WO2022105301A1 - Asiaticoside-chitosan-sodium-alginate microsphere, and preparation method therefor and use thereof - Google Patents
Asiaticoside-chitosan-sodium-alginate microsphere, and preparation method therefor and use thereof Download PDFInfo
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- WO2022105301A1 WO2022105301A1 PCT/CN2021/109745 CN2021109745W WO2022105301A1 WO 2022105301 A1 WO2022105301 A1 WO 2022105301A1 CN 2021109745 W CN2021109745 W CN 2021109745W WO 2022105301 A1 WO2022105301 A1 WO 2022105301A1
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- Prior art keywords
- chitosan
- asiaticoside
- sodium alginate
- solution
- acetic acid
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- 239000004005 microsphere Substances 0.000 title claims abstract description 69
- 239000000661 sodium alginate Substances 0.000 title claims abstract description 51
- 229940005550 sodium alginate Drugs 0.000 title claims abstract description 51
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 85
- 229920001661 Chitosan Polymers 0.000 claims abstract description 64
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 27
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 14
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- QCYLIQBVLZBPNK-UHFFFAOYSA-N asiaticoside A Natural products O1C(C(=O)C(C)C)=CC(C)C(C2(C(OC(C)=O)CC34C5)C)C1CC2(C)C3CCC(C1(C)C)C45CCC1OC1OCC(O)C(O)C1O QCYLIQBVLZBPNK-UHFFFAOYSA-N 0.000 claims description 36
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- 229940022757 asiaticoside Drugs 0.000 claims description 33
- WYQVAPGDARQUBT-FGWHUCSPSA-N Madecassol Chemical compound O([C@@H]1[C@@H](CO)O[C@H]([C@@H]([C@H]1O)O)OC[C@H]1O[C@H]([C@@H]([C@@H](O)[C@@H]1O)O)OC(=O)[C@]12CC[C@H]([C@@H]([C@H]1C=1[C@@]([C@@]3(CC[C@H]4[C@](C)(CO)[C@@H](O)[C@H](O)C[C@]4(C)[C@H]3CC=1)C)(C)CC2)C)C)[C@@H]1O[C@@H](C)[C@H](O)[C@@H](O)[C@H]1O WYQVAPGDARQUBT-FGWHUCSPSA-N 0.000 claims description 32
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5021—Organic macromolecular compounds
- A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7024—Esters of saccharides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5089—Processes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
Definitions
- the invention relates to the technical field of biomedical materials, in particular to asiaticoside-chitosan-sodium alginate microspheres, a preparation method and applications thereof.
- Asiaticoside is a triterpenoid saponin compound, which is the main active component of the traditional Chinese medicine Centella asiatica. It has excellent effects on promoting wound healing and resisting hypertrophic scars. In addition, the researchers induced mice with collagen. Arthritis experiments have found that asiaticoside can inhibit the production of arthritis in mice. The specific mechanism of action may be through inhibiting the expression of COX-2, reducing the expression and release of inflammatory factors such as IL-6 and TNF- ⁇ , so as to inhibit the production of arthritis. The proliferation of lymphocytes, thereby inhibiting the occurrence of immune responses.
- asiaticoside can inhibit the expression of COX-2, inhibit the expression and secretion of inflammatory factors such as MPO, and promote the expression and activity of inflammatory protective factors such as HO-1, thereby promoting the Inhibit the occurrence of inflammatory response; at the same time, asiaticoside can also inhibit the accumulation of heating medium PGE2, so as to achieve the effect of inhibiting lipopolysaccharide-induced fever in rats and leading to inflammatory response.
- asiaticoside can promote fibroblast proliferation and extracellular matrix synthesis during wound healing, and increase the content of hydroxyproline, improve epidermal growth, collagen and skin growth, and promote chicken Angiogenesis in the embryonic choriourea membrane model.
- AS is mostly used for the prevention and treatment of aging-related chronic inflammation and the diseases caused by it in the form of skin coating, especially for allergic diseases, abnormal skin pigmentation, excessive formation of skin blood vessels or inflammation Sexual laceration, and the prevention and treatment of regulating the homeostasis of skin tissue.
- Chitosan is a polycationic polysaccharide containing a large number of amino groups obtained by deacetylation of chitin. It has good biocompatibility, non-toxicity and biodegradability.
- Sodium alginate (SA) is a polyanionic polysaccharide containing carboxyl groups extracted from marine organisms brown algae, with good bioadhesion and biodegradability.
- the polyelectrolyte complex formed by the amino group of chitosan and the carboxyl group of sodium alginate attracted by positive and negative charges has the functions of hemostasis, anti-inflammatory and promoting wound repair.
- Sodium alginate-chitosan microspheres is a new type of preparation with sustained release.
- the release rate of the drug By controlling the release rate of the drug, it can achieve the purpose of slow and controlled release and prolong the drug effect, and at the same time protect the drug from enzymatic degradation. It can improve the bioavailability of drugs; at the same time, drugs with low therapeutic index, poor water solubility and unstable properties can be encapsulated in it, so that the drugs can be concentrated in the target area, enhance the curative effect and reduce the toxic and side effects.
- the application of microspheres prepared by traditional emulsion cross-linking method is limited due to the use of toxic cross-linking agents such as glutaraldehyde.
- AS can use natural sodium alginate-chitosan microspheres as a carrier and use non-toxic glyceraldehyde as a new cross-linking agent, it is expected to reduce the toxicity of the microspheres, and achieve its continuous administration to lesions, synergistic efficacy, And reduce the pain of the patient caused by the sudden release of the drug and repeated administration, thereby reducing the adverse drug reaction.
- the invention provides an asiaticoside-chitosan- The preparation method of sodium alginate microspheres.
- the present invention chooses to use sodium alginate-chitosan microspheres to encapsulate AS, and adopts the emulsification cross-linking method to prepare asiaticoside-sodium alginate-chitosan microspheres, aiming to integrate AS and sodium alginate-chitosan
- the performance advantages of AS can provide a certain method basis for proposing new AS dosage forms.
- the present invention provides the following scheme:
- the invention provides a preparation method of asiaticoside-chitosan-sodium alginate microspheres, comprising the following steps: preparing asiaticoside-chitosan-sodium alginate microspheres by an emulsification cross-linking method, freeze-drying That's it.
- the preparation method specifically includes the following steps:
- step (c) the asiaticoside-chitosan acetic acid solution containing calcium chloride is slowly and uniformly added dropwise to the colostrum formed in step (b), stirred, and fully emulsified to form an emulsion;
- step (d) slowly and uniformly add glyceraldehyde solution dropwise to the emulsion obtained in step (c) (it takes about 5 minutes to add 1 ml), after cross-linking and solidification, centrifuge, remove the supernatant, and wash the precipitate with petroleum ether in turn, without Washed with water ethanol, vacuum freeze-dried to obtain light yellow powder of asiaticoside-sodium alginate-chitosan microspheres.
- the volume fraction of the glacial acetic acid aqueous solution in step (a) is 2%, the mass ratio of anhydrous calcium chloride and chitosan is 4:1, and the mass of chitosan and madecassoside mixed The ratio is 1:0.2-0.6.
- the volume ratio of sodium alginate and oil phase in step (b) is 1:6.
- the volume ratio of the calcium chloride-containing asiaticoside-chitosan acetic acid solution to colostrum in step (c) is 1:7.
- the volume ratio of glyceraldehyde in step (d) to the emulsion obtained in step (c) is 1:40.
- the molecular weight of the chitosan is 300-400 kDa, and the degree of deacetylation is 90%.
- the present invention also provides madecassoside-chitosan-sodium alginate microspheres prepared by the method for preparing the asiaticoside-chitosan-sodium alginate microspheres.
- the present invention also provides the application of the asiaticoside-chitosan-sodium alginate microspheres as wound healing materials.
- the asiaticoside-chitosan-sodium alginate microspheres prepared by the present invention have a good slow-release effect
- the madecassoside-chitosan-sodium alginate microspheres prepared by the present invention are small and uniform, with small degree of adhesion, high roundness, and narrow particle size range, and the particle size range is measured by a laser particle size analyzer, Using wet dispersion technology, mechanical stirring makes the microspheres evenly disperse in the water phase, ultrasonic high-frequency vibration makes the agglomerated microspheres fully dispersed, and electromagnetic circulation pump makes the microspheres evenly distributed in the whole circulation system.
- the asiaticoside-chitosan-sodium alginate microspheres prepared by the present invention have high drug loading and encapsulation efficiency
- the asiaticoside-chitosan-sodium alginate microspheres prepared by the present invention have good biocompatibility and are biodegradable;
- the asiaticoside-chitosan-sodium alginate microsphere preparation process prepared by the present invention has mild reaction conditions, simple operation, easy to obtain microspheres with round appearance, high yield, and easy industrial production.
- Fig. 1 is the microsphere particle size distribution figure prepared by embodiment 1;
- Fig. 2 is the electron microscope image of the microsphere prepared in Example 1-microsphere magnified 5k times the electron microscope image;
- Fig. 3 is the electron microscope image of the microsphere prepared in Example 1-microsphere magnified 10k times the electron microscope image;
- Fig. 4 is the time-release curve of microspheres prepared in Example 1;
- FIG. 5 is a time-cumulative release percentage curve of the microspheres prepared in Example 1.
- the sodium alginate of the invention is of food grade or pharmaceutical grade, with a molecular weight of 100 kDa; the molecular weight of chitosan is 300-400 kDa, and the degree of deacetylation is 90%.
- M t VC i + ⁇ C i-1 V sample (wherein, M t is the cumulative release amount, C i is the release concentration of asiaticoside in the ith sampling; V is the first sampling The previous volume was 50 mL in this experiment; C i-1 corresponds to the concentration of asiaticoside at the sampling point before the sampling point at time i; V sample is the volume of each sampling, which is 5 mL in this experiment.)
- the microspheres of the present invention are uniform in size, small in degree of adhesion, and high in roundness.
- the in vitro release of the asiaticoside-sodium alginate-chitosan microspheres prepared by the present invention is divided into two stages: burst release and sustained release, and the released amount of asiaticoside in the first 5 hours Small, a burst of asiaticoside occurred at 6h, and the cumulative release percentage was (43.79 ⁇ 2.07)%.
- asiaticoside began to be released into the body at a slow rate, and the average hourly release of asiaticoside was 0.77% for 7-24h.
- the cumulative release percentage in 24h was (57.65 ⁇ 1.32)%
- the average daily release of asiaticoside was 0.92% in 2-7d
- the cumulative release percentage in 7d was (90.03 ⁇ 0.84)%.
- a laser particle size analyzer was used to measure the particle size range of the microspheres prepared in the best example.
- Wet dispersion technology was used to disperse the microspheres uniformly in the water phase by mechanical stirring.
- Drug loading (DL) [the amount of drug in the microspheres - the weight of the microspheres]*100%;
- Encapsulation efficiency (EE) [the amount of drug in the microspheres - the dose of drug] * 100%;
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Abstract
An asiaticoside-chitosan-sodium-alginate microsphere, a preparation method therefor and the use thereof. The microsphere is prepared by using an emulsion cross-linking method and is finally obtained by means of freeze-drying. The preparation method comprises the following steps: 1) respectively preparing a sodium alginate solution and an asiaticoside-chitosan acetic acid solution; 2) slowly and uniformly adding the sodium alginate solution to an oil phase in a dropwise manner, and stirring same at a high speed to form a primary emulsion; 3) slowly and uniformly adding the asiaticoside-chitosan acetic acid solution to the primary emulsion in a dropwise manner, stirring same, and fully emulsifying same; and 4) slowly and uniformly adding a glyceraldehyde solution in a dropwise manner for cross-linking and curing, performing centrifugation, sequentially washing a precipitate with petroleum ether and anhydrous ethanol, and performing vacuum freeze-drying to obtain a light yellow asiaticoside-sodium-alginate-chitosan microsphere powder.
Description
本发明涉及生物医用材料技术领域，特别是涉及一种积雪草苷-壳聚糖-海藻酸钠微球、制备方法及其应用。The invention relates to the technical field of biomedical materials, in particular to asiaticoside-chitosan-sodium alginate microspheres, a preparation method and applications thereof.
积雪草苷(Asiaticoside，AS)是一种三萜皂苷类化合物，是中药积雪草的主要活性成分，具有促进创伤愈合、抗肥厚性瘢痕的优良作用；此外，研究人员通过胶原诱导小鼠关节炎的实验发现，积雪草苷能够抑制小鼠关节炎的产生，具体作用机制可能是通过抑制COX-2的表达，降低IL-6、TNF-α等炎症因子的表达和释放，以抑制淋巴细胞的增殖，从而抑制免疫反应的发生。研究人员在脂多糖诱导大鼠发热的实验中发现积雪草苷能够通过抑制COX-2的表达，抑制MPO等炎症因子的表达和分泌，促进HO-1等炎症保护因子的表达和活性，从而抑制炎症反应的发生；同时，积雪草苷还能够抑制制热介质PGE2的积累，从而达到抑制脂多糖诱导大鼠发热导致炎症反应发生的作用。此外，已有研究还表明积雪草苷可以在创伤愈合过程中促进成纤维细胞增生以及细胞外基质的合成，并且使羟基脯氨酸含量增加，改善表皮生长、胶原蛋白和皮肤生长，促进鸡胚绒毛脲囊膜模型血管新生。基于以上诸多研究，当前AS多以皮肤涂布给药的方式用于防治与衰老相关的慢性炎症和由此引起的疾病，尤其用于过敏性疾病、皮肤色素沉着异常、皮肤血管过度形成或炎性裂伤，以及调节皮肤组织内稳态的防治。Asiaticoside (AS) is a triterpenoid saponin compound, which is the main active component of the traditional Chinese medicine Centella asiatica. It has excellent effects on promoting wound healing and resisting hypertrophic scars. In addition, the researchers induced mice with collagen. Arthritis experiments have found that asiaticoside can inhibit the production of arthritis in mice. The specific mechanism of action may be through inhibiting the expression of COX-2, reducing the expression and release of inflammatory factors such as IL-6 and TNF-α, so as to inhibit the production of arthritis. The proliferation of lymphocytes, thereby inhibiting the occurrence of immune responses. In the experiment of lipopolysaccharide-induced fever in rats, the researchers found that asiaticoside can inhibit the expression of COX-2, inhibit the expression and secretion of inflammatory factors such as MPO, and promote the expression and activity of inflammatory protective factors such as HO-1, thereby promoting the Inhibit the occurrence of inflammatory response; at the same time, asiaticoside can also inhibit the accumulation of heating medium PGE2, so as to achieve the effect of inhibiting lipopolysaccharide-induced fever in rats and leading to inflammatory response. In addition, studies have also shown that asiaticoside can promote fibroblast proliferation and extracellular matrix synthesis during wound healing, and increase the content of hydroxyproline, improve epidermal growth, collagen and skin growth, and promote chicken Angiogenesis in the embryonic choriourea membrane model. Based on many of the above studies, currently AS is mostly used for the prevention and treatment of aging-related chronic inflammation and the diseases caused by it in the form of skin coating, especially for allergic diseases, abnormal skin pigmentation, excessive formation of skin blood vessels or inflammation Sexual laceration, and the prevention and treatment of regulating the homeostasis of skin tissue.
壳聚糖(CS)是由甲壳素脱乙酰化后得到的含有大量氨基的聚阳离子多糖，具有良好的生物相容性、无毒、可生物降解性。海藻酸钠(SA)是从海洋生物褐藻中提取的含有羧基的聚阴离子多糖，具有良好的生物黏性、可生物降解性。壳聚糖的氨基和海藻酸钠的羧基通过正负电荷吸引形成的聚电解质复合物，具有止血、抗炎、促进创面修复的作用。而海藻酸钠-壳聚糖微球则是一种具有缓释作用的新型制剂，通过控制药物的释放速度从而达到缓控释和延长药效的目的，同时保护药物免受酶降解的影响，起到提高药物生物利用度的作用；同时，还可将治疗指数低、水溶性差、性质不稳定的药物包载其内，以使药物浓集于靶区，增强疗效，降低毒副作用。但传统的乳化交联法制备的微球由于使用了戊二醛等有毒的交联剂，使得其应用受到限制。若AS能以天然海藻酸钠-壳聚糖微球为载体，使用无毒的甘油醛作为新型交联剂，有望减小微球的毒性，并实现其对病灶的持续给药，协同疗效，并降低药物突释而反复给药对病人带来的痛苦，进而减小药物不良反应。Chitosan (CS) is a polycationic polysaccharide containing a large number of amino groups obtained by deacetylation of chitin. It has good biocompatibility, non-toxicity and biodegradability. Sodium alginate (SA) is a polyanionic polysaccharide containing carboxyl groups extracted from marine organisms brown algae, with good bioadhesion and biodegradability. The polyelectrolyte complex formed by the amino group of chitosan and the carboxyl group of sodium alginate attracted by positive and negative charges has the functions of hemostasis, anti-inflammatory and promoting wound repair. Sodium alginate-chitosan microspheres is a new type of preparation with sustained release. By controlling the release rate of the drug, it can achieve the purpose of slow and controlled release and prolong the drug effect, and at the same time protect the drug from enzymatic degradation. It can improve the bioavailability of drugs; at the same time, drugs with low therapeutic index, poor water solubility and unstable properties can be encapsulated in it, so that the drugs can be concentrated in the target area, enhance the curative effect and reduce the toxic and side effects. However, the application of microspheres prepared by traditional emulsion cross-linking method is limited due to the use of toxic cross-linking agents such as glutaraldehyde. If AS can use natural sodium alginate-chitosan microspheres as a carrier and use non-toxic glyceraldehyde as a new cross-linking agent, it is expected to reduce the toxicity of the microspheres, and achieve its continuous administration to lesions, synergistic efficacy, And reduce the pain of the patient caused by the sudden release of the drug and repeated administration, thereby reducing the adverse drug reaction.
发明内容SUMMARY OF THE INVENTION
本发明针对目前积雪草苷的生物利用度低以及微球的制备方法的不足，即使用戊二醛等有毒交联剂引起的微球毒性，提供一种积雪草苷-壳聚糖-海藻酸钠微球的制备方法。本发明选择用海藻酸钠-壳聚糖微球包载AS，采用乳化交联法制备积雪草苷-海藻酸钠-壳聚糖微球，旨在整合AS与海藻酸钠-壳聚糖的性能优势，为提出AS新的给药剂型提供一定的方法依据。Aiming at the low bioavailability of asiaticoside and the deficiency of the preparation method of microspheres at present, the invention provides an asiaticoside-chitosan- The preparation method of sodium alginate microspheres. The present invention chooses to use sodium alginate-chitosan microspheres to encapsulate AS, and adopts the emulsification cross-linking method to prepare asiaticoside-sodium alginate-chitosan microspheres, aiming to integrate AS and sodium alginate-chitosan The performance advantages of AS can provide a certain method basis for proposing new AS dosage forms.
为实现上述目的，本发明提供了如下方案：For achieving the above object, the present invention provides the following scheme:
本发明提供一种积雪草苷-壳聚糖-海藻酸钠微球的制备方法，包括以下步骤：采用乳化交联法制备积雪草苷-壳聚糖-海藻酸钠微球，冷冻干燥即可。The invention provides a preparation method of asiaticoside-chitosan-sodium alginate microspheres, comprising the following steps: preparing asiaticoside-chitosan-sodium alginate microspheres by an emulsification cross-linking method, freeze-drying That's it.
作为本发明的进一步改进，所述制备方法，具体包括以下步骤：As a further improvement of the present invention, the preparation method specifically includes the following steps:
(a)将壳聚糖加入到体积分数为1-3％的冰醋酸溶液中溶解壳聚糖，制备壳聚糖醋酸溶液；用无水乙醇溶解积雪草苷得到积雪草苷溶液；将壳聚糖水溶液与积雪草苷溶液混合，搅匀，待乙醇挥发完，加入无水氯化钙，即得含氯化钙的积雪草苷-壳聚糖醋酸溶液；(a) adding chitosan to a glacial acetic acid solution with a volume fraction of 1-3% to dissolve chitosan to prepare a chitosan acetic acid solution; dissolving asiaticoside with absolute ethanol to obtain an asiaticoside solution; The chitosan aqueous solution is mixed with the asiaticoside solution, stirred evenly, and after the ethanol has evaporated, anhydrous calcium chloride is added to obtain a calcium chloride-containing asiaticoside-chitosan acetic acid solution;
(b)用纯净水溶解海藻酸钠，制得海藻酸钠水溶液；将配制好的海藻酸钠溶液缓慢均匀地滴加到油相(29.25ml液体石蜡和0.75mlSpan80的混合溶液)中，以500-800rpm的速度搅拌20-40min形成初乳；(b) dissolving sodium alginate with purified water to obtain an aqueous solution of sodium alginate; slowly and evenly add the prepared sodium alginate solution dropwise to the oil phase (mixed solution of 29.25ml liquid paraffin and 0.75ml Span80), add 500 -800rpm stirring for 20-40min to form colostrum;
(c)将含氯化钙的积雪草苷-壳聚糖醋酸溶液缓慢均匀地滴加到步骤(b)形成的初乳中，搅拌，充分乳化，形成乳液；(c) the asiaticoside-chitosan acetic acid solution containing calcium chloride is slowly and uniformly added dropwise to the colostrum formed in step (b), stirred, and fully emulsified to form an emulsion;
(d)在步骤(c)得到的乳液中缓慢均匀地滴加甘油醛溶液(加1ml大约需要5分钟)，交联固化后，离心，除去上清液，沉淀物依次用石油醚洗涤、无水乙醇洗涤，真空冷冻干燥，即得积雪草苷-海藻酸钠-壳聚糖微球的浅黄色粉末。(d) slowly and uniformly add glyceraldehyde solution dropwise to the emulsion obtained in step (c) (it takes about 5 minutes to add 1 ml), after cross-linking and solidification, centrifuge, remove the supernatant, and wash the precipitate with petroleum ether in turn, without Washed with water ethanol, vacuum freeze-dried to obtain light yellow powder of asiaticoside-sodium alginate-chitosan microspheres.
作为本发明的进一步改进，步骤(a)中冰醋酸水溶液的体积分数为2％，无水氯化钙与壳聚糖的质量比为4：1，壳聚糖与积雪草苷混合的质量比例为1∶0.2-0.6。As a further improvement of the present invention, the volume fraction of the glacial acetic acid aqueous solution in step (a) is 2%, the mass ratio of anhydrous calcium chloride and chitosan is 4:1, and the mass of chitosan and madecassoside mixed The ratio is 1:0.2-0.6.
作为本发明的进一步改进，步骤(b)中海藻酸钠和油相的体积比为1∶6。As a further improvement of the present invention, the volume ratio of sodium alginate and oil phase in step (b) is 1:6.
作为本发明的进一步改进，步骤(c)中含氯化钙的积雪草苷-壳聚糖醋酸溶液与初乳的体积比为1∶7。As a further improvement of the present invention, the volume ratio of the calcium chloride-containing asiaticoside-chitosan acetic acid solution to colostrum in step (c) is 1:7.
作为本发明的进一步改进，步骤(d)中甘油醛与步骤(c)得到的乳液的体积比为1∶40。As a further improvement of the present invention, the volume ratio of glyceraldehyde in step (d) to the emulsion obtained in step (c) is 1:40.
作为本发明的进一步改进，所述壳聚糖分子量300-400kDa、脱乙酰度90％。As a further improvement of the present invention, the molecular weight of the chitosan is 300-400 kDa, and the degree of deacetylation is 90%.
本发明还提供一种由所述的积雪草苷-壳聚糖-海藻酸钠微球制备方法制备得到的积雪草苷-壳聚糖-海藻酸钠微球。The present invention also provides madecassoside-chitosan-sodium alginate microspheres prepared by the method for preparing the asiaticoside-chitosan-sodium alginate microspheres.
本发明还提供所述的积雪草苷-壳聚糖-海藻酸钠微球在作为创伤愈合材料中的应用。The present invention also provides the application of the asiaticoside-chitosan-sodium alginate microspheres as wound healing materials.
本发明公开了以下技术效果：The present invention discloses the following technical effects:
(1)本发明制备的积雪草苷-壳聚糖-海藻酸钠微球具有良好的缓释作用；(1) the asiaticoside-chitosan-sodium alginate microspheres prepared by the present invention have a good slow-release effect;
(2)采用温和无毒的交联剂，不仅使材料具有无毒安全的性能，且提高了均匀度，载药量、包封率高；(2) The use of mild and non-toxic cross-linking agent not only makes the material non-toxic and safe, but also improves the uniformity, high drug loading and encapsulation efficiency;
(3)本发明制备的积雪草苷-壳聚糖-海藻酸钠微球小均一，黏连程度小，圆整度高，粒径范围窄，采用激光粒度分析仪测其粒径范围，利用湿法分散技术，机械搅拌使微球在水相中均匀散开，超声高频震荡使团聚的微球充分分散，电磁循环泵使微球在整个循环体系中 均匀分布，测试结果显示：微球的粒径多数都分布在3.122-4.366μm之间，D
10＝3.122μm，D
50＝3.819μm,D
90＝4.366μm，Sp＝0.326；
(3) the madecassoside-chitosan-sodium alginate microspheres prepared by the present invention are small and uniform, with small degree of adhesion, high roundness, and narrow particle size range, and the particle size range is measured by a laser particle size analyzer, Using wet dispersion technology, mechanical stirring makes the microspheres evenly disperse in the water phase, ultrasonic high-frequency vibration makes the agglomerated microspheres fully dispersed, and electromagnetic circulation pump makes the microspheres evenly distributed in the whole circulation system. The test results show that: The particle sizes of the balls are mostly distributed between 3.122-4.366μm, D 10 =3.122μm, D 50 =3.819μm, D 90 =4.366μm, Sp = 0.326;
(4)本发明制备的积雪草苷-壳聚糖-海藻酸钠微球载药量、包封率高；(4) the asiaticoside-chitosan-sodium alginate microspheres prepared by the present invention have high drug loading and encapsulation efficiency;
(5)本发明制备的积雪草苷-壳聚糖-海藻酸钠微球生物相容性好，可生物降解；(5) the asiaticoside-chitosan-sodium alginate microspheres prepared by the present invention have good biocompatibility and are biodegradable;
(6)本发明制备的积雪草苷-壳聚糖-海藻酸钠微球制备工艺反应条件温和，操作简单，易得到外观圆整的微球，且产率较高，易于工业化生产。(6) The asiaticoside-chitosan-sodium alginate microsphere preparation process prepared by the present invention has mild reaction conditions, simple operation, easy to obtain microspheres with round appearance, high yield, and easy industrial production.
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings required in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some of the present invention. In the embodiments, for those of ordinary skill in the art, other drawings can also be obtained according to these drawings without creative labor.
图1为实施例1制备的微球粒径分布图；Fig. 1 is the microsphere particle size distribution figure prepared by embodiment 1;
图2为实施例1制备的微球电镜图-微球放大5k倍电镜图；Fig. 2 is the electron microscope image of the microsphere prepared in Example 1-microsphere magnified 5k times the electron microscope image;
图3为实施例1制备的微球电镜图-微球放大10k倍电镜图；Fig. 3 is the electron microscope image of the microsphere prepared in Example 1-microsphere magnified 10k times the electron microscope image;
图4为实施例1制备的微球时间-释放量曲线；Fig. 4 is the time-release curve of microspheres prepared in Example 1;
图5为实施例1制备的微球时间-累计释放百分数曲线。FIG. 5 is a time-cumulative release percentage curve of the microspheres prepared in Example 1. FIG.
现详细说明本发明的多种示例性实施方式，该详细说明不应认为 是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。Various exemplary embodiments of the present invention will now be described in detail, which detailed description should not be construed as a limitation of the invention, but rather as a more detailed description of certain aspects, features, and embodiments of the invention.
应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。另外，对于本发明中的数值范围，应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。It should be understood that the terms described in the present invention are only used to describe particular embodiments, and are not used to limit the present invention. Additionally, for numerical ranges in the present disclosure, it should be understood that each intervening value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated value or intervening value in that stated range is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates. Although only the preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials in connection with which the documents are referred. In the event of conflict with any incorporated document, the content of this specification controls.
在不背离本发明的范围或精神的情况下，可对本发明说明书的具体实施方式做多种改进和变化，这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the present invention. The description and examples of the present invention are exemplary only.
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等，均为开放性的用语，即意指包含但不限于。As used herein, "comprising," "including," "having," "containing," and the like, are open-ended terms, meaning including but not limited to.
本发明所述海藻酸钠为食品级或医药级，分子量100kDa；壳聚糖分子量300-400kDa、脱乙酰度90％。The sodium alginate of the invention is of food grade or pharmaceutical grade, with a molecular weight of 100 kDa; the molecular weight of chitosan is 300-400 kDa, and the degree of deacetylation is 90%.
实施例1Example 1
(a)称取1g壳聚糖加入到100mL体积分数为2％的冰醋酸溶液中溶解壳聚糖，制备-壳聚糖醋酸溶液；将0.6g的积雪草苷用无水乙醇溶解得到积雪草苷溶液；将上述二者溶液混合，搅匀，待乙醇挥发净，加入2g的无水氯化钙，即得含氯化钙的积雪草苷-壳聚糖醋酸溶液；用纯净水溶解1g的海藻酸钠，制得海藻酸钠水溶液；(a) Weigh 1 g of chitosan and add it to 100 mL of 2% glacial acetic acid solution to dissolve chitosan to prepare - chitosan acetic acid solution; dissolve 0.6 g of asiaticoside in absolute ethanol to obtain a Madecassoside solution; mix the above two solutions, stir evenly, and after the ethanol is volatilized, add 2g of anhydrous calcium chloride to obtain a calcium chloride-containing asiaticoside-chitosan acetic acid solution; use purified water Dissolve 1 g of sodium alginate to obtain an aqueous solution of sodium alginate;
(b)将配制好的海藻酸钠溶液缓慢均匀地滴加到油相中，800rpm搅拌30min形成初乳；(b) the prepared sodium alginate solution is slowly and uniformly added dropwise to the oil phase, and stirred at 800rpm for 30min to form colostrum;
(c)将含氯化钙的积雪草苷-壳聚糖醋酸溶液缓慢均匀地滴加到初乳中，以1000转高速搅拌，充分乳化1h；(c) The asiaticoside-chitosan acetic acid solution containing calcium chloride is slowly and evenly added dropwise to the colostrum, stirred at a high speed of 1000 rpm, and fully emulsified for 1h;
(d)缓慢均匀地滴加1ml的甘油醛溶液，交联固化1h后，以1000rpm离心10min，除去上清液，沉淀物用石油醚洗涤两次，无水乙醇洗涤一次，真空冷冻干燥，即得积雪草苷-海藻酸钠-壳聚糖微球的浅黄色粉末。(d) Slowly and evenly add 1 ml of glyceraldehyde solution dropwise, after 1 h of cross-linking and solidification, centrifuge at 1000 rpm for 10 min, remove the supernatant, wash the precipitate twice with petroleum ether, once with absolute ethanol, and freeze-dry in vacuo, that is, The pale yellow powder of asiaticoside-sodium alginate-chitosan microspheres was obtained.
实施例2Example 2
(a)称取1g壳聚糖加入到100mL体积分数为1％的冰醋酸溶液中溶解壳聚糖，制备壳聚糖醋酸溶液；将0.4g的积雪草苷用无水乙醇溶解得到积雪草苷溶液；将上述二者溶液混合，搅匀，待乙醇挥发净，加入2g的无水氯化钙，即得含氯化钙的积雪草苷-壳聚糖醋酸溶液；用纯净水溶解1g的海藻酸钠，制得海藻酸钠水溶液；(a) Weigh 1 g of chitosan and add it to 100 mL of glacial acetic acid solution with a volume fraction of 1% to dissolve chitosan to prepare a chitosan acetic acid solution; dissolve 0.4 g of asiaticoside in absolute ethanol to obtain snow asiatica oxaloside solution; mix the above two solutions, stir evenly, and after the ethanol is volatilized, add 2 g of anhydrous calcium chloride to obtain a calcium chloride-containing asiaticoside-chitosan acetic acid solution; dissolve in purified water 1 g of sodium alginate to obtain an aqueous solution of sodium alginate;
(b)将配制好的海藻酸钠溶液缓慢均匀地滴加到油相中，500rpm搅拌40min形成初乳；(b) the prepared sodium alginate solution is slowly and evenly added dropwise to the oil phase, and the colostrum is formed by stirring at 500rpm for 40min;
(c)将含氯化钙的积雪草苷-壳聚糖醋酸溶液缓慢均匀地滴加到初乳中，以3000转高速搅拌，充分乳化1h；(c) adding the asiaticoside-chitosan acetic acid solution containing calcium chloride slowly and uniformly to the colostrum, stirring at a high speed of 3000 rpm, and fully emulsifying for 1 h;
(d)缓慢均匀地滴加1ml的甘油醛溶液，交联固化1h后，以1000rpm离心10min，除去上清液，沉淀物用石油醚洗涤两次，无水乙醇洗涤一次，真空冷冻干燥，即得积雪草苷-海藻酸钠-壳聚糖微球的浅黄色粉末。(d) Slowly and evenly add 1 ml of glyceraldehyde solution dropwise, after 1 h of cross-linking and solidification, centrifuge at 1000 rpm for 10 min, remove the supernatant, wash the precipitate twice with petroleum ether, once with absolute ethanol, and freeze-dry in vacuo, that is, The pale yellow powder of asiaticoside-sodium alginate-chitosan microspheres was obtained.
实施例3Example 3
(a)称取1g壳聚糖加入到100mL体积分数为3％的冰醋酸溶液中溶解壳聚糖，制备壳聚糖醋酸溶液；将0.6g的积雪草苷用无水乙醇溶解得到积雪草苷溶液；将上述二者溶液混合，搅匀，待乙醇挥发净，加入2g的无水氯化钙，即得含氯化钙的积雪草苷-壳聚糖醋酸溶液；用纯净水溶解1.5g的海藻酸钠，制得海藻酸钠水溶液；(a) Weigh 1 g of chitosan and add it to 100 mL of glacial acetic acid solution with a volume fraction of 3% to dissolve chitosan to prepare a chitosan acetic acid solution; dissolve 0.6 g of asiaticoside in absolute ethanol to obtain snow asiatica oxaloside solution; mix the above two solutions, stir evenly, and after the ethanol is volatilized, add 2 g of anhydrous calcium chloride to obtain a calcium chloride-containing asiaticoside-chitosan acetic acid solution; dissolve in purified water 1.5g of sodium alginate to obtain an aqueous solution of sodium alginate;
(b)将配制好的海藻酸钠溶液缓慢均匀地滴加到油相中，700rpm搅拌20min形成初乳；(b) the prepared sodium alginate solution is slowly and uniformly added dropwise to the oil phase, and stirred at 700rpm for 20min to form colostrum;
(c)将含氯化钙的积雪草苷-壳聚糖醋酸溶液缓慢均匀地滴加到初乳中，以3000转高速搅拌，充分乳化1h；(c) adding the asiaticoside-chitosan acetic acid solution containing calcium chloride slowly and uniformly to the colostrum, stirring at a high speed of 3000 rpm, and fully emulsifying for 1 h;
(d)缓慢均匀地滴加2ml的甘油醛溶液，交联固化1h后，以1000rpm离心10min，除去上清液，沉淀物用石油醚洗涤两次，无水乙醇洗涤两次，真空冷冻干燥，即得积雪草苷-海藻酸钠-壳聚糖微球的浅黄色粉末。(d) Slowly and uniformly add 2 ml of glyceraldehyde solution dropwise, after cross-linking and solidifying for 1 hour, centrifuge at 1000 rpm for 10 min, remove the supernatant, wash the precipitate twice with petroleum ether, twice with absolute ethanol, and freeze-dried in vacuo. That is, the light yellow powder of asiaticoside-sodium alginate-chitosan microspheres is obtained.
实施例4Example 4
(a)称取1g壳聚糖加入到100mL体积分数为2％的冰醋酸溶液中溶解壳聚糖，制备壳聚糖醋酸溶液；将0.2g的积雪草苷用无水乙醇溶解得到积雪草苷溶液；将上述二者溶液混合，搅匀，待乙醇挥发净，加入2g的无水氯化钙，即得含氯化钙的积雪草苷-壳聚糖醋酸溶液；用纯净水溶解0.5g的海藻酸钠，制得海藻酸钠水溶液；(a) Weigh 1 g of chitosan and add it to 100 mL of 2% glacial acetic acid solution to dissolve chitosan to prepare chitosan acetic acid solution; dissolve 0.2 g of asiaticoside in absolute ethanol to obtain snow asiatica oxaloside solution; mix the above two solutions, stir evenly, and after the ethanol is volatilized, add 2 g of anhydrous calcium chloride to obtain a calcium chloride-containing asiaticoside-chitosan acetic acid solution; dissolve in purified water 0.5g of sodium alginate to obtain an aqueous solution of sodium alginate;
(b)将配制好的海藻酸钠溶液缓慢均匀地滴加到油相中，800rpm搅拌20min形成初乳；(b) the prepared sodium alginate solution is slowly and uniformly added dropwise to the oil phase, and stirred at 800rpm for 20min to form colostrum;
(c)将含氯化钙的积雪草苷-壳聚糖醋酸溶液缓慢均匀地滴加到初乳中，以3000转高速搅拌，充分乳化1h；(c) adding the asiaticoside-chitosan acetic acid solution containing calcium chloride slowly and uniformly to the colostrum, stirring at a high speed of 3000 rpm, and fully emulsifying for 1h;
(d)缓慢均匀地滴加1ml的甘油醛溶液，交联固化1h后，以1000rpm离心10min，除去上清液，沉淀物用石油醚洗涤两次，无水乙醇洗涤一次，真空冷冻干燥，即得积雪草苷-海藻酸钠-壳聚糖微球的浅黄色粉末。(d) Slowly and evenly add 1 ml of glyceraldehyde solution dropwise, after 1 h of cross-linking and solidification, centrifuge at 1000 rpm for 10 min, remove the supernatant, wash the precipitate twice with petroleum ether, once with absolute ethanol, and freeze-dry in vacuo, that is, The pale yellow powder of asiaticoside-sodium alginate-chitosan microspheres was obtained.
精确称取实施例1积雪草苷-海藻酸钠-壳聚糖微球100mg加入到50mL磷酸盐缓冲液中，置于(37±1)℃恒温水浴中，以100r-min匀速振荡，分别于0.5h、1h、2h、3h、4h、5h、6h、12h、24h、2d、3d、5d、7d取出溶出液5mL，并同时加入等量同温的新鲜磷酸盐缓冲溶液，溶出液以4000r-min离心5min后，取上清液，以空白微球(海藻酸钠-壳聚糖微球)溶出液的上清液为空白对照，计算不同时间点积雪草苷的累计释放量、累计释放百分数；Accurately weigh 100 mg of asiaticoside-sodium alginate-chitosan microspheres in Example 1, add them to 50 mL of phosphate buffer, place them in a (37±1) ℃ constant temperature water bath, and shake them at a constant speed of 100 r-min, respectively. At 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 12h, 24h, 2d, 3d, 5d, and 7d, take out 5 mL of the dissolution solution, and add an equal amount of fresh phosphate buffer solution at the same temperature at the same time. After centrifugation for 5 min, the supernatant was taken, and the supernatant of the dissolution solution of blank microspheres (sodium alginate-chitosan microspheres) was used as blank control to calculate the cumulative release of asiaticoside at different time points. release percentage;
累计释放量计算公式：M
t＝VC
i+ΣC
i-1V
样(其中，M
t为累计释放量，C
i为第i次取样时积雪草苷的释放浓度；V为第一次取样前的体 积，本实验中为50mL；C
i-1对应于i时间取样点的前一个取样点时积雪草苷的浓度；V样为每次取样体积，本实验中为5mL。)
Cumulative release calculation formula: M t =VC i +ΣC i-1 V sample (wherein, M t is the cumulative release amount, C i is the release concentration of asiaticoside in the ith sampling; V is the first sampling The previous volume was 50 mL in this experiment; C i-1 corresponds to the concentration of asiaticoside at the sampling point before the sampling point at time i; V sample is the volume of each sampling, which is 5 mL in this experiment.)
累计释放百分数Q计算公式：Q＝M
t-体系中所含积雪草苷含量*100％
Cumulative release percentage Q calculation formula: Q = M t - asiaticoside content in the system * 100%
从图2和图3可以看出，本发明的微球大小均一，黏连程度小，圆整度高。从图4和图5可以看出，本发明制备的积雪草苷-海藻酸钠-壳聚糖微球的体外释放分为突释和缓释两个阶段，前5h积雪草苷释放量较小，在6h时发生一次突释，累计释放百分数达(43.79±2.07)％，此后积雪草苷开始以缓慢的速度释放到体外，7-24h平均每小时释放积雪草苷0.77％，于24h累计释放百分数达(57.65±1.32)％，2-7d平均每天释放积雪草苷0.92％，于7d累计释放百分数达(90.03±0.84)％。It can be seen from Fig. 2 and Fig. 3 that the microspheres of the present invention are uniform in size, small in degree of adhesion, and high in roundness. As can be seen from Figure 4 and Figure 5, the in vitro release of the asiaticoside-sodium alginate-chitosan microspheres prepared by the present invention is divided into two stages: burst release and sustained release, and the released amount of asiaticoside in the first 5 hours Small, a burst of asiaticoside occurred at 6h, and the cumulative release percentage was (43.79±2.07)%. After that, asiaticoside began to be released into the body at a slow rate, and the average hourly release of asiaticoside was 0.77% for 7-24h. The cumulative release percentage in 24h was (57.65±1.32)%, the average daily release of asiaticoside was 0.92% in 2-7d, and the cumulative release percentage in 7d was (90.03±0.84)%.
采用激光粒度分析仪测最优实施例制备的微球粒径范围，利用湿法分散技术，机械搅拌使微球在水相中均匀散开，超声高频震荡使团聚的微球充分分散，电磁循环泵使微球在整个循环体系中均匀分布，从图1的结果可以看出：微球的粒径多数都分布在3.122-4.366μm之间，D
10＝3.122μm，D
50＝3.819μm,D
90＝4.366μm，Sp＝0.326。
A laser particle size analyzer was used to measure the particle size range of the microspheres prepared in the best example. Wet dispersion technology was used to disperse the microspheres uniformly in the water phase by mechanical stirring. The circulating pump makes the microspheres evenly distributed in the whole circulation system. It can be seen from the results in Figure 1 that the particle sizes of the microspheres are mostly distributed between 3.122-4.366μm, D 10 =3.122μm, D 50 =3.819μm, D 90 =4.366 μm, Sp=0.326.
精密称取干燥至恒重的空白微球50mg，置于10mL容量瓶中，加入0.6mL 5％曲拉通X-100溶液破乳后，再加入8mL甲醇溶液，震荡摇匀，超声3h，用甲醇定容后浸泡24h，得到空白微球的甲醇浸出液；同理操作得到最优实施例积雪草苷-海藻酸钠-壳聚糖微球的甲醇浸出液。以空白微球的甲醇浸出液为参比溶液，测定在205nm下积雪草 苷-海藻酸钠-壳聚糖微球的甲醇浸出液的吸收值，代入线性回归方程内计算积雪草苷-海藻酸钠-壳聚糖微球中AS的浓度。计算公式如下：Accurately weigh 50 mg of blank microspheres dried to constant weight, place them in a 10 mL volumetric flask, add 0.6 mL of 5% Triton X-100 solution to break the demulsification, add 8 mL of methanol solution, shake well, sonicate for 3 h, and use The methanol leaching solution of blank microspheres was obtained after immersion in methanol for 24 hours; the same operation was performed to obtain the methanol leaching solution of the best example of asiaticoside-sodium alginate-chitosan microspheres. Taking the methanol leaching solution of blank microspheres as the reference solution, the absorption value of the methanol leaching solution of asiaticoside-alginate-chitosan microspheres at 205 nm was determined, and substituted into the linear regression equation to calculate asiaticoside-alginic acid Concentration of AS in sodium-chitosan microspheres. Calculated as follows:
载药量(DL)＝[微球中药物量-微球重量]*100％；Drug loading (DL) = [the amount of drug in the microspheres - the weight of the microspheres]*100%;
包封率(EE)＝[微球中药物量-投药量]*100％；Encapsulation efficiency (EE) = [the amount of drug in the microspheres - the dose of drug] * 100%;
计算结果：平均载药量为12.98％，平均包封率为95.84％，对应的RSD值分别为1.54％、2.23％。Calculation results: the average drug loading was 12.98%, the average encapsulation efficiency was 95.84%, and the corresponding RSD values were 1.54% and 2.23%, respectively.
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。The above-mentioned embodiments are only to describe the preferred modes of the present invention, but not to limit the scope of the present invention. Without departing from the design spirit of the present invention, those of ordinary skill in the art can make various modifications to the technical solutions of the present invention. Variations and improvements should fall within the protection scope determined by the claims of the present invention.
Claims (9)
- 一种积雪草苷-壳聚糖-海藻酸钠微球的制备方法，其特征在于，包括以下步骤：采用乳化交联法制备积雪草苷-壳聚糖-海藻酸钠微球，冷冻干燥即可。A method for preparing asiaticoside-chitosan-sodium alginate microspheres, comprising the following steps: preparing asiaticoside-chitosan-sodium alginate microspheres by an emulsification cross-linking method, freezing Just dry.
- 根据权利要求1所述的一种积雪草苷-壳聚糖-海藻酸钠微球的制备方法，其特征在于，具体包括以下步骤：The preparation method of a kind of asiaticoside-chitosan-sodium alginate microsphere according to claim 1, is characterized in that, specifically comprises the following steps:(a)将壳聚糖加入到体积分数为1-3％的冰醋酸溶液中溶解壳聚糖，制备壳聚糖醋酸溶液；用无水乙醇溶解积雪草苷得到积雪草苷溶液；将壳聚糖醋酸溶液与积雪草苷溶液混合，搅匀，待乙醇挥发完，加入无水氯化钙，即得含氯化钙的积雪草苷-壳聚糖醋酸溶液；(a) adding chitosan to a glacial acetic acid solution with a volume fraction of 1-3% to dissolve chitosan to prepare a chitosan acetic acid solution; dissolving asiaticoside with absolute ethanol to obtain an asiaticoside solution; The chitosan acetic acid solution and the madecassoside solution are mixed, stirred evenly, and after the ethanol has evaporated, anhydrous calcium chloride is added to obtain the madecassoside-chitosan acetic acid solution containing calcium chloride;(b)用纯净水溶解海藻酸钠，制得海藻酸钠水溶液；将配制好的海藻酸钠溶液缓慢均匀地滴加到油相中，以500-800rpm的速度搅拌20-40min形成初乳；(b) dissolving sodium alginate with purified water to obtain an aqueous solution of sodium alginate; the prepared sodium alginate solution is slowly and uniformly added dropwise to the oil phase, and the colostrum is formed by stirring at a speed of 500-800rpm for 20-40min;(c)将含氯化钙的积雪草苷-壳聚糖醋酸溶液缓慢均匀地滴加到步骤(b)形成的初乳中，搅拌，充分乳化，形成乳液；(c) the asiaticoside-chitosan acetic acid solution containing calcium chloride is slowly and uniformly added dropwise to the colostrum formed in step (b), stirred, and fully emulsified to form an emulsion;(d)在步骤(c)得到的乳液中均匀地滴加甘油醛溶液，交联固化后，离心，除去上清液，沉淀物依次用石油醚洗涤、无水乙醇洗涤，真空冷冻干燥，即得积雪草苷-海藻酸钠-壳聚糖微球的浅黄色粉末。(d) uniformly adding glyceraldehyde solution dropwise to the emulsion obtained in step (c), after cross-linking and solidifying, centrifuging, removing the supernatant, and washing the precipitate with petroleum ether and anhydrous ethanol in turn, and vacuum freeze-drying, namely The pale yellow powder of asiaticoside-sodium alginate-chitosan microspheres was obtained.
- 根据权利要求2所述的一种积雪草苷-壳聚糖-海藻酸钠微球的制备方法，其特征在于，步骤(a)中冰醋酸水溶液的体积分数为2％，无水氯化钙与壳聚糖的质量比为4：1，壳聚糖与积雪草苷混合的质量比为1∶0.2-0.6。The preparation method of a kind of asiaticoside-chitosan-sodium alginate microspheres according to claim 2, is characterized in that, in step (a), the volume fraction of glacial acetic acid aqueous solution is 2%, and anhydrous chlorination The mass ratio of calcium to chitosan is 4:1, and the mass ratio of chitosan to asiaticoside is 1:0.2-0.6.
- 根据权利要求2所述的一种积雪草苷-壳聚糖-海藻酸钠微球的制备方法，其特征在于，步骤(b)中海藻酸钠和油相的体积比为1∶6。The preparation method of a kind of asiaticoside-chitosan-sodium alginate microspheres according to claim 2, is characterized in that, in step (b), the volume ratio of sodium alginate and oil phase is 1:6.
- 根据权利要求2所述的一种积雪草苷-壳聚糖-海藻酸钠微球的制备方法，其特征在于，步骤(c)中含氯化钙的积雪草苷-壳聚糖醋酸溶液与初乳的体积比为1∶7。The preparation method of a kind of asiaticoside-chitosan-sodium alginate microspheres according to claim 2, is characterized in that, in step (c), the asiaticoside-chitosan acetic acid containing calcium chloride The volume ratio of solution to colostrum was 1:7.
- 根据权利要求2所述的一种积雪草苷-壳聚糖-海藻酸钠微球的制备方法，其特征在于，步骤(d)中甘油醛溶液与步骤(c)得到的乳液的体积比为1∶40。The preparation method of a kind of asiaticoside-chitosan-sodium alginate microspheres according to claim 2, is characterized in that, in step (d), the volume ratio of the emulsion obtained by glyceraldehyde solution and step (c) is obtained 1:40.
- 根据权利要求2所述的一种积雪草苷-壳聚糖-海藻酸钠微球的制备方法，其特征在于，所述壳聚糖分子量300-400kDa、脱乙酰度90％。The method for preparing asiaticoside-chitosan-sodium alginate microspheres according to claim 2, wherein the chitosan has a molecular weight of 300-400 kDa and a degree of deacetylation of 90%.
- 一种如权利要求1-7任一项所述的积雪草苷-壳聚糖-海藻酸钠微球制备方法制备得到的积雪草苷-壳聚糖-海藻酸钠微球。A madecassoside-chitosan-sodium alginate microsphere prepared by the method for preparing the asiaticoside-chitosan-sodium alginate microsphere according to any one of claims 1-7.
- 一种如权利要求8所述的积雪草苷-壳聚糖-海藻酸钠微球在作为创伤愈合材料中的应用。An application of the asiaticoside-chitosan-sodium alginate microspheres as claimed in claim 8 as a wound healing material.
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| CN115337229A (en) * | 2021-12-07 | 2022-11-15 | 博汇美萃生物工程技术(广东)有限公司 | Preparation method and application of composition for scalp care |
| CN115463052A (en) * | 2022-09-22 | 2022-12-13 | 武汉纺织大学 | Preparation method of polymer microsphere sunscreen composition embedded with organic ultraviolet absorbent and sunscreen agent |
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