AU2020101591A4

AU2020101591A4 - A thermosensitive gel, preparation method and application thereof

Info

Publication number: AU2020101591A4
Application number: AU2020101591A
Authority: AU
Inventors: Jing Chen; Li Chen; Li Cheng; Xincai Hao; Bailing Wang; Hao Wu; Changli Zeng; Yongheng ZHAO; Tao Zheng; Xi ZHENG
Original assignee: Hubei Jinshuiyuan Biotechnology Co Ltd; Hubei University of Medicine
Current assignee: Hubei Jinshuiyuan Biotechnology Co Ltd; Hubei University of Medicine
Priority date: 2020-07-31
Filing date: 2020-07-31
Publication date: 2020-09-10
Anticipated expiration: 2028-07-31

Abstract

The invention provides a preparation method of bletilla striata polysaccharide / bioglass temperature sensitive gel and application thereof. The raw material formula of the gel preparation is composed of the main medicinal bletilla striata polysaccharide and the bioactive glass, the temperature-sensitive gel matrix poloxamer, the stabilizer glycerin, the skin absorption promoter DMSO, the preservative hydroxyphenethyl ester and the water for injection. In that invention, the combination of bletilla striata polysaccharide / bioactive glass and the temperature-sensitive in-situ gel is apply to the treatment of diabetic foot ulcer. In addition, that gel is in a liquid state in vitro, and can quickly change into a gel-like semi-solid after bee administered to the ulcer wound surface, so that the drug can be retained on the ulcer surface for a long time. At the same time, combined with the repair effect of bletilla striata polysaccharide and bioactive glass, the purpose of wound healing was achieved.

Description

AUSTRALIA

PATENTS ACT 1990

PATENT SPECIFICATION FOR THE INVENTION ENTITLED:

A thermosensitive gel, preparation method and application thereof

The invention is described in the following statement:-

A thermosensitive gel, preparation method and application thereof

TECHNICAL FIELD

[0001] The invention belongs to the technical field of medicine, in particular to a bletilla striata polysaccharide / bioglass temperature-sensitive gel and its preparation method and application.

BACKGROUND

[0002] Diabetes can lead to severe complications in different systems, with 15% to 25% of diabetic patients eventually developing chronic non-curative diabetic foot ulcers (DFUs). DFUs, divided into ischemic type, neural type and mixed type, is ulceration and gangrene state caused by the decrease of arterial perfusion in the lower extremity and the microcirculation disorder of the foot, because of neuropathy, microvascular and macrovascular lesions of the foot. It is one of the most common complications of diabetes mellitus, mainly manifested as numbness of lower limbs, coldness, abnormal sensation, formation of ulcer and gangrene, and ulcer is difficult to heal and easy to infect. Once DFUs are formed, the risk of wound deterioration is further increased, and the lower limb amputation risk is 40 times higher than that of normal people. According to statistics, more than 85% of amputations are caused by DFUs, which have great impact on the quality of life of patients, even threatening the life, and cause a heavy burden to the patient's family and public health.

[0003] That clinical treatment of DFUs is facing the follow problems: 0 debridement (such as autolysis, mechanical debriding, maggot biological debridment) can remove necrosis, infected tissue and stimulate the growth of new granulation tissue. However, it is difficult for clinicians to master its adaptation and contraindication. @ Interventional therapy can effectively improve the blood flow perfusion of the blood vessels in the lower limbs of diabetic foot, but it also has the problems of intimal hyperplasia, thrombosis and re-stenosis and occlusion. @ Stem cell transplantation can regenerate the blood vessels of ischemic limbs, improve the blood supply of the affected limbs, and promote the healing of ulcer wounds. Foreign countries have begun to have curative effect, but less in China. 4Some scholars have proposed the "five-ring therapy", meaning to control blood sugar, circulate blood, use anti-inflammatory, change medicine, support symptomatic treatment. Most of the current research has focused on the use of biologically active substances (antimicrobial agents, protein drugs, living cells, etc.) to promote wound healing in diabetes mellitus. Developed therapeutic products give little consideration to the special pathological environment of the diabetic wound, and the therapeutic effect was still unsatisfactory.

[0004] Therefore, appropriate products and dosage forms are sought to make the drug stay at the ulcer site for a long time, so as to change the special pathological environment of diabetic foot ulcer wound surface, increase the expression or activity of growth factors in the wound area of patients with DFUs ,which is a major problem in the treatment of diabetic foot ulcers.

SUMMARY

[0005] In order to solve that technical problems, the invention provides a bletilla striata polysaccharide / bioglass thermosensitive gel agent, which is used for treating diabetic foot ulcer by combine the bletilla striata polysaccharide/ bioglass and the thermosensitive in-situ gel. In addition, that gel is in a liquid state in vitro, the gel is quickly changed into a gel-like semi-solid after the drug is administered to the ulcer wound surface, so that the drug can be retained on the ulcer surface for a long time. At the same time, combined with the repair effect of bletilla striata polysaccharide and bioactive glass, the purpose of wound healing was achieved.

[0006] One of the technical solutions of the present invention is a bletilla striata polysaccharide / bioglass temperature-sensitive gel having a gelation temperature of 32 2 ° C. By mass percentage , the raw material comprises bletilla striata polysaccharides of

4.0-7.0% t, bioactive glass of 0.5%, heat-sensitive gel base 19-28%, stabilize 0.5- 5 %, skin absorption accelerator 0.1%, preservative 0.01%, and balance of water for injection.

[0007] Preferably, the temperature-sensitive gel matrix is selected from one or any combination of polosham, chitosan, and poly-N-isopropylacrylamide.

[0008] Preferably, the polosham is composed of polosham 407 and 188. wherein by mass of the percentage, the polosham 407 is 18-24%, polosham 188 is 14%.

[0009] Preferably, the stabilizer is glycerol, the skin absorption promoter is DMSO, the preservative is hydroxyphenylethyl, the water for injection is one of purified water, physiological saline, and a buffer solution having a pH of 3 to 8, or any combination thereof.

[0010] Preferably, the preparation method of bletilla striata polysaccharide comprises the following steps: bletilla striata tubers were crushed, sieved, added with distilled water, stirred evenly, and placed overnight. After ultrasonic extraction, heating by water bath, precipitating the lower layer of the solution adding in water, and then heating in the water bath again. Combining the two solutions, centrifuging, collecting the supernatant, suction filtering, and concentrating the supernatant under reduced pressure, immersing in alcohol twice, combining filter residue, drying, dissolving in water, filtering with microporous filter membrane, concentrating and freeze-drying filtrate to obtain bletilla striata polysaccharide.

[0011] Preferably, the preparation method of bletilla striata polysaccharide comprises the following steps: crushing and sieving the dried bletilla striata tubers, adding with distilled water, stirring evenly, and placing it overnight. The next day, distilled water was added until the material-liquid ratio was 1:20, ultrasonic extraction was conducted at room temperature for 30min, and the average power was 80W. Then heating it directly in a 70 °C water bath for 1.5h. The lower layer of the solution was added with water to 1000ml. Then the solution was heated at 70 °C for 1h. The solution was combined twice and centrifuged at 4000rpm for 25min to collect the supernatant. The supernatant was concentrated to 1 / 3 of its original volume at 70 °C to form a viscous shape of about 600 ml. 85% ethanol of 3 times volume was added and placed overnight at 4 °C. The next day, the supernatant was filtered and alcohol precipitated twice. The filter residue was combined and dried in an oven at 50 °C. After drying, it was dissolved in water and filtered with 0. 2 2 m microporous membrane. The filtrate was concentrated and freeze dried to obtain bletilla polysaccharide.

[0012] In the second technical proposal of the present invention, the preparation method of the bletilla striata polysaccharide / bioglass temperature-sensitive gel comprises the following steps:

[0013] Heat-sensitive gel matrix, bletilla striata polysaccharide are uniformly mixed with 1 / 3 of water for injection, placed in refrigerator, completely dissolved and fully swelled as that first mixture. The Bioglass is ground evenly in the stabilizer to obtain a second mixture; Mix the first mixture, the second mixture, and the skin absorption promoter, preservative with the remaining water for injection to obtain a bletilla striata polysaccharide / bioglass thermosensitive gel.

[0014] Preferably, the cold storage conditions is 0-4°C, and time is 24 h.

[0015] The third technical proposal of the present invention is the application of the above-mentioned bletilla striata polysaccharide / bioglass thermosensitive gel, which is used for treating diabetic foot ulcer.

[0016] In comparison with that prior art, the present invention has the follow beneficial effects:

[0017] (1)In that invention takes bletilla striata polysaccharide as main medicine, and adds a certain amount of bioactive glass, stabilizer, skin absorption promoter and preservative in combination with the matrix of the gel to prepare a temperature-sensitive gel of bletilla striata polysaccharide / bioglass and the gel has a gelation temperature of 32± 2 C. In vitro liquid state, that medicine gels rapidly into a gel-like semi-solid after being administer to the ulcer wound surface, so that the medicine can be retained on the ulcer surface for a long time. At the same time, combined with the repair effect of bletilla striata polysaccharide and bioactive glass, the purpose of wound healing was achieved.

[0018] (2) Bletilla striata polysaccharides (BSPS) is a kind of glucose with mannan obtained from bletilla striata tuber by water extraction and alcohol precipitation. It has the following pharmacological activities: (1) promoting the expression and activity of growth factors and wound healing. Wound healing is a process of cell proliferation, differentiation, migration and apoptosis, involving hemostasis, inflammation, proliferation and tissue remodeling. Once the protective barrier of the epidermis and dermis is destroyed, the physiological wound healing process proceeds immediately. BSPS can promote the growth of human umbilical vein endothelial cells and the expression of vascular endothelial growth factor. The mechanism of action is related to the stimulation of macrophages to produce increased levels of inducible nitric oxide synthase (iNOS), TNF-a, interleukin-1 (IL- I) mRNA, and can increase the production of these cytokines, improve that expression of epidermal growth factor, alleviate inflammatory reaction and promote wound healing. BSPS can promote proliferation of vascular endothelial cells and fibroblasts in a dose-dependent manner, increase the number of wound macrophages in wound tissues, increase hydroxyproline content, and enhance the activity of cell growth factors. It can increase the secretion of extracellular matrix protein and promote the wound healing. @ Treating diabetic ulcer. BSPS can significantly promote the wound healing of streptozotocin-induced diabetic rat model, effectively stimulate the infiltration of inflammatory cells, promote the formation of epithelial tissue, the proliferation of fibroblasts (FB) and the synthesis of collagen and increase the synthesis and release of hydroxyproline (OHP), so as to promote the wound healing of diabetic ulcer. @ Treat bums and scalds. BSPS could significantly shorten the healing time of deep II degree scalded wound in rats. One week after administration, the serum levels of LPS, TNF-a and IL-6 were significantly decreased in rats. The positive expression rate of Toll-like receptor 4 in rat peripheral blood was significantly decreased. ®Treat gastrointestinal ulcer. BSPS has protective effects on cold-water-stressed mouse gastric mucosa injury, ethanol-induced mouse gastric mucosal injury and ligated rat gastric mucosa injury. It can inhibit stress gastric ulcer in a dose-dependent manner. The mechanism of anti-gastric ulcer may be related to strengthening of the gastric mucosal barrier and defense function, reducing of the injury of attack factors to gastric mucosa, promoting of the proliferation of local gastric mucosal epithelial cells of gastric ulcer, and strengthening of the repair of damaged tissues. BSPS could significantly reduce gastric ulcer induced by absolute alcohol in mice, decrease gastric ulcer caused by acetic acid in rats, decrease MDA content in serum and increase SOD content in ulcer rats. In addition, BSPS can promote that heal of gastric ulcer in rats with acute gastric ulcer induce by pyloric ligation, chronic gastric ulcer induced by acetic acid and gastric mucosal injury induced by ethanol. In addition, that mechanism may be relate to the regulation of the expression of the p65 protein of nuclear transcription factorKB (NF-KB), or the inhibition of tumor necrosis factor-a (TNF-a) and nuclear transcription factors KB and upregulation of interleukin-10 (IL-10) levels. BSPS can promote the repair of intestinal mucosa in mice with ulcerative colitis induced by oxazolone, inhibit inflammation and restore immune balance. The mechanism may be related to the inhibition of TNF-a, NF-KB and the up-regulation of IL-10. Hemostasis. BSPs is the pharmacodynamic material basis of Bletilla striata coagulation. The mechanism may be by increasing the platelet aggregation rate, increasing the content of plasminogen activator inhibitor (PAI-i), decreasing the t-PA / PAI-lratio, improving the pathological changes of lung and stomach tissues to exert the function of hemostasis. In addition, that BSPS is sticky in texture, and adheres to the bleeding wound surface and fuses with the wound surface to produce the hemostatic effect of physical obstruction, and also can promote platelet aggregation, accelerate hemostasis, and its hemostatic effects and hemostatic time are similar to thrombin. It has no influence on the whole body coagulation function and has no obvious adverse reaction. It can be used as a local hemostatic drug to treat gastrointestinal bleeding and stress ulcer bleeding. @ Inhibit bacterial growth. BSPS has obvious antibacterial effect on staphylococcus aureus, and can be used to treat burns, reduce the bleeding of burns, in the same time it can inhibit staphylococci growth, and promote the healing of bum wounds. BSPS is soluble in hot water and has strong viscosity and has no side effects, while it has good biodegradability and biocompatibility.

[0019] (3)Bioactive glass is a kind of special glass which has good binding ability to bone tissue and soft tissue. It was developed by Professor Hench in 1969, and its main components include Si02, CaO, Na20 and P20. The biological active glass can remarkably promote the wound healing, and its mechanism is that in the body fluid environment, ions with high surface activity such as Na + and Ca2 + are firstly dissolved out, and ion exchange occurs with H + in body fluid, and the Si-O-Si chemical bond are broken, the irregular network is dissolved, and Si-OH is formed on the glass surface. The soluble silicon is released in the form of silanol to form a gel layer rich in SiO2. The soluble Ca2+ and P043- in the body fluid were adsorbed by SiO2 gel layer and formed hydroxyapatite cementation layer through crystallization process. Soluble silicon has the metabolic and structural action of connective tissue at the molecular level, and after the bioglass is dissolved, the increase of local Si concentration can promote the intracellular response of cell metabolism, stimulate an autosecretory response of the wound healing factor and all cells involved in wound repair at the wound curing factor. All the cells involved in wound healing were stimulated by wound healing factors to accelerate the growth and division, and accumulated on the surface of the material to form the hydroxyapatite cement layer, so that the new tissue could smoothly crawl and cover the whole wound.

[0020] (4) In the bletilla striata polysaccharide / bioglass temperature-sensitive in-situ gelling agent prepared by the present invention, glycerin is used as a stabilizer to increase the viscosity of the preparation, increase the wettability of the bioactive glass, and prevent the sedimentation of bioactive glass.

[0021] (5) In-situ gel is a kind of semi-solid gel preparation which is non-chemically cross-linked in response to the change of environment after being administered in solution. In addition, it combines the advantages of solution and gel, and has the advantages of simple preparation process, convenient use and strong affinity of mucosa tissue. It can be used as a drug reservoir to prolong the residence time in drug delivery sites. Polosham 407 has surface activity and can increase the solubility of bletilla striata polysaccharide, and its unique PEO-PPO-PEO triblock structure enables it to prolong the residence time of bletilla striata polysaccharide at the administration site while facilitating its application which is in favor of improving that bioavailability and curative effect. In that invention, the polosham 407 and the peloxamer 188 are used as in-situ gel carrier material. After compounding in a certain proportion, the polosham 407 and the peloxamer 188 are mixed with injection water in certain proportion, so that the gel temperature is 322°C, and the long time gel can be changed into gel like semisolid at the surface temperature, so the drug can stay on the ulcer surface for a long time, improve the special pathological environment of the wound, and combine the repair effect of bletillae rhizoma polysaccharide and bioactive glass to achieve the purpose of healing the wound.

BRIEF DESCRIPTION OF THE FIGURES

[0022] FIG. 1 is a graph of cumulative release of a bletilla striata polysaccharide/ bioactive glass thermosensitive gel prepared in Example 1 and a solution of bletilla striata polysaccharides prepared in example 3;

[0023] FIG. 2 is a standard graph of glucose control;

[0024] FIG. 3 is a graph showing the results of healing of the ulcer surface of diabetic rats using the temperature-sensitive gel of bletilla striata polysaccharide / bioactive glass prepared in Example 1 and the gel of Examples 2, 3 and 4.

DESCRIPTION OF THE INVENTION

[0025] Various exemplary embodiments of the present invention will now be described in detail, which should not be construed as limiting the invention, but rather as a more detailed description of certain aspects, features, and embodiments thereof.

[0026] It is to be understood that the terms set forth in the present invention are merely for the purpose of describing particular embodiments and are not intended to limit the invention. In addition, for the numerical ranges in the present invention, it is to be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any statement value or range of statements and any other statement values or intermediate values within said range is also included within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the ranges.

[0027] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as would normally be understood by one of ordinary skill in the art to which the invention pertains. Although only preferred methods and materials have been described herein, any method and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. All documents referred to in this specification are incorporated by reference to disclose and describe methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the contents of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made in the specific embodiments of the present description without departing from the scope or spirit of the invention. Other embodiments resulting from the description of the invention will be apparent to the skilled person. The specification and embodiments of the present application are exemplary only.

[0029] As for the words "including," "include," and "have," or "contain," etc., used herein, are open expressions, that is, they mean including but not limited to.

Example 1

[0030] (1)Preparation of bletilla striata polysaccharide: 50g of dried bletilla striata were crushed, sieved, added distilled water, stirred and placed overnight. Distilled water was added on the next day until the material ratio was 1: 20, ultrasonic extraction was carried out for 30 min at room temperature, and average power was 80W. Heating directly in a water bath at 70 °C for 1.5h, adding water to 1000ml in that lower layer of the solution, heating in the water bath for 1 h at 70°C again. Combining the solutions twice, centrifuging for 25min at 4000 rpm, collecting the supernatant, and filtering with suction. In 70 °C, that supernatant is concentrated under reduce pressure to 1 / 3 of the original volume into a viscous state of about 600ml. Add in 3-fold volume of 85% ethanol, and leave at 4 °C overnight, filter by suction the next day, precipitat by alcohol twice, combine with filter residue, and get dried in a 50°C oven, After drying, it was dissolved in water, filtered through a 0.22pm microporous filter membrane, and the filtrate was concentrated and freeze-dried to obtain bletilla striata polysaccharide.

[0031] (2) 20g of polosham 407, 2g of polysacchrides 188and 5g of polysaccharose are uniformly dispersed on the surface of 60g of purified water. After full stirring, it is still placed in a refrigerator at 0-4 °C for 24h, completely dissolved and fully swelled as the first mixture.

[0032] (3) Stirring and dissolving 0.5 g of bioactive glass and 3g of glycerin as a second mixture;

[0033] (4) Adding the second mixture to the first mixture while stirring, adding 0.1 g of DMSO, 0.01 g of hydroxyphenethyl ester, finally adding purified water to 100 g, and stirring to obtain a thermosensitive gel for treating diabetic foot ulcer.

Example 2

[0034] This example is the same as example 1 except that bletilla striata polysaccharide were not added; Example 3

[0035] This example is the same as example 1 except that no bioactive glass was added; Example 4

[0036] This example is the same as example 1, except that the bioactive glass and bletilla striata polysaccharide were not added, only a temperature-sensitive gel matrix polosham was added. Effect example 1

[0037] Hair was removed from the abdomen of mice with 8% Na2S solution. After 24 hours, the mice were killed, the abdominal skin was stripped, washed with normal saline, stored in normal saline at 4 °C, and the isolated skin was prepared. The isolated skin was fixed between a supply chamber and a receiving chamber of a modified Franz diffusion cell, and the products prepared in Examples 1 and 3 were uniformly coated on the skin surface, respectively and the receiving cell was added with 10% ethanol-phosphate buffer. Drain the air bubbles so that one side of the dermis is in full contact with the receiving liquid. Water bath temperature is 32 °C and magnetic stirring speed is 200 r/ min. Take out 2 ml of receiving medium at 1, 3, 6, 9, 12, 15, 18, 24, 30 h respectively, and add the same amount of fresh receiving medium. After the taken-out receiving medium is filtered through a 0.45tm micro-pore filtration membrane, the permeation quantity of bletilla striata polysaccharide is measured and calculated according to the "polysaccharide content determination" method, the concentration is converted according to a standard curve, and the release rate is calculated. The results are shown in FIG. 1, which shows that the release rate of the bletilla striata polysaccharide / bioactive glass thermosensitive gel of the present invention is significantly lower than that of the solution, showing a significant slow-release effect. The bletilla striata polysaccharide/ bioglass thermosensitive gel of the present invention is capable of skin delivery and long term release. In that invention, the skin drug delivery mode is adopt, so that the drug plays a good slow-release role in the drug use part, and the drug can be prevented from extravasation and long time release.

[0038] Determination of polysaccharide content:

[0039] Standard curve drawing

[0040] Accurately weigh 0.0054g of glucose standard, dissolve in water, make up to ml, and mix well to obtain 0.108 mg / ml of standard glucose solution. Take 8 10ml clean test tubes and add 0.10ml, 0.20ml, 030ml, 40ml, 50ml, 0. 60ml, 0.8ml, 1.0ml of standard glucose solution respectively, and add them to 1.00ml with distilled water. Add 3.00ml of anthrone sulfate solution to each test tube, shake it carefully and thoroughly, place it in boiling water bath at 100 °C for 10mmin, take out the test tube and cool it to room temperature. The absorbance was measured at 620nm with the blank solution as reference. Draw a standard curve with the glucose concentration as the abscissa and the absorbance A as the ordinate, as shown in Figure 2. The regression equation is A = 0.38C-0.0103, R2= 0.9992. The linear range of glucose concentration is 0.27-2.7tg / ml.

[0041] Polysaccharide content determination

[0042] Add 0.50ml of receiving liquid into the clean tube, add 1.00ml of distilled water to each tube, shake carefully and thoroughly with sulfuric acid anthrone solution 3.00ml, put it in boiling water at 100 °C for 10mmin, take out the tube and cool it to room temperature. The absorbance was measured at 620nm with the blank solution as reference. The concentration of bletilla striata polysaccharide was calculated according to the standard curve and the release rate was calculated.

Application example 1

[0043] Sixty healthy SD rats were taken to have adaptive feeding for one week, then fasten for 14 hours and take intraperitoneal injection of streptozotocin (STZ, 50 mg - kg 1) to establish the diabetic model. After 72 hours, if the random blood glucose level is more than 16.7 mmol - L-1, the model is considered to be successful. After 4 weeks, diabetic ulcer models were made by shaving the back hair under anesthesia with pentobarbital sodium (35 mg - kg-1), sterilizing, and cutting four round full-thickness skin wounds, deep to subcutaneous with a diameter of 1.8 cm on the back with a special wound maker. After hemostasis, the rats were naturally exposed, and each rat was injected with 5 ml of Ringer's test solution for resuscitation. In that manufacture of the diabetic ulcer model, the wound surface were treated with gel matrix, bioactive glass gel, bletilla striata polysaccharide gel and bletilla striata polysaccharides / bioactive glass gels, respectively. The wound healing area and the proliferation of fibroblasts (FB) were observed and detected at the same time point on the 3rd, 7th and 14th days after injury. The wound healing of diabetic rats after administration is shown in FIG. 3. In that third day after the administration, the wounds of bletilla striata polysaccharide / bioactive glass thermosensitive gel began to heal, and in the seventh day, the wound was significantly less than that of the control group. In the fourteenth day, the wounds of the rats treated with bletilla striata polysaccharide / bioactive glass thermosensitive gel were basically healed, but the wounds in the group treated with bletilla striata polysaccharide thermosensitive gelatin were not completely healed. The effect on the FB proliferation of the wound surface was shown in Table 1. The results showed that the bletilla striata polysaccharide / bioactive glass thermosensitive gel group promoted the growth of fibroblasts in the diabetic ulcer wound surface. On the 3rd and 7th day, it was significantly higher than that of the bletilla striata polysaccharide thermosensitive gel group, and on the 14th day, there was a decrease in the number of cells composed of white, polysaccharide / bioactive glass thermosensitive gels.

[0044] Table 1 Effects of bletilla striata polysaccharide / bioactive glass thermosensitive gel on FB proliferation in diabetic ulcer rats (x ±s, n = 6) Group 3d 7d 14d Pure matrix group 7.1+2.2 26+4.5 55+3.4 Bioactive glass group 7.8+3.7 33+2.8 43+1.2 Bletilla striata polysaccharide 8.3+1.6 35+3.6 41+2.8 thermosensitive gel group Bletilla striata polysaccharide/ 10.2+2.3 40+2.9 37+4.1 bioactive glass thermosensitive gel group

[0045] The gel obtained by the method for preparing the bletilla striata polysaccharide/ bioglass thermosensitive gel according to the embodiment of the present invention is liquid when stored and refrigerated in the refrigerator. Since P407 and P188 are thermosensitive materials, when administered to the ulcer surface, if that temperature reaches 32 °C, the gel is rapidly changed into a gel semi-solid, so that the medicine can stay on the surface of the ulcer for a long time, and the special pathological environment of the wound surface is improved. At the same time, it can combine with repair effect of the bletilla striata polysaccharide and bioactive glass to achieve wound healing. Example 4

[0046] This example is the same with example 1, except that bletilla striata polysaccharide were not added. When the therapeutic effect on diabetic foot ulcer was verify in the same manner as in application example 1, it was found that, in that pure matrix group, a large amount of wound surface still exist after 14 day; the wound surface begins to heal after the biological active glass is added alone; bletilla striata polysaccharide is added alone, and only a few wounds are not healed after the medicine is applied; It is proved that when bioactive glass and rhizoma bletillae polysaccharides are used alone, the therapeutic effect of diabetic foot ulcers is not as good as that of mixed use. Example 1 has good therapeutic effect, which is achieved by synergistic effect of rhizoma bletillae polysaccharide, bioactive glass and thermosensitive gel.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. The bletilla striata polysaccharide / bio glass thermosensitive gel comprising a gel

temperature of 32± 2C, and by mass percentage, the raw material comprises bletilla striata

polysaccharide 3. 0-7. 0%, biological active glass 0. 5%, thermosensitive gel matrix 19-28%,

stabilizing agent 0.5- 5 %, skin absorption accelerator 0.1%, preservative 0.01%, and balance of

water for injection.

2. The bletilla striata polysaccharide / bio glass thermosensitive gel, according to claim 1

wherein the temperature-sensitive gel matrix is selected from one or any combination of

polosham, chitosan, and poly-n-isopropylacrylamide.

3. The bletilla striata polysaccharide / bioglass temperature sensitive gel according to claim 2,

wherein the polosham is composed of poloxam 407 and 188, wherein, in terms of mass

percentage, the percentage by mass of polosham 407 is 18-24%, and the proportion by mass is

1-4% for polosham 188.

4. The bletilla striata polysaccharide / bioglass temperature sensitive gel according to claim 1,

wherein the stabilizer is glycerin, the skin absorption promoter is DMSO, and the preservative

is phenethyl paraben, the water for injection is one or any combination of purified water,

physiological saline, and a buffer solution having a pH of 3 to 8.

5. The bletilla striata polysaccharide / bioglass temperature-sensitive gel according to claim 1,

wherein the method for preparing comprises the steps: bletilla striata tubers were crushed,

sieved, added with distilled water, stirred evenly, and placed overnight; after ultrasonic

extraction, heating by water bath, precipitating the lower layer of the solution adding in water,

and then heating in the water bath again; combining the two solutions, centrifuging, collecting

the supernatant, suction filtering, and concentrating the supernatant under reduced pressure,

immersing in alcohol twice, combining filter residue, drying, dissolving in water, filtering with microporous filter membrane, concentrating and freeze-drying filtrate to obtain bletilla striata polysaccharide.

6. The bletilla striata polysaccharide / bioglass thermosensitive gel according to claim 5

wherein the method for preparing bletilla striata polysaccharide comprises the steps of crushing

and sieving the dried bletilla striata tubers, adding with distilled water, stirring evenly, and

placing it overnight; the next day, distilled water was added until the material-liquid ratio was

1:20, ultrasonic extraction was conducted at room temperature for 30min, and the average

power was 80W; then heating it directly in a 70 °C water bath for 1.5h. The lower layer of the

solution was added with water to 1000ml; then the solution was heated at 70 °C for lh; the

solution was combined twice and centrifuged at 4000rpm for 25min to collect the supernatant;

the supernatant was concentrated to 1/ 3 of its original volume at 70 °C to form a viscous shape

of about 600 ml; 85% ethanol of 3 times volume was added and placed overnight at 4 °C; the

next day, the supernatant was filtered and alcohol precipitated twice; the filter residue was

combined and dried in an oven at 50 °C; after drying, it was dissolved in water and filtered with

0.22 m microporous membrane; the filtrate was concentrated and freeze-dried to obtain

bletilla polysaccharide.

7. A method for preparing a bletilla striata polysaccharide / bioglass thermosensitive gel

according to any one of claims I to 6, comprising the steps of:

heat-sensitive gel matrix, bletilla striata polysaccharide are uniformly mixed with 1 / 3 of water

for injection, placed in refrigerator, completely dissolved and fully swelled as thatfirst mixture;

the Bioglass is ground evenly in the stabilizer to obtain a second mixture; mix the first mixture,

the second mixture, and the skin absorption promoter, preservative with the remaining water

for injection to obtain a bletilla striata polysaccharide / bioglass thermosensitive gel.

8. The method for preparing the bletilla striata polysaccharide / bioglass temperature-sensitive

gel according to claim 7, wherein the gel is refrigerated at 0-4C for 24 hours.

9. The use of a bletilla striata polysaccharide / bioglass temperature sensitive gel according to

any one of claims 1 to 6, wherein the bletilla striata polysaccharide / bioglass thermosensitive

gel is a drug for the treatment of diabetic foot ulcers.

Figure 1 -1/3- accumulative releasing degree(%) 31 Jul 2020 2020101591

Absorbance A -2/3-

Figure 2

-3/3- 31 Jul 2020

Pure matrix group 2020101591

Bioactive glass group

Bletilla striata polysaccharide thermosensitive gel group

Bletilla striata polysaccharide / bioactive glass thermosensitive gel group

Figure 3