WO2019227744A1 - 一种壳寡糖颗粒剂及其制备方法 - Google Patents

一种壳寡糖颗粒剂及其制备方法 Download PDF

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WO2019227744A1
WO2019227744A1 PCT/CN2018/103511 CN2018103511W WO2019227744A1 WO 2019227744 A1 WO2019227744 A1 WO 2019227744A1 CN 2018103511 W CN2018103511 W CN 2018103511W WO 2019227744 A1 WO2019227744 A1 WO 2019227744A1
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chitosan
povidone
granules
oligosaccharide
chitooligosaccharide
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French (fr)
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苏政权
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广东药科大学
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    • 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
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1635—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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
    • A61K31/7016—Disaccharides, e.g. lactose, lactulose
    • 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
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • 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
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/722—Chitin, chitosan
    • 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
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1617—Organic compounds, e.g. phospholipids, fats
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents

Definitions

  • the invention belongs to the technical field of medicine, and particularly relates to a chitosan granule and a preparation method thereof.
  • Chitosan oligosaccharides is a degradation product of deacetylation of chitosan. It is not only the only positively charged cationic basic amino oligosaccharide in nature, but also an animal cellulose. Chitosan is easily soluble in water, has good bioavailability in the human body, and is a safe and non-toxic substance with rich biologically active functions, including anti-tumor, hypolipidemic, antioxidant, anti-inflammatory, and neuroprotective and many more. Kang et al.
  • a mixture of chitooligosaccharide and protamine can reduce pancreatic lipase activity, thereby regulating lipid metabolism in animals; in vitro animal experiments have shown that the mixture can also reduce serum TG, TC and LDL-C. Concentration, increase the concentration of HDL-C, improve lipid metabolism in the liver and feces, thereby reducing the risk of cardiovascular diseases.
  • chitooligosaccharide can reduce the concentration of TC in the serum of hyperlipidemic mice, increase the concentration of HDL-C, and reduce the content of both AST and ALT in the serum, thereby slowing liver damage. Or lesions, with the dual role of lipid-lowering and liver protection.
  • COSM chito-oligosaccharides
  • Orlistat the only OTC weight loss drug currently on the market
  • Simvastatin a currently used lipid-lowering drug
  • chitosan has a low molecular weight and is highly susceptible to moisture absorption. Its solution has strong reducing properties, and it is prone to oxidation reactions when exposed to air, and its shelf life is short.
  • COSM chitosan oligosaccharide particles
  • the object of the present invention is to provide a chitooligosaccharide granule.
  • the chitooligosaccharide tablet provided by the present invention has a smooth appearance and a high content of active pharmaceutical ingredients. Good, stable physical and chemical properties, long shelf life.
  • the chitooligosaccharide granules according to the present invention contain chitooligosaccharide, icariin, povidone, and aspartam and are prepared by the following method: a certain amount of povidone is dissolved in an appropriate amount of anhydrous Ethanol, stir to completely dissolve povidone to obtain povidone anhydrous ethanol solution; add chitosan, icariin and aspartam to the configured povidone anhydrous ethanol solution and stir It is homogeneous. Finally, the mixture is granulated, dried, granulated, and divided into portions.
  • the weight ratio of chitosan to icariin is 50-100: 1; preferably, the weight ratio is 75: 1.
  • the weight ratio of povidone to chitooligosaccharide is 1: 24-45; preferably, the weight ratio is 1:30.
  • the weight ratio of aspartame to chitosan is 1: 150-160; preferably, the weight ratio is 1: 155.
  • the concentration of the povidone anhydrous ethanol solution is 1.5-4%, and preferably, the concentration is 3%.
  • the chitosan granules have an average molecular weight of chitosan ⁇ 3000 Da.
  • This product is a granule. After inspection of the natural properties of multiple batches of samples, they are all brown to tan particles, with a slightly sweet taste, uniform particles, and consistent color, without moisture absorption, softening, and agglomeration. The results are shown in Figure 1.
  • the experimental method was determined in accordance with the provisions of Appendix IC of the 2015 Chinese Pharmacopoeia, and the particle size was determined by the double sieving method.
  • Granulate according to the determined ratio of auxiliary materials accurately weigh three oligochitosan granules, each 30g, weigh a certain weight, place them in No. 1 sieve, place No. 5 sieve below, and go back and forth horizontally.
  • the experiment was carried out according to the method.
  • the particle size is shown in Table 1.
  • the particle size range of the three granules is between 7% and 8%, none of which exceeds 15%, which complies with the pharmacopoeia.
  • the chitosan granule sample is placed in a flat weighing bottle that has been dried to constant weight under the same conditions, accurately weighed, and dried to constant weight in an oven at a specified temperature.
  • Constant weight refers to a weight difference of less than 0.3 mg after chitosan sugar granule samples have been dried or ignited twice in a row.
  • the second and subsequent weighings after drying to constant weight shall be carried out after drying for 1 hour under the specified conditions.
  • the drying temperature is generally 105 ° C.
  • the measurement results of the weight loss on drying are shown in Table 4.
  • Chitosan granules can be hydrolyzed to D-glucosamine under acidic conditions and have reducing properties. Therefore, the content of chitosan granules can be determined indirectly by measuring reducing sugars.
  • the principle of the phenol-sulfuric acid method is: sugar compounds are dehydrated under the action of concentrated sulfuric acid to produce furfural, and then a condensation reaction with phenol generates an orange-red compound, which has a maximum absorption at a wavelength of 488 nm.
  • the phenol-sulfuric acid method is relatively easy to operate, has good repeatability and high accuracy, and is also a method for determining sugars included in the pharmacopoeia. Therefore, the phenol-sulfuric acid method is used to determine the content of chitosan granules.
  • Phenol solution Take 100.0 g of phenol, add 0.05 g of sodium bicarbonate and 0.10 g of aluminum flakes, and collect the 182 ° C fraction by distillation to obtain recrystallized phenol. Weigh 80.0 g of recrystallized phenol and add 20.0 g of distilled water to obtain a phenol solution with a concentration of 80%, and store it in a refrigerator protected from light. Immediately before use, take 37.5mL of 80% phenol solution to 500mL to prepare a 6% phenol solution.
  • Glucosamine hydrochloride standard solution Weigh precisely 1.2038 g of glucosamine hydrochloride and dissolve it in distilled water to make it 100mL, so as to obtain a mother liquid with a concentration of 10mg / mL glucosamine.
  • Chitosan granule test solution Weigh precisely the dried chitosan granule solution, dissolve it with distilled water and make up to 100mL, and prepare a certain concentration of chitosan granule test solution.
  • the average content of chitooligosaccharide granules was 93.64% and RSD was 0.91%.
  • the results are shown in the analysis: The RSD value of the measurement result is less than 2%, which indicates that the method has good reproducibility.
  • the present invention mainly studies the preparation of chitooligosaccharide granules. Because COSM condenses into agglomerates when it meets water, it cannot be ground and granulated, so it is ideal to control the ambient temperature at 20-25 °C and humidity ⁇ 40%. Chitosan granules use absolute ethanol as a wetting agent and 3% PVP as a binder.
  • Chitosan granules have uniform particles, consistent color and luster, no moisture absorption, softening, and agglomeration, which meet the requirements of the Pharmacopoeia; the moisture content is 6.19%, which meets the requirements of health food; the content of chitosan granules is measured by the phenol-sulfuric acid method It was determined that the average content of chitosan granules was 93.64%, and the RSD was 0.91%, which met the set requirements.
  • the present invention finds that chito-oligosaccharide has a synergistic weight-loss effect after adding a small amount of icariin, and the granules dissolve quickly, and at the same time, the stability of chito-oligosaccharide granules can be effectively improved.
  • the oligochitosan granules of the present invention have a simple formula, readily available raw materials, simple preparation method, stable process, and can be popularized and applied.
  • FIG. 1 is an external view of a chitooligosaccharide granule prepared by the present invention.
  • FIG. 2 is a GLC standard curve diagram for the determination of chitooligosaccharide granules by the phenol sulfuric acid method of the present invention.
  • Preparation process Dissolve 2 g of povidone in 100 g of absolute ethanol and stir to completely dissolve povidone to obtain a solution of povidone in absolute ethanol; 90 g of chitooligosaccharide, 0.9 g of icariin, and 0.6 g of arabin Spartan was added to the prepared povidone anhydrous ethanol solution, stirred well, and finally the mixture was granulated, dried at 50 ° C, sieved with a 14-mesh sieve, sieved, and packed to obtain.
  • Preparation process Dissolve 4 g of povidone in 100 g of absolute ethanol and stir to completely dissolve povidone to obtain a solution of povidone anhydrous ethanol; 96 g of chitooligosaccharide, 1.92 g of icariin, and 0.6 g of arganin Spartan was added to the prepared povidone anhydrous ethanol solution, stirred well, and finally the mixture was granulated, dried at 50 ° C, sieved with a 14-mesh sieve, sieved, and packed to obtain.
  • Preparation process Dissolve 3.1 g of povidone in 100 g of absolute ethanol and stir to completely dissolve povidone to obtain a solution of povidone anhydrous ethanol; 93 g of chitooligosaccharide, 1.24 g of icariin and 0.6 g Aspartame was added to the prepared povidone absolute ethanol solution, stirred well, and finally the mixture was granulated, dried at 50 ° C, sieved with a 14 mesh sieve, sieved, and packed to obtain.
  • the preparation method is the same as that in Example 3 of the present invention.
  • the preparation method is the same as that in Example 3 of the present invention.
  • the preparation method is the same as Example 3 of the present invention.
  • the preparation method is the same as that in Example 3 of the present invention.
  • Verification example 2 stability test of chitosan oligosaccharide particles
  • the accelerated test and stabilization of the chitooligosaccharide granules prepared in Examples 1-3 and Comparative Examples 1-4 of the present invention were conducted with reference to the XIXC guidelines for stability testing of APIs and pharmaceutical preparations in Appendix 2 of the Chinese Pharmacopoeia (2010 edition) Sex test.
  • the inspection items include appearance characteristics, chitosan content, and related substances. All inspection items that meet the requirements of the Pharmacopoeia are considered as passing, and one or more items that do not meet the requirements of the Pharmacopoeia are considered as failing. The results are shown in Tables 12 and 13.
  • the physical and chemical properties of the chitosan oligosaccharide granules of Examples 1-3 of the present invention are stable and the shelf life is long.
  • the stability of the chitooligosaccharide granules prepared by the present invention is better than The ratio and the longer shelf life indicate that the composition and the ratio of the invention are reasonable and can effectively improve the stability of the chitosan granules.
  • the experiment consists of 6 groups:
  • Chitosan granule dosage high, medium and low doses were administered by gavage at 12 times, 6 times, and 3 times the human recommended dose (6g / 60kg), namely 1200mg / kg.d, 600mg / kg.d 300mg / kg.d.
  • the dose of the group of Comparative Example 4 was administered orally at 6 times the recommended human dose (6g / 60kg), that is, 600mg / kg.d.
  • Orlistat dose twice the human dose. Orlistat's recommended oral dose for humans is 0.36g / d, which translates to a rat dose of 37.5mg / kg, so twice the rat's gavage dose is 75mg / kg.d.
  • Each rat was administered gavage at 1mL / 100g body weight, and the model control group and blank control group were given equal volume of distilled water.
  • the test sample was given for at least 6 weeks and not more than 10 weeks.
  • model phosphorus of the model feed must meet the national standards for maintaining feed.
  • mice After the end of the adaptation period, they were randomly divided into two groups according to body weight. Ten rats were given a maintenance feed as a blank control group, and 120 rats were given a high-calorie model feed. Record the amount of feed, the amount of food leftover, and the amount of food leftover every week, and weigh once.
  • model control group 70 obese sensitive rats were randomly divided into 7 groups according to body weight, which were model control group, blank control group, positive control group and 4 sample groups. Record the food intake, food intake, and food intake daily, and weigh yourself once a week. Model control group, positive control group and 4 sample groups were given high-calorie model feed, while blank control group was given maintenance feed. Each administration group was administered with different doses of test samples by gavage. The model control group and blank control group were given the same amount of the corresponding solvent. The test sample was given for 8 weeks, but not more than 10 weeks. The experimental results are shown in Table 14.
  • the body weight was measured every two weeks from the second week after the administration, and the results showed that from the fourth week, compared with the model control group, the blank control group, the orlistat group, and the comparative example 4 group
  • the body weight of rats in the three experimental groups of low, medium, and high doses was significantly lower than that of the model control group (P ⁇ 0.05), and the three experimental groups of low, medium, and high doses had no significant difference with the blank control group and orlistat Significant difference (P> 0.05), and starting from the 6th week, there was a significant difference between the medium-dose group of chitosan granules and the group of Comparative Example 4 (both dosing doses were the same) (P ⁇ 0.05). It indicates that the combined application of chitooligosaccharide and icariin has a good weight-loss effect on obese mice, and it can be confirmed that the two substances in the present invention have significant synergistic effects. Works best.
  • Lee's index is similar to human body mass index BMI, which mainly reflects the degree of body obesity.

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Abstract

一种壳寡糖颗粒剂及其制备方法,含有壳寡糖、淫羊藿苷元、聚维酮、阿司帕坦，由如下方法制备而成:将壳寡糖、淫羊藿苷元和阿司帕坦加入到配置好的聚维酮无水乙醇溶液中，搅拌均匀，最后将此混合物制粒即得。

Description

一种壳寡糖颗粒剂及其制备方法 技术领域
本发明属于医药技术领域，具体涉及一种壳寡糖颗粒剂及其制备方法。
背景技术
壳寡糖(chitosan oligosaccharides，COS)是壳聚糖脱乙酰化的降解产物，不仅是自然界中唯一带正电荷的阳离子碱性氨基低聚糖，还是一种动物性纤维素。壳寡糖极易溶于水，在人体内生物利用度较好，是一种安全无毒并且具有丰富的生物活性功能的物质，包括了抗肿瘤、降血脂、抗氧化、抗炎症、神经保护等等。Kang NH等人发现，壳寡糖和protamine的混合物能够降低胰脂肪酶的活性，从而调节动物体内的脂质代谢；体外动物实验说明了该混合物还能够减少血清中TG、TC和LDL-C的浓度，提高HDL-C的浓度，改善肝脏、粪便中的脂质代谢，从而降低心血管等疾病的风险。同时，有另一研究表明，壳寡糖能降低高脂血症小鼠血清中TC的浓度，提高HDL-C的浓度，还减少了血清中AST和ALT两者的含量，从而减缓肝脏的损伤或病变，具有降脂和护肝的双重作用。
发明内容
发明人发现平均分子量≤3000Da壳寡糖(COSM)具有优良的减肥降血脂活性。体内研究表明，COSM水溶性好，生物利用度高，减肥功效与Orlistat(市场上目前唯一的一个OTC减肥药)相当、降血脂功效与Simvastatin(目前常用的降脂药物)相当。同时，壳寡糖分子量低，极易吸潮，其溶液具有强还原性，暴露在空气中易发生氧化反应，保质期较短。因此，有必要研发一种剂型从而提高壳寡糖的稳定性。本发明将COSM制成壳寡糖颗粒(COSMG)，对其制备工艺进行研究，并建立高脂肥胖大鼠模型，对其减肥活性进行研究。
为了克服现有技术的不足，本发明的目的在于提供一种壳寡糖颗粒剂，本发明提供的壳寡糖片剂外观光洁，同时其药物活性成分含量高，溶出性能好，服用方便，口感好，理化性质稳定，保质期长。
为了实现上述目的，本发明的技术方案如下：
本发明所述的壳寡糖颗粒剂，含有壳寡糖、淫羊藿苷元、聚维酮、阿司帕坦， 由如下方法制备而成：将一定量的聚维酮溶解于适量无水乙醇，搅拌，使聚维酮完全溶解，得到聚维酮无水乙醇溶液；将壳寡糖、淫羊藿苷元和阿司帕坦加入到配置好的聚维酮无水乙醇溶液中，搅拌均匀，最后将此混合物制粒，干燥，整粒，分装即得。
如上所述的壳寡糖颗粒剂，壳寡糖与淫羊藿苷元的重量比为50-100:1；优选地，重量比为75:1。
如上所述的壳寡糖颗粒剂，聚维酮与壳寡糖的重量比为1:24-45；优选地，重量比为1:30。
如上所述的壳寡糖颗粒剂，阿司帕坦与壳寡糖的重量比为1:150-160；优选地，重量比为1:155。
如上所述的壳寡糖颗粒剂，聚维酮无水乙醇溶液的浓度为1.5-4％，优选地，浓度为3％。
如上所述壳寡糖颗粒剂，壳寡糖平均分子量≤3000Da。
质量标准研究
1性状
本品为颗粒剂。经多批样品的自然属性检查，均为褐色至棕褐色颗粒，味微甜，颗粒均勻，色泽一致，无吸潮、软化、结块等现象。结果见图1。
2粒度
实验方法按2015版《中国药典》附录IC项下规定，采用双筛分法测定颗粒粒度。按确定的辅料配比进行制粒，精密称取壳寡糖颗粒剂三份，每份30g，称定重量，分别放置在一号筛网中，下方放置五号筛网，左右往返，水平过筛，过筛边轻叩筛网3min。取无法过一号筛网和能过五号筛网的大颗粒及细粉末(X)，精密称取其重量，计算其占总重量的百分比。按照方法进行实验，颗粒粒度，结果见表1。
表1 壳寡糖颗粒剂粒度测定结果
Figure PCTCN2018103511-appb-000001
由表中实验结果可知，三份颗粒剂粒度范围在7％-8％之间，均未超过15％，符合药典规定。
3溶化性
实验方法按2015版《中国药典》附录IC项下规定，取本发明得到的颗粒三份，每份1袋(10g)，分别加200mL热水，快速搅拌5min，立刻观察。按照该方法进行实验，观察颗粒溶化情况。结果见表2。
表2 颗粒溶化性考察结果
序号 溶化性
1 颗粒全部溶化，无浑浊现象
2 颗粒全部溶化，无浑浊现象
3 颗粒全部溶化，无浑浊现象
由表中实验结果可知所制得颗粒全部溶化，无浑浊现象，符合药典规定。
4水分测定
称取约1.0g壳寡糖颗粒剂样品放置于红外水分测定仪的托盘上，合上仪器盖子，启动程序。干燥程序完成后，仪器操作面板显示的读数即为样品水分含量，记下读数。结果见表3。
表3 壳寡糖颗粒剂水分测定
Figure PCTCN2018103511-appb-000002
由表中实验结果可知所制得壳寡糖颗粒剂平均含水量为6.19％，小于10％，符合要求。
5干燥失重
本法适用于受热稳定及水分易蒸发的样品。将壳寡糖颗粒剂样品置于已在相同条件下干燥至恒重的扁形称量瓶内，精密称定，于烘箱内在规定温度下干燥至恒重。"恒重"是指壳寡糖颗粒剂样品连续两次干燥或炽灼后的重量差异在0.3mg以下的重量。干燥至恒重的第二次以及以后各次称重均应在规定条件下继续干燥1小时后进行。干燥温度一般为105℃。干燥失重测定结果见表4。
表4 壳寡糖颗粒剂干燥失重
Figure PCTCN2018103511-appb-000003
由表4可知，壳寡糖颗粒剂干燥失重平均值为1.49％，未超过2％，符合药典规定。
6含量测定
6.1苯酚硫酸含量测定法的建立
壳寡糖颗粒剂在酸性条件下可以水解成为D-氨基葡萄糖，具有还原性，因此可以利用测定还原糖的方法间接测定壳寡糖颗粒剂的含量。苯酚-硫酸法原理为：糖类化合物在浓硫酸作用下脱水生成糠醛，然后与苯酚发生缩合反应生成一种橙红色化合物，在488nm波长下有最大吸收。苯酚-硫酸法操作相对简便，重复性好，准确度较高，同时也是药典收录的测定糖类的方法，因此选用苯酚-硫酸法测定壳寡糖颗粒剂的含量。
6.2溶液的配制
苯酚溶液：取100.0g苯酚，加0.05g碳酸氢钠和0.10g铝片，蒸馏收集182℃馏分即得重结晶苯酚。称取重结晶苯酚80.0g，加20.0g蒸馏水，即得浓度为80％的苯酚溶液，避光保存于冰箱中。临用前取80％苯酚溶液37.5mL定容至500mL配制成6％的苯酚溶液，现配现用，注意避光。盐酸氨基葡萄糖标准溶液：精密称取1.2038g盐酸氨基葡萄糖，用蒸馏水溶解定容到100mL，即得氨基葡萄糖浓度为10mg/mL的母液。
壳寡糖颗粒剂待测溶液：精密称取干燥的壳寡糖颗粒剂，用蒸馏水溶解并定容到100mL，配制一定浓度的壳寡糖颗粒剂待测溶液。
6.3标准曲线的建立
准确吸取一定体积的氨基葡萄糖母液，配成一系列浓度梯度的溶液，以浓度为横坐标，吸光度为纵坐标，绘制标准曲线。结果见表5和图2。
表5 GLC标准曲线数据
浓度 0 25 50 75 100 125 150 175
(μg/mL)                
吸光度 0.056 0.076 0.101 0.121 0.143 0.169 0.192 0.211
6.4壳寡糖颗粒剂样品含量的测定
取壳寡糖颗粒剂待测溶液2.0mL于25mL比色管中，依次加入6％的重结晶苯酚溶液1.0mL，浓硫酸5.0mL，轻轻摇匀，沸水浴45min，反应完全后于488nm下测定吸光度。将测定值代入标准曲线方程进行计算。测定结果见表6。
表6 壳寡糖颗粒剂样品含量测定
Figure PCTCN2018103511-appb-000004
实验测得壳寡糖颗粒剂平均含量为93.64％，RSD为0.91％，符合设定要求。
6.5精密度试验
取壳寡糖颗粒剂待测溶液2mL于25mL比色管中，依次加入5％的重结晶苯酚溶液1ml,浓硫酸5mL，轻轻摇匀，沸水浴45min,反应完全后于488nm下平行测定6次吸光度。计算含量，RSD值。精密度结果见表7。
表7 壳寡糖颗粒剂样品精密度
Figure PCTCN2018103511-appb-000005
结果分析：在该量测定条件下，RSD值小于2％，表明本方法精密度良好。
7稳定性考察
精密吸取批号为20170928的壳寡糖颗粒剂为待测溶液，在0、2、4、6、8、10、12小时内每隔两小时利用苯酚硫酸法进行含量测定，计算平均值和RSD值。测定结果见表8。
表8 壳寡糖颗粒剂样品稳定性
Figure PCTCN2018103511-appb-000006
Figure PCTCN2018103511-appb-000007
结果分析：壳寡糖颗粒剂在12小时内的含量测定值稳定。样品溶液从制备至测试一般在12小时内即可全部完成。
8重现性考察
精密称取同一批供试品6份，利用苯酚硫酸法平行测定含量，计算平均值和RSD值。结果见表9。
表9 壳寡糖颗粒剂样品重现性
Figure PCTCN2018103511-appb-000008
结果见分析：测定结果的RSD值小于2％，表明此方法重现性良好。
9加样回收率考察
精密称取已知含量的壳寡糖颗粒剂样品6份，向其中加入定量的标准物质，测定含量后计算加标回收率和RSD值。
实验测得壳寡糖颗粒剂加标回收率平均值为99.19％，RSD值为0.78％，如表10所示。由结果可知该测定方法准确度高。
表10 壳寡糖颗粒剂加标回收率实验
Figure PCTCN2018103511-appb-000009
Figure PCTCN2018103511-appb-000010
10实验结论与讨论
本发明主要研究了壳寡糖颗粒剂的制备。由于COSM遇水凝结成块，无法研磨制粒，所以将环境温度控制在20-25℃，湿度≤40％较理想。壳寡糖颗粒剂使用无水乙醇作为润湿剂，采用3％的PVP作为黏合剂。壳寡糖颗粒剂颗粒均勻，色泽一致，无吸潮、软化、结块等现象，符合药典要求；水分含量为6.19％，符合保健食品要求；利用苯酚-硫酸法对壳寡糖颗粒剂进行含量测定，壳寡糖颗粒剂平均含量为93.64％，RSD为0.91％，符合设定要求。
本发明的技术方案具有如下优点：
1)具有前体剂型的优点吸收快、显效迅速。
2)服用方便，稳定性高，口感好。
3)飞散性、附着性、聚集性、吸湿性等均较小。
4)本发明发现壳寡糖添加少量淫羊藿苷元后具有协同减肥作用，并且使颗粒剂溶出迅速，同时可有效提高壳寡糖颗粒剂的稳定性。
5)本发明壳寡糖颗粒剂的配方简单、原料易得，同时制备方法简易，工艺稳定，可推广应用。
附图说明
图1为本发明制备得到的壳寡糖颗粒剂外观图。
图2为本发明苯酚硫酸法测定壳寡糖颗粒剂的GLC标准曲线图。
具体实施方式
以下通过具体实施例进一步描述本发明，但所述实施例并不以任何方式限定本发明专利保护的范围。
实施例1
壳寡糖 90g
聚维酮 2g
淫羊藿苷元 0.9g
阿司帕坦 0.6g
无水乙醇 100g
制备工艺：将2g聚维酮溶解于100g无水乙醇，搅拌，使聚维酮完全溶解，得到聚维酮无水乙醇溶液；将90g壳寡糖、0.9g淫羊藿苷元和0.6g阿司帕坦加入到配置好的聚维酮无水乙醇溶液中，搅拌均匀，最后将此混合物制粒，50℃干燥，14目筛整粒，筛分，包装即得。
实施例2
壳寡糖 96g
聚维酮 4g
淫羊藿苷元 1.92g
阿司帕坦 0.6g
无水乙醇 100g
制备工艺：将4g聚维酮溶解于100g无水乙醇，搅拌，使聚维酮完全溶解，得到聚维酮无水乙醇溶液；将96g壳寡糖、1.92g淫羊藿苷元和0.6g阿司帕坦加入到配置好的聚维酮无水乙醇溶液中，搅拌均匀，最后将此混合物制粒，50℃干燥，14目筛整粒，筛分，包装即得。
实施例3
壳寡糖 93g
聚维酮 3.1g
淫羊藿苷元 1.24g
阿司帕坦 0.6g
无水乙醇 100g
制备工艺：将3.1g聚维酮溶解于100g无水乙醇，搅拌，使聚维酮完全溶解，得到聚维酮无水乙醇溶液；将93g壳寡糖、1.24g淫羊藿苷元和0.6g阿司帕坦加入到配置好的聚维酮无水乙醇溶液中，搅拌均匀，最后将此混合物制粒，50℃干 燥，14目筛整粒，筛分，包装即得。
对比实施例1
壳寡糖 93g
聚维酮 3.1g
淫羊藿苷元 1.24g
阿司帕坦 0.6g
蒸馏水 100g
制备方法同本发明实施例3。
对比实施例2
壳寡糖 93g
聚维酮 6g
淫羊藿苷元 1.24g
阿司帕坦 0.6g
无水乙醇 100g
制备方法同本发明实施例3。
对比实施例3
壳寡糖 93g
聚维酮 3.1g
淫羊藿苷元 1.24g
阿司帕坦 1.2g
无水乙醇 100g
制备方法同本发明实施例3。
对比实施例4
壳寡糖 93g
聚维酮 3.1g
阿司帕坦 0.6g
无水乙醇 100g
制备方法同本发明实施例3。
验证例1 本发明壳寡糖颗粒剂溶出度的测定
溶出度测定取样品6片，照中国药典附录溶出度测定第二法，以pH6.8磷酸盐缓冲液为溶出介质，体积为500ml，转速50r/min，温度37℃，经15分钟时，取样，测定其吸光度，限度为标示量的80％。实验结果见表11。
表11 颗粒剂溶出度测定结果
组别 5min溶出度(％) 15min溶出度(％)
实施例1 99.6 99.9
实施例2 99.6 99.9
实施例3 99.8 99.9
对比实施例1 72.5 92.4
对比实施例2 89.3 97.3
对比实施例3 82.4 96.2
对比实施例4 92.4 98.1
从表1中可知，本发明实施例溶出迅速；对比实施例1，蒸馏水代替乙醇，溶出最差；对比实施例2，加大聚维酮的含量，制备的颗粒剂在磷酸盐中溶出慢；对比实施例3，加大阿司帕坦的量，溶出较本发明慢；对比实施例4没有添加淫羊藿苷元，溶出较本发明慢。
验证例2 本发明壳寡糖颗粒剂稳定性试验
参考《中国药典》(2010年版)二部附录XIXC原料药与药物制剂稳定性试验指导原则，对本发明实施例1-3和对比例1-4制得的壳寡糖颗粒剂进行加速试验和稳定性试验。考察项目包括外观性状、壳寡糖含量和有关物质，所有考察项目均符合药典规定则视为及格，有一项或以上项目不符合药典要求则视为不及格。结果见表12和表13。
表12 壳寡糖颗粒剂加速实验结果
Figure PCTCN2018103511-appb-000011
注：“+”表示合格；“—”表示不合格。
表13 壳寡糖颗粒剂长期实验结果
Figure PCTCN2018103511-appb-000012
注：“+”表示合格；“—”表示不合格。
由表12和表13可知，本发明实施例1-3壳寡糖颗粒剂的理化性质稳定，保质期长。通过对比例1-4制备的壳寡糖颗粒剂与本发明实施例1-3制备的壳寡糖颗粒剂稳定性试验结果对比，本发明制备得到的壳寡糖颗粒剂的稳定性优于对比例，保质期更长，说明本发明的组成和配比合理，可有效提高壳寡糖颗粒剂的稳定性。
实验例1 本发明壳寡糖颗粒剂降低体重的药效学试验研究
1.1实验动物
选用雄性大鼠，适应期结束时，体重100±10g，每组10只。
1.2剂量分组及受试样品给予时间
实验共设6组，分别为：
1)空白组
2)模型组
3)奥利司他组(阳性组)
4)壳寡糖颗粒剂高剂量组
5)壳寡糖颗粒剂中剂量组
6)壳寡糖颗粒剂低剂量组
7)对比实施例4组
壳寡糖颗粒剂剂量：高、中、低剂量分别按人体推荐剂量(6g/60kg)的12倍、6倍、3倍灌胃给药，即1200mg/kg.d，600mg/kg.d，300mg/kg.d。
对比实施例4组剂量按人体推荐剂量(6g/60kg)的6倍灌胃给药，即600mg/kg.d。
奥利司他剂量：按照人体剂量的2倍给药。Orlistat人体口服推荐剂量为0.36g/d，换算成大鼠的剂量为37.5mg/kg，所以大鼠灌胃2倍的剂量为75mg/kg.d。
计算方法：大鼠剂量＝人剂量/60*6.25
每只大鼠按1mL/100g体重进行灌胃，模型对照组和空白对照组给予等体积的蒸馏水。受试样品给予时间至少给予6周，不超过10周。
1.3高热量模型饲料
在维持饲料中添加15.0％蔗糖、15.0％猪油，适量的酪蛋白、磷酸氢钙、石粉等。除了粗脂肪外，模型饲料的水分、粗蛋白、粗脂肪、粗纤维、粗灰分、钙、磷、钙：磷均要达到维持饲料的国家标准。
1.4实验步骤
1.2.4.1肥胖模型法
1)适应期：于屏障系统下大鼠喂饲维持饲料观察5-7天。
2)造模期：
适应期结束后按体重随机分成2组，10只大鼠给予维持饲料作为空白对照组，120只大鼠给予高热量模型饲料。每周记录给食量、撒食量、剩食量，称量体重1次。
喂养2周后，给予高热量饲料的120只大鼠按体重增重排序，淘汰体重增重较低的肥胖抵抗大鼠。将筛选出的60只肥胖敏感大鼠再给予高热量饲料6周，空白对照组同时给予维持饲料。
3)受试样品给予：
造模期结束后，70只肥胖敏感大鼠按体重随机分成7组，分别为模型对照 组、空白对照组、阳性对照组和4个样品组。每天记录给食量、摄食量、剩食量，每周称量体重1次。模型对照组、阳性对照组和4个样品组给予高热量模型饲料，空白对照组给予维持饲料。各给药组灌胃给予不同剂量的受试样品，模型对照组和空白对照组给予等量的相应溶剂，受试样品给予时间8周，不超过10周。实验结果见表14。
表14 不同实验组对小鼠体重的影响(单位：g)
Figure PCTCN2018103511-appb-000013
注：与模型对照组比较，*为P<0.05；与对比实施例4组比较，△为P＜0.05。
由表14可知，从给药后第2周开始每隔2周称体重，结果显示，从第4周开始，与模型对照组比较，空白对照组、奥利司他组、对比实施例4组和低、中、高剂量三个试验组大鼠体重均明显低于模型对照组(P<0.05)，而且低、中、高剂量三个试验组与空白对照组和奥利司他组比较无显著性差异(P>0.05)，并且从第6周开始，壳寡糖颗粒剂中剂量组与对比实施例4组(二者给药剂量相同)比较有显著性差异(P<0.05)，这说明壳寡糖与淫羊藿苷元联合应用后对于肥胖小鼠具有很好的减肥功效，由此可以证实本发明中的2种物质具有显著的协同效果，在三个剂量中，中剂量组的作用效果最好。
实验例2 本发明壳寡糖颗粒剂对肥胖大鼠Lee’s指数的影响
Figure PCTCN2018103511-appb-000014
Lee's指数和人的体重指数BMI类似，主要反映机体肥胖程度。
表15 不同实验组对肥胖大鼠Lee’s指数的影响
组别 Lee’s指数(实验开始前) Lee’s指数(实验结束后)
空白对照组 297.8 * 301.2 *
模型对照组 322.7 318.4
奥利司他组(阳性组) 321.4 301.3 *
壳寡糖颗粒剂低剂量组 319.6 308.2 *
壳寡糖颗粒剂中剂量组 320.5 303.1 *△
壳寡糖颗粒剂高剂量组 320.1 306.7 *
对比实施例4组 321.2 310.4 *
注：与模型对照组比较，*为P<0.05
由表15可知，实验结束后空白对照组、对比实施例4组、奥利司他组和低、中、高剂量三个试验组大鼠的Lee’s指数均明显低于模型对照组(P<0.05)，而且低、中、高剂量三个试验组之间以及试验组与空白对照组和奥利司他组之间差异均无显著性意义(P>0.05)，此外壳寡糖颗粒剂中剂量组与对比实施例4组(二者给药剂量相同)比较有显著性差异(P<0.05)，这说明壳寡糖与淫羊藿苷元联合应用后对于肥胖小鼠具有很好的减肥功效，由此可以证实本发明中的2种物质具有显著的协同效果，本发明壳寡糖颗粒剂中剂量组肥胖指数改善在统计学上优于壳寡糖颗粒剂低、高剂量组。

Claims (8)

  1. 一种壳寡糖颗粒剂，其特征在于，含有壳寡糖、淫羊藿苷元、聚维酮、阿司帕坦，由如下方法制备而成：将一定量的聚维酮溶解于适量无水乙醇，搅拌，使聚维酮完全溶解，得到聚维酮无水乙醇溶液；将壳寡糖、淫羊藿苷元和阿司帕坦加入到配置好的聚维酮无水乙醇溶液中，搅拌均匀，最后将此混合物制粒，干燥，整粒，分装即得；聚维酮与壳寡糖的重量比为1:24-45；阿司帕坦与壳寡糖的重量比为1:150-160。
  2. 根据权利要求1所述的壳寡糖颗粒剂，其特征在于，所述颗粒剂中壳寡糖与淫羊藿苷元的重量比为50-100:1。
  3. 根据权利要求1所述的壳寡糖颗粒剂，其特征在于，所述颗粒剂中壳寡糖与淫羊藿苷元的重量比为75:1。
  4. 根据权利要求1所述的壳寡糖颗粒剂，其特征在于，聚维酮与壳寡糖的重量比为1:30。
  5. 根据权利要求1所述的壳寡糖颗粒剂，其特征在于，阿司帕坦与壳寡糖1:155。
  6. 根据权利要求1所述的壳寡糖颗粒剂，其特征在于，所述的聚维酮无水乙醇溶液的浓度为1.5-4％。
  7. 根据权利要求4所述的壳寡糖颗粒剂，其特征在于，所述的聚维酮无水乙醇溶液的浓度为3％。
  8. 根据权利要求1所述壳寡糖颗粒剂，其特征在于，所述壳寡糖平均分子量≤3000Da。
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