixing Eucommia leaf powder and water for 1 to 20 hours. The mixing method can be standing for any one of 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, and 20 hours, or a range between any two of them.

[0030] In some embodiments, the alcohol precipitation step comprises: mixing the crude polysaccharide solution of Eucommia ulmoides leaves with an ethanol solution, and then collecting the precipitate.

[0031] In some embodiments, the volume ratio of the eucommia leaf crude polysaccharide solution to the ethanol solution is 1:(2-6). The volume ratio can be any one of 1:2, 1:3, 1:4, 1:5, 1:6 or a range between any two of them.

[0032] In some embodiments, the ethanol solution is pre-cooled ethanol, and low temperature can accelerate the precipitation of polysaccharides.

[0033] In some embodiments, the precooling refers to precooling to 4-8°C.

[0034] In some embodiments, the alcohol precipitation further comprises: drying the precipitate to obtain the final crude polysaccharide of Eucommia ulmoides leaves.

[0035] In some embodiments, the preparation method further comprises: purifying the crude polysaccharide from Eucommia ulmoides leaves.

[0036] In some embodiments, the purification comprises: subjecting the crude polysaccharide from Eucommia ulmoides leaves to anion column chromatography to obtain purified crude polysaccharide from Eucommia ulmoides leaves;

[0037] In some embodiments, the anion column comprises: a DEAE anion column.

[0038] In some embodiments, the eluent used in the chromatography comprises: NaCl solutions with different concentration gradients.

[0039] In some embodiments, the different concentration gradients include: 0 mol / L, 0.12-0.14 mol / L, 0.23-0.27 mol / L, 0.48-0.52 mol / L, 0.8-1.2 mol / L and 1.8-2.2 mol / L.

[0040] In some embodiments, the purification further comprises: dialyzing the eluted component obtained after the anion column chromatography, performing ethanol precipitation, and taking the precipitate for ultrafiltration;

[0041] In some embodiments, the ultrafiltration includes: ultrafiltration with an ultrafiltration membrane of a first molecular weight, after removing substances with a molecular weight less than the first molecular weight, filtering with an ultrafiltration membrane of a second molecular weight to obtain a eucommia leaf polysaccharide with a molecular weight between the first molecular weight and the second molecular weight; wherein the first molecular weight is 20-40K, specifically any one of 20K, 22K, 24K, 26K, 28K, 30K, 32K, 34K, 36K, 38K, 40K or any two thereof. The second molecular weight is 70-90K, specifically any one of 70K, 72K, 74K, 76K, 78K, 80K, 82K, 84K, 86K, 88K, 90K or any two thereof. After testing, the purified polysaccharide has a high purity, and the molecular weight is within the range of 40-70KD, and the limited range is more conducive to obtaining a polysaccharide compound with high purity.

[0042] On the other hand, an embodiment of the present invention further provides an Eucommia ulmoides extract, which is prepared by the preparation method described in any of the aforementioned embodiments.

[0043] On the other hand, an embodiment of the present invention further provides use of the Eucommia ulmoides extract prepared by the preparation method described in any of the aforementioned embodiments in the preparation of a product for promoting skin wound healing.

[0044] In some embodiments, promoting skin wound healing includes: promoting skin wound healing in diabetic patients.

[0045] In some embodiments, the diabetes includes: any one of type I and type II;

[0046] In some embodiments, the wound comprises at least one of a burn, an incision, a laceration, an abrasion, a puncture wound, and an ulcer.

[0047] In addition, an embodiment of the present invention further provides a product for promoting skin wound healing, the main ingredient of which includes: the Eucommia ulmoides extract described in any of the above embodiments.

[0048] In some embodiments, the product may further include: auxiliary materials.

[0049] Optionally, the auxiliary material may be: gelatin solution, polyvinyl alcohol, alginate. The mass concentration of gelatin in the gelatin solution may be 2% to 5%, specifically any one of 2%, 3%, 4%, 5% or a range between any two. When the auxiliary material is a gelatin solution, the product is an eucommia polysaccharide gel dry film dressing, and the preparation method thereof may be: mixing the gelatin solution with the eucommia leaf polysaccharide described in any of the above embodiments in a volume ratio of (2 to 6): 1, standing and drying after mixing to obtain a dry film dressing.

[0050] In some embodiments, the volume ratio of the gelatin solution to the eucommia leaf polysaccharide may be any one of 2:1, 3:1, 4:1, 5:1 and 6:1 or a range between any two of them.

[0051] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0052] Example 1

[0053] A method for preparing eucommia leaf polysaccharide, comprising:

[0054] (1) Grinding the dried Eucommia ulmoides leaves into powder using a Chinese medicine grinder and passing the powder through a 200-mesh sieve to obtain Eucommia ulmoides leaf powder;

[0055] (2) adding 10 times the mass volume of distilled water to the above-obtained Eucommia leaf powder, soaking for 4 hours, heating and stirring at 100° C. for 2 hours, filtering to obtain a supernatant and a residue, adding an equal mass volume of distilled water to the residue, extracting twice, combining the supernatants and concentrating to 1 / 20 of the volume of the original solution, i.e., the Eucommia leaf water extract;

[0056] (3) adding the above-mentioned Eucommia leaf water extract to an ethanol-ammonium sulfate two-phase aqueous system (the mass concentration of ethanol in each 100 mL is 27%, and the mass concentration of ammonium sulfate is 18%) for extraction, collecting the lower ammonium sulfate phase, placing it at 4° C. to allow salt crystallization, and then dialyzing it in a 3500KD dialysis bag for 2 days, i.e., the Eucommia leaf crude polysaccharide solution;

[0057] (4) adding 4 times the volume of pre-cooled (4° C.) 95% ethanol solution to the crude polysaccharide solution obtained above, collecting the precipitate after alcohol precipitation, and freeze-drying to obtain the crude polysaccharide of Eucommia ulmoides leaves;

[0058] (5) The crude polysaccharide of Eucommia ulmoides leaf was prepared into a solution with a concentration of 50 mg / mL using distilled water, centrifuged, and the supernatant was slowly added to a DEAE anion column, and 0 mol / L, 0.1 mol / L, 0.25 mol / L, 0.5 mol / L, 1.0 mol / L, and 2.0 mol / L sodium chloride aqueous solution was used as the mobile phase, and each group of fractions was collected respectively. After dialysis, precooled (4°C) ethanol was added respectively, and the precipitate was collected. The precipitate of the water component (0 mol / L) was the largest, so the polysaccharide in the water component was selected for ultrafiltration segmentation using an ultrafiltration membrane; first, a 30K (first molecular weight) ultrafiltration membrane was used to remove polysaccharides with small molecular weight, and then an 80K (second molecular weight) ultrafiltration membrane was used for ultrafiltration, so that Eucommia ulmoides leaf polysaccharide with a molecular weight of 30K-80K and a certain molecular weight fragment could be obtained. The ultrafiltrate was concentrated and freeze-dried to obtain the purified Eucommia ulmoides leaf polysaccharide.

[0059] Example 2

[0060] A method for preparing eucommia leaf polysaccharide is substantially the same as that of Example 1, except that the time for each heating, stirring and extraction in step (2) is 1 hour.

[0061] Example 3

[0062] A method for preparing eucommia leaf polysaccharide is substantially the same as that of Example 1, except that the temperature of each heating and stirring extraction in step (2) is 80°C.

[0063] Example 4

[0064] A method for preparing eucommia leaf polysaccharide is substantially the same as that of Example 1, except that in step (2), the temperature of each heating and stirring extraction is 80° C. and the time is 1 hour.

[0065] Example 5

[0066] A method for preparing eucommia leaf polysaccharide is substantially the same as that of Example 1, except that the material-liquid ratio (eucommia leaf powder / filter residue: distilled water) in step (2) of heating extraction is 1:5.

[0067] Example 6

[0068] A method for preparing eucommia leaf polysaccharide is substantially the same as that of Example 5, except that the time for each heating and stirring extraction in step (2) is 1 hour.

[0069] Example 7

[0070] A method for preparing eucommia leaf polysaccharide is substantially the same as that of Example 5, except that the temperature of each heating and stirring extraction in step (2) is 80°C.

[0071] Example 8

[0072] A method for preparing eucommia leaf polysaccharide is substantially the same as that of Example 5, except that in step (2), the temperature of each heating and stirring extraction is 80° C. and the time is 1 hour.

[0073] Example 9

[0074] A method for preparing eucommia leaf polysaccharide is substantially the same as that of Example 1, except that the mobile phase in step (5): the sodium chloride concentration is 0 mol / L.

[0075] Example 10

[0076] A method for preparing eucommia leaf polysaccharide is substantially the same as that of Example 1, except that the mobile phase in step (5): the sodium chloride concentration is 0.1 mol / L.

[0077] Embodiment 11

[0078] A method for preparing eucommia leaf polysaccharide is substantially the same as that of Example 1, except that the mobile phase in step (5): the sodium chloride concentration is 0.25 mol / L.

[0079] Example 12

[0080] A method for preparing eucommia leaf polysaccharide is substantially the same as that of Example 1, except that the mobile phase in step (5): the sodium chloride concentration is 0.5 mol / L.

[0081] Example 13

[0082] A method for preparing eucommia leaf polysaccharide is substantially the same as that of Example 1, except that the mobile phase in step (5): the sodium chloride concentration is 1 mol / L.

[0083] Embodiment 14

[0084] A method for preparing eucommia leaf polysaccharide is substantially the same as that of Example 1, except that the mobile phase in step (5): the sodium chloride concentration is 2 mol / L.

[0085] Embodiment 15

[0086] A method for preparing eucommia leaf polysaccharide is substantially the same as that of Example 1, except that the first molecular weight is 15K.

[0087] Example 16

[0088] A method for preparing eucommia leaf polysaccharide is substantially the same as that of Example 1, except that the first molecular weight is 20K.

[0089] Embodiment 17

[0090] A method for preparing eucommia leaf polysaccharide is substantially the same as that of Example 1, except that the first molecular weight is 40K.

[0091] Embodiment 18

[0092] A method for preparing eucommia leaf polysaccharide is substantially the same as that of Example 1, except that the first molecular weight is 50K.

[0093] Embodiment 19

[0094] A method for preparing eucommia leaf polysaccharide is substantially the same as that of Example 1, except that the second molecular weight is 60K.

[0095] Embodiment 20

[0096] A method for preparing eucommia leaf polysaccharide is substantially the same as that of Example 1, except that the second molecular weight is 70K.

[0097] Embodiment 21

[0098] A method for preparing eucommia leaf polysaccharide is substantially the same as that of Example 1, except that the second molecular weight is 90K.

[0099] Embodiment 22

[0100] A method for preparing eucommia leaf polysaccharide is substantially the same as that of Example 1, except that the second molecular weight is 100K.

[0101] Embodiment 23

[0102] A method for preparing eucommia leaf polysaccharide is substantially the same as that of Example 1, except that in step (5), only an ultrafiltration membrane with a first molecular weight is used for interception.

[0103] Embodiment 24

[0104] A method for preparing eucommia leaf polysaccharide is substantially the same as that of Example 1, except that in step (5), only an ultrafiltration membrane with a second molecular weight is used for interception.

[0105] Embodiment 25

[0106] Preparation of Eucommia polysaccharide gel dry film dressing:

[0107] Weigh 0.8 g of the Eucommia polysaccharide powder in Example 1, add ddH2O, make the volume to 40 ml, shake evenly on a shaker, mix evenly with the 4% gelatin solution in Comparative Example 1 at a volume ratio of 1:4, fill into a mold, place at 37°C and 10% humidity for 24 h, dry to form a film to obtain a dry film dressing, and store at room temperature for use.

[0108] Embodiment 26

[0109] Preparation of Eucommia polysaccharide gel dry film dressing:

[0110] Weigh 0.8 g of the Eucommia polysaccharide powder in Example 1, add ddH2O, make the volume to 40 ml, shake evenly on a shaker, mix evenly with the 4% gelatin solution in Comparative Example 1 at a volume ratio of 1:5, fill into a mold, place at 37°C and 10% humidity for 24 h, dry to form a film to obtain a dry film dressing, and store at room temperature for use.

[0111] Embodiment 27

[0112] Preparation of Eucommia polysaccharide gel dry film dressing:

[0113] Weigh 0.8 g of the Eucommia polysaccharide powder in Example 1, add ddH2O, make the volume to 40 ml, shake evenly on a shaker, mix evenly with the 4% gelatin solution in Comparative Example 1 at a volume ratio of 1:6, fill into a mold, place at 37°C and 10% humidity for 24 h, dry to form a film to obtain a dry film dressing, and store at room temperature for use.

[0114] Embodiment 28

[0115] Preparation of Eucommia polysaccharide gel dry film dressing:

[0116] Weigh 0.8 g of the Eucommia polysaccharide powder in Example 1, add ddH2O, make the volume to 40 ml, shake evenly on a shaker, mix evenly with the 4% gelatin solution in Comparative Example 1 at a volume ratio of 1:3, fill into a mold, place at 37°C and 10% humidity for 24 h, dry to form a film to obtain a dry film dressing, and store at room temperature for use.

[0117] Comparative Example 1

[0118] Preparation of 4% gelatin solution: weigh 1.00 g of gelatin powder into a 50 ml centrifuge tube, add ddH2O, dilute to 25 ml, mix well, and store in a refrigerator at 4°C for later use;

[0119] Preparation of gelatin dry film dressing: fill into a mold, place at 37°C and 10% humidity for 24 hours, dry to form a film to obtain a dry film dressing, and store at room temperature for later use.

[0120] Comparative Example 2

[0121] Preparation of PDGF gel dry film dressing:

[0122] Weigh 0.8 g of PDGF powder, add ddH2O, make the volume to 40 ml, shake evenly on a shaker, mix evenly with the 4% gelatin solution in Comparative Example 1 at a volume ratio of 1:4, fill into a mold, place at 37°C and 10% humidity for 24 hours, dry to form a film to obtain a dry film dressing, and store at room temperature for use.

[0123] Comparative Example 3

[0124] A method for preparing eucommia leaf polysaccharide is substantially the same as that of Example 1, except that step (3) is omitted.

[0125] Test Example 1

[0126] The polysaccharide extraction rate and purity of Examples 1 to 24 were verified.

[0127] Test process

[0128] Crude polysaccharide extraction rate (%) = dry weight of extract (g) / Bletilla striata tuber powder (g); Purity (%) refers to the number of milligrams of sugar contained in 1g of extract. The specific implementation steps include: weigh 50mg of glucose dried to constant weight at 105 degrees, add 500ml of distilled water, and prepare a 0.1mg / mL standard solution, dilute with distilled water to 0, 0.02, 0.04, 0.06, 0.08, 0.1mg / mL, then add 300μL of 6% phenol and 1.5mL sulfuric acid respectively, shake well, let stand for 10 minutes, detect absorbance at 490nm, and draw a standard curve with glucose content as the horizontal axis and OD value as the vertical axis. Take a sample and prepare it to 1mg / mL, repeat the above operation, measure the absorbance at 490nm, and bring it into the standard curve to calculate the total sugar content of the sample. Purity (%) = m 糖 / m 样品 *100, where m 糖 Refers to milligrams of sugar, m 样品 Refers to the number of milligrams of sample.

[0129] The test results are shown in Table 1.

[0130] Table 1. Polysaccharide extraction rate and purity

[0131]

[0132]

[0133] Test Example 2

[0134] Animal wound healing test: The full-thickness skin defect model of diabetic mice commonly used in the literature was used to evaluate the wound healing performance of the hydrogels of the embodiments and comparative examples in animals.

[0135] Specific steps: A wound with a diameter of 10 mm was formed on the back skin of the mouse using a hole punch. The same amount of Example 25 (V 杜仲 :V 4％明胶 =1:4), Comparative Example 1 (4% gelatin) and Comparative Example 2 (V PDGF :V 4％明胶 =1:4) hydrogel was made into a circular wound dressing (10 mm in diameter and 1 mm in thickness), and the skin defect of mice in the control group was not treated. Photos of the wound site were taken and recorded on days 0, 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 (corresponding to D0, D1, D3, D5, D7, D9, D11, D13, D15, D17, and D19, respectively).

[0136] from Figure 1It can be seen that the skin wounds of mice covered with the polysaccharide group of Example 1 healed faster than the wounds of mice covered with the hydrogel group of Comparative Example 1, the PDGF gelatin group of Comparative Example 2, and the untreated group, which indicates that the Eucommia polysaccharide prepared in Example 1 has better wound repair function as a wound dressing.

[0137] Figure 2 The wound healing rate of mice within 2 weeks of the Eucommia polysaccharide prepared in Examples 1-8 (Schemes 1 to 8) also shows that the Eucommia polysaccharide prepared in Example 1 has good wound repair function as a wound dressing.

[0138] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing an eucommia extract, characterized in that: It includes: The crude polysaccharide of Eucommia leaf is extracted from the water extract of Eucommia leaf by using a two-phase aqueous system formed by an ethanol aqueous solution and an ammonium sulfate aqueous solution, and the polysaccharide solution phase is taken, and after standing to allow ammonium sulfate to crystallize and precipitate, the crude polysaccharide solution of Eucommia leaf is obtained by dialysis; in the two-phase aqueous system, the mass concentration of ethanol in each 100 mL of the solution is 20%-30%, and the mass concentration of ammonium sulfate is 10%-20%; The obtained crude polysaccharide solution of Eucommia ulmoides leaves is subjected to alcohol precipitation to obtain crude polysaccharide of Eucommia ulmoides leaves; The preparation method further comprises: purifying the crude polysaccharide from eucommia leaves; the purification comprises: subjecting the crude polysaccharide from eucommia leaves to anion column chromatography to obtain purified crude polysaccharide from eucommia leaves; The anion column includes: a DEAE anion column; the eluent used in the column chromatography includes: a NaCl solution with different concentration gradients; the different concentration gradients include: 0 mol / L, 0.12-0.14 mol / L, 0.23-0.27 mol / L, 0.48-0.52 mol / L, 0.8-1.2 mol / L and 1.8-2.2 mol / L; The purification further comprises: dialyzing the eluted component obtained after the anion column chromatography, performing ethanol precipitation, and ultrafiltration of the precipitate of the water component; The ultrafiltration comprises: performing ultrafiltration with an ultrafiltration membrane of a first molecular weight, removing substances with a molecular weight less than the first molecular weight, and then filtering with an ultrafiltration membrane of a second molecular weight to obtain eucommia leaf polysaccharides with a molecular weight between the first molecular weight and the second molecular weight; wherein the first molecular weight is 20-40K and the second molecular weight is 70-90K.

2. The method for preparing the Eucommia ulmoides extract according to claim 1, characterized in that: The dialysis comprises: using a dialysis bag with a molecular weight of 3000-4000 KD for interception.

3. The method for preparing the Eucommia ulmoides extract according to claim 1, characterized in that: The dialysis time is 1 to 3 days.

4. The method for preparing the Eucommia ulmoides extract according to claim 1, characterized in that: The standing temperature is 0-5°C.

5. The method for preparing the Eucommia ulmoides extract according to claim 1, characterized in that: The method for extracting the eucommia leaf water extract comprises: heating and extracting a mixture of eucommia leaf powder and water, and concentrating the supernatant obtained after the extraction to obtain the eucommia leaf water extract.

6. The method for preparing the Eucommia ulmoides extract according to claim 5, characterized in that: The temperature of the heating extraction is 80-100° C., and the time is 1-2 hours.

7. The method for preparing the Eucommia ulmoides extract according to claim 5, characterized in that: The heating extraction is performed 1 to 3 times.

8. The method for preparing the Eucommia ulmoides extract according to claim 5, characterized in that: In the mixed solution, the volume ratio of eucommia leaf powder to water is 1: (5-10).

9. The method for preparing the Eucommia ulmoides extract according to claim 5, characterized in that: The mixed solution of eucommia leaf powder and water is obtained by mixing the eucommia leaf powder and water for 1 to 20 hours.

10. The method for preparing the Eucommia ulmoides extract according to claim 1, characterized in that: The alcohol precipitation step comprises: mixing the crude polysaccharide solution of Eucommia ulmoides leaves with an ethanol solution, and then collecting the precipitate.

11. The method for preparing the Eucommia ulmoides extract according to claim 10, characterized in that: The volume ratio of the eucommia leaf crude polysaccharide solution to the ethanol solution is 1:(2-6).

12. The method for preparing the Eucommia ulmoides extract according to claim 10, characterized in that: The ethanol solution is a precooled ethanol solution.

13. The method for preparing the Eucommia ulmoides extract according to claim 12, characterized in that: The precooling refers to precooling to 4-8°C.

14. The method for preparing the Eucommia ulmoides extract according to claim 10, characterized in that: The alcohol precipitation further comprises: drying the precipitate to obtain the final eucommia leaf crude polysaccharide.

15. An eucommia extract, characterized in that It is prepared by the preparation method according to any one of claims 1 to 14.

16. Use of the eucommia extract obtained by the preparation method according to any one of claims 1 to 14 or the eucommia extract according to claim 15 in the preparation of a product for promoting skin wound healing.

17. The use according to claim 16, characterized in that The promoting skin wound healing comprises: promoting skin wound healing in diabetic patients.

18. The use according to claim 17, characterized in that The diabetes mellitus includes any one of type I and type II.

19. The use according to claim 16, characterized in that: The wound comprises at least one of a burn, an incision, a laceration, an abrasion, a puncture wound and an ulcer.

20. A product for promoting skin wound healing, characterized in that: The main ingredients include: the eucommia extract according to claim 15.

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