CN118027236A - Method for flash-type efficient extraction of Cordyceps militaris polysaccharide with high hypoglycemic activity and application thereof - Google Patents

Method for flash-type efficient extraction of Cordyceps militaris polysaccharide with high hypoglycemic activity and application thereof Download PDF

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CN118027236A
CN118027236A CN202410158285.5A CN202410158285A CN118027236A CN 118027236 A CN118027236 A CN 118027236A CN 202410158285 A CN202410158285 A CN 202410158285A CN 118027236 A CN118027236 A CN 118027236A
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张扬
王晓兰
杨祖根
郑丽雪
刘星雨
刘文烜
顾芸竹
谢宇星
李雅怡
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Abstract

本发明属于天然产物制备和应用领域,具体涉及一种利用闪式提取高效制备高降糖活性蚕蛹虫草多糖的方法与用途。本发明所提供的闪式提取技术能高效制备蚕蛹虫草多糖,具有多糖得率高(18.3±1.82%)、对α‑葡萄糖苷酶的抑制率高(83.2±7.64%)、能耗低及提取时间极短(仅需1min)的优势,显著优于热水提取、酶辅助提取、微波辅助提取等方法。利用该技术提取纯化的蛹虫草纯化多糖同样具有较强的降糖活性,在5mg/mL浓度时,对α‑葡萄糖苷酶的抑制率达到了99.4±1.25%,显著高于同浓度的阿卡波糖。本发明技术方案提取纯化的蚕蛹虫草纯化多糖可用于口服液、胶囊剂、片剂等降糖功能产品的制备。

The present invention belongs to the field of natural product preparation and application, and is particularly related to a method and use for efficiently preparing high hypoglycemic activity silkworm pupa cordyceps polysaccharides using flash extraction. The flash extraction technology provided by the present invention can efficiently prepare silkworm pupa cordyceps polysaccharides, with high polysaccharide yield (18.3 ± 1.82%), high inhibition rate of α-glucosidase (83.2 ± 7.64%), low energy consumption and extremely short extraction time (only 1min), significantly better than hot water extraction, enzyme-assisted extraction, microwave-assisted extraction and other methods. The purified polysaccharides of Cordyceps militaris extracted and purified using this technology also have strong hypoglycemic activity. At a concentration of 5mg/mL, the inhibition rate of α-glucosidase reached 99.4 ± 1.25%, which is significantly higher than acarbose at the same concentration. The purified polysaccharides of silkworm pupa cordyceps extracted and purified by the technical solution of the present invention can be used for the preparation of hypoglycemic functional products such as oral liquids, capsules, and tablets.

Description

一种闪式高效提取高降糖活性蚕蛹虫草多糖的方法与用途A method and application for the high-efficiency extraction of silkworm chrysalis and Cordyceps sinensis polysaccharides with high hypoglycemic activity

技术领域Technical Field

本发明属于天然产物制备领域,具体涉及一种利用闪式提取高效制备高降糖活性蚕蛹虫草多糖的方法与用途。The invention belongs to the field of natural product preparation, and specifically relates to a method and use for efficiently preparing silkworm chrysalis cordyceps polysaccharide with high hypoglycemic activity by flash extraction.

背景技术Background technique

蚕蛹虫草(Cordyceps militaris),简称蛹虫草,又称北冬虫夏草,属麦角菌科虫草属真菌,原产于我国,2009年被国家卫生部批准为新资源食品,是一种具有较高价值的药食两用真菌。蚕蛹虫草富含多糖、虫草素、虫草酸等多种生物活性物质。其中,多糖是蚕蛹虫草重要的活性成分,具有增强机体免疫力、抗肿瘤、降血糖等广泛的药理作用(FoodBioscience,2022,45:101503),在生物医药与食品等领域有较高的应用价值。Cordyceps militaris, also known as Cordyceps militaris, is a fungus belonging to the genus Cordyceps of the family Clavicipitaceae. It is native to my country and was approved as a new resource food by the Ministry of Health in 2009. It is a high-value medicinal and edible fungus. Cordyceps militaris is rich in polysaccharides, cordycepin, cordycepic acid and other bioactive substances. Among them, polysaccharides are important active ingredients in Cordyceps militaris, which have a wide range of pharmacological effects such as enhancing immunity, anti-tumor, and lowering blood sugar (Food Bioscience, 2022, 45: 101503), and have high application value in the fields of biomedicine and food.

蚕蛹虫草多糖的含量少、提取率不高、提取难度大,成为制约产业化开发的瓶颈问题。目前,蚕蛹虫草多糖的提取方法主要有热水浸提法(食品科学,2017,38(14):91-96;CN106749742A;CN116496424A)、超声波辅助法(食品研究与开发,2023,44(11):159-165;CN107501428A)、微波辅助法(江苏农业学报,2009,25(5):1143-1150)等。然而,这些方法存在时间长、能耗高、提取率低等缺点。The low content, low extraction rate and great difficulty of silkworm pupa cordyceps polysaccharide have become bottleneck problems restricting industrial development. At present, the main extraction methods of silkworm pupa cordyceps polysaccharide include hot water extraction method (Food Science, 2017, 38 (14): 91-96; CN106749742A; CN116496424A), ultrasonic assisted method (Food Research and Development, 2023, 44 (11): 159-165; CN107501428A), microwave assisted method (Journal of Jiangsu Agricultural Sciences, 2009, 25 (5): 1143-1150), etc. However, these methods have the disadvantages of long time, high energy consumption and low extraction rate.

闪式提取技术是利用高速剪切头形成高强度的剪切力,具有极高的破壁效率,并利用强力搅拌,使溶剂快速渗透,有效成分迅速扩散,具有提取时间短、温度低、无辐射风险的优点(Trends in Food Science&Technology,2021,112:581-591)。目前,利用闪式提取技术制备蚕蛹虫草多糖还未见报道。Flash extraction technology uses a high-speed shear head to form a high-intensity shear force, has a very high wall-breaking efficiency, and uses strong stirring to allow the solvent to penetrate quickly and the effective ingredients to diffuse rapidly. It has the advantages of short extraction time, low temperature, and no radiation risk (Trends in Food Science & Technology, 2021, 112: 581-591). At present, there are no reports on the preparation of silkworm Cordyceps polysaccharides using flash extraction technology.

以糖尿病为首的代谢性疾病依然是危害公众健康的重要因素之一。《中国2型糖尿病防治指南》报道:我国2型糖尿病的发病率为10.4%,此外60岁以上患病率高于20%,而未诊断患者就占总数的63%。尽管磺脲类、双胍类等化学降糖药在调控血糖、减少并发症方面发挥了重要作用,但长期服用存在的不良反应大、抵抗等问题始终无法解决。相比化学降糖药,天然降糖产品以其安全性高、适合长期服用等特点逐渐被人们所认可。抑制α-葡萄糖苷酶进而减少葡萄糖摄入是多糖降糖最为重要的机制,已上市的寡糖类降糖药物阿卡波糖及其类似物就是经典的α-葡萄糖苷酶抑制剂。朱振元等(现代食品科技,2014,30(12):55-60)发现利用热水浸提法制备的蚕蛹虫草多糖可以抑制α-葡萄糖苷酶活力,但并未考察不同提取参数及方法是否对活性有影响。Metabolic diseases, led by diabetes, remain one of the major factors endangering public health. The Guidelines for the Prevention and Treatment of Type 2 Diabetes in China reported that the incidence of type 2 diabetes in my country is 10.4%. In addition, the prevalence of people over 60 years old is higher than 20%, and undiagnosed patients account for 63% of the total. Although chemical hypoglycemic drugs such as sulfonylureas and biguanides have played an important role in regulating blood sugar and reducing complications, the adverse reactions and resistance of long-term use have never been solved. Compared with chemical hypoglycemic drugs, natural hypoglycemic products are gradually being recognized by people for their high safety and suitability for long-term use. Inhibition of α-glucosidase and thus reduction of glucose intake is the most important mechanism of polysaccharide hypoglycemic. The oligosaccharide hypoglycemic drugs acarbose and its analogs that have been marketed are classic α-glucosidase inhibitors. Zhu Zhenyuan et al. (Modern Food Science and Technology, 2014, 30(12):55-60) found that the Cordyceps sinensis polysaccharide prepared by hot water extraction can inhibit the activity of α-glucosidase, but did not investigate whether different extraction parameters and methods have an effect on the activity.

处理电压是闪式提取重要的技术参数,人们往往关注电压对多糖得率的影响(食品工业,2020,41(11):42-45;食品研究与开发,2023,44(9):164-170),而忽视了电压对多糖生物学活性的影响。本发明通过探索不同提取电压对蚕蛹虫草多糖得率及降糖活性的影响,提供了一种利用闪式提取高效制备高降糖活性蚕蛹虫草多糖的方法与用途。Processing voltage is an important technical parameter for flash extraction. People often focus on the effect of voltage on polysaccharide yield (Food Industry, 2020, 41(11):42-45; Food Research and Development, 2023, 44(9):164-170), but ignore the effect of voltage on the biological activity of polysaccharides. The present invention provides a method and use for efficiently preparing silkworm pupa cordyceps polysaccharides with high hypoglycemic activity by flash extraction by exploring the effects of different extraction voltages on the yield and hypoglycemic activity of silkworm pupa cordyceps polysaccharides.

发明内容Summary of the invention

本发明的目的在于提供一种利用闪式提取高效制备高降糖活性蚕蛹虫草多糖的方法。相比传统热水提取法、酶辅助提取法、微波辅助提取法和超声辅助提取法,利用本发明所提供的闪式提取技术制备的蚕蛹虫草多糖的得率高、能耗低、提取时间短;且利用该技术制备纯化的蚕蛹虫草多糖具有极高的α-葡萄糖苷酶抑制活性,表现出优良的降糖功能。The purpose of the present invention is to provide a method for efficiently preparing silkworm pupa cordyceps polysaccharides with high hypoglycemic activity using flash extraction. Compared with traditional hot water extraction, enzyme-assisted extraction, microwave-assisted extraction and ultrasound-assisted extraction, the silkworm pupa cordyceps polysaccharides prepared using the flash extraction technology provided by the present invention have high yield, low energy consumption and short extraction time; and the silkworm pupa cordyceps polysaccharides prepared and purified using this technology have extremely high α-glucosidase inhibitory activity and show excellent hypoglycemic function.

根据本发明的第一个方面,本发明提供了一种利用闪式提取高效制备蚕蛹虫草多糖的方法,以蚕蛹虫草脱脂粉为原料,在水中浸泡,然后置于闪式提取器中,在一定电压下处理;经离心、浓缩上清液、乙醇沉淀、冷冻干燥得蚕蛹虫草多糖。According to the first aspect of the present invention, the present invention provides a method for efficiently preparing silkworm pupa cordyceps polysaccharides by flash extraction, wherein silkworm pupa cordyceps defatted powder is used as raw material, soaked in water, then placed in a flash extractor, and treated under a certain voltage; the silkworm pupa cordyceps polysaccharides are obtained by centrifugation, concentrating the supernatant, ethanol precipitation, and freeze drying.

优选的,所述蚕蛹虫草脱脂粉由石油醚脱脂,具体步骤为:将新鲜蚕蛹虫草于60℃烘干至恒重,粉碎后过40目筛制成蚕蛹虫草粉备用;加入石油醚避光脱脂12h,减压抽滤得蚕蛹虫草脱脂粉。Preferably, the defatted powder of silkworm pupa and Cordyceps is defatted by petroleum ether, and the specific steps are: drying fresh silkworm pupa and Cordyceps at 60° C. to constant weight, crushing and passing through a 40-mesh sieve to prepare silkworm pupa and Cordyceps powder for later use; adding petroleum ether to defatting in the dark for 12 hours, and filtering under reduced pressure to obtain the defatted powder of silkworm pupa and Cordyceps.

优选的,所述一定的处理电压为70~150V,进一步优选110V;更优选地,采用西安太康生物科技有限公司型号为JHBE-20A的闪式提取器,处理电压设置为110V。Preferably, the certain processing voltage is 70-150V, more preferably 110V; more preferably, a flash extractor model JHBE-20A produced by Xi'an Taikang Biotechnology Co., Ltd. is used, and the processing voltage is set to 110V.

优选的,所述利用闪式提取高效制备蚕蛹虫草多糖的方法具体步骤为:将蚕蛹虫草脱脂粉以1:40g/mL的料液比在水中浸泡2h,然后置于闪式提取器中(西安太康生物科技有限公司型号为JHBE-20A),在电压110V下处理1min;结束后离心取上清液,将上清液浓缩至原体积1/4得浓缩液,加入浓缩液3倍体积的无水乙醇于4℃沉淀12h,沉淀物经冷冻干燥得蚕蛹虫草多糖;该条件下,蚕蛹虫草多糖得率为18.3±1.82%、5mg/mL时对α-葡萄糖苷酶的抑制率为83.2±7.64%,显著优于传统热水提取法(多糖得率:10.7±0.26%;5mg/mL时对α-葡萄糖苷酶的抑制率:31.5±4.81%)、酶辅助提取法(多糖得率:10.7±0.93%;5mg/mL时对α-葡萄糖苷酶的抑制率:35.4±2.95%)、微波辅助提取法(多糖得率:14.4±2.03%;5mg/mL时对α-葡萄糖苷酶的抑制率:48.6±5.11%)和超声辅助提取法(多糖得率:13.6±1.53%;5mg/mL时对α-葡萄糖苷酶的抑制率:50.7±4.69%)。Preferably, the method for efficiently preparing silkworm pupa cordyceps polysaccharide by flash extraction specifically comprises the following steps: soaking silkworm pupa cordyceps defatted powder in water at a solid-liquid ratio of 1:40 g/mL for 2 hours, then placing it in a flash extractor (model JHBE-20A of Xi'an Taikang Biotechnology Co., Ltd.), and treating it at a voltage of 110 V for 1 minute; centrifuging and taking the supernatant after the end, concentrating the supernatant to 1/4 of the original volume to obtain a concentrated solution, adding 3 times the volume of anhydrous ethanol to precipitate at 4°C for 12 hours, and freeze-drying the precipitate to obtain silkworm pupa cordyceps polysaccharide; under this condition, the yield of silkworm pupa cordyceps polysaccharide is 18.3±1.82%, and the inhibition rate of α-glucosidase at 5 mg/mL is 83.2±7. 64%, which is significantly better than traditional hot water extraction (polysaccharide yield: 10.7±0.26%; inhibition rate of α-glucosidase at 5 mg/mL: 31.5±4.81%), enzyme-assisted extraction (polysaccharide yield: 10.7±0.93%; inhibition rate of α-glucosidase at 5 mg/mL: 35.4±2.95%), microwave-assisted extraction (polysaccharide yield: 14.4±2.03%; inhibition rate of α-glucosidase at 5 mg/mL: 48.6±5.11%) and ultrasound-assisted extraction (polysaccharide yield: 13.6±1.53%; inhibition rate of α-glucosidase at 5 mg/mL: 50.7±4.69%).

本发明所述的蚕蛹虫草多糖的制备方法中,还包括对制备出的蚕蛹虫草多糖进行纯化,采用Sevag试剂脱蛋白,除去残留的Sevag试剂后在蒸馏水中透析,透析液冷冻干燥后得脱蛋白多糖,经二乙氨基乙基(DEAE)纤维素色谱柱纯化得蚕蛹虫草纯化多糖;具体方法为:采用Sevag试剂(氯仿:正丁醇=4:1,体积比)脱蛋白3次;通过离心除去变性的蛋白和残留的Sevag试剂后在蒸馏水中透析3次(透析袋截留分子量为7000Da、间隔12h更换一次蒸馏水);透析液冷冻干燥后得脱蛋白多糖。称取100mg脱蛋白多糖溶于20mL去离子水中,上样至二乙氨基乙基(DEAE)纤维素色谱柱(2.6cm×60cm);用0.5mol/L的NaCl溶液进行缓慢洗脱,流速0.5mL/min,每管2mL;采用苯酚-硫酸法测定每管洗脱液的吸光度,当多糖全部洗脱下来后合并洗脱液,浓缩后透析3次(方法同上),冷冻干燥得蚕蛹虫草纯化多糖。The preparation method of silkworm pupa cordyceps polysaccharide of the present invention also comprises the steps of purifying the prepared silkworm pupa cordyceps polysaccharide, deproteinizing by using Sevag reagent, dialyzing in distilled water after removing residual Sevag reagent, freeze-drying the dialyzate to obtain deproteinized polysaccharide, and purifying by using a diethylaminoethyl (DEAE) cellulose chromatographic column to obtain purified silkworm pupa cordyceps polysaccharide; the specific method is: deproteinizing by using Sevag reagent (chloroform: n-butanol=4:1, volume ratio) for 3 times; removing denatured protein and residual Sevag reagent by centrifugation, dialyzing in distilled water for 3 times (dialysis bag molecular weight cutoff is 7000Da, distilled water is replaced once every 12 hours); freeze-drying the dialyzate to obtain the deproteinized polysaccharide. Weigh 100 mg of deproteinized polysaccharide and dissolve it in 20 mL of deionized water, then load it onto a diethylaminoethyl (DEAE) cellulose chromatographic column (2.6 cm × 60 cm); slowly elute with 0.5 mol/L NaCl solution at a flow rate of 0.5 mL/min, 2 mL per tube; determine the absorbance of the eluate in each tube by the phenol-sulfuric acid method, and when all the polysaccharides have been eluted, combine the eluates, concentrate them, and dialyze them three times (the same method as above), and freeze-dry them to obtain the purified polysaccharide of silkworm chrysalis.

根据本发明的第二个方面,本发明提供了一种蚕蛹虫草纯化多糖,所述的蚕蛹虫草纯化多糖中总糖含量为83.5±1.46%、蛋白含量为7.26±2.05%、糖醛酸含量为4.16±0.68%、硫酸基含量为3.71±0.69%;所述的蚕蛹虫草纯化多糖由阿拉伯糖、半乳糖、葡萄糖、木糖、半乳糖醛酸、葡萄糖醛酸以31.6:21.6:274:23.6:1.00:3.56的摩尔比构成;所述的蚕蛹虫草纯化多糖的数均分子量(Mn)为1.85kDa、重均分子量(Mw)为5.09kDa。According to the second aspect of the present invention, the present invention provides a purified polysaccharide of silkworm pupa and Cordyceps militaris, wherein the total sugar content of the purified polysaccharide of silkworm pupa and Cordyceps militaris is 83.5±1.46%, the protein content is 7.26±2.05%, the uronic acid content is 4.16±0.68%, and the sulfate content is 3.71±0.69%; the purified polysaccharide of silkworm pupa and Cordyceps militaris is composed of arabinose, galactose, glucose, xylose, galacturonic acid, and glucuronic acid in a molar ratio of 31.6:21.6:274:23.6:1.00:3.56; the number average molecular weight (Mn) of the purified polysaccharide of silkworm pupa and Cordyceps militaris is 1.85kDa, and the weight average molecular weight (Mw) is 5.09kDa.

根据本发明的第三个方面,本发明提供了所述蚕蛹虫草纯化多糖在制备降糖功能产品中的用途;可用于制备口服液、胶囊、片剂等制剂。According to the third aspect of the present invention, the present invention provides the use of the purified polysaccharide of silkworm chrysalis and Cordyceps militaris in the preparation of a product with hypoglycemic function; it can be used to prepare oral liquids, capsules, tablets and other preparations.

本发明具有如下有益效果:The present invention has the following beneficial effects:

①本发明提供的闪式提取技术在缩短提取时间、减低能耗、保护多糖结构方面具有显著优势,能极大提高蚕蛹虫草多糖的得率及降糖活性,显著优于热水提取法等现有技术。① The flash extraction technology provided by the present invention has significant advantages in shortening the extraction time, reducing energy consumption, and protecting the polysaccharide structure. It can greatly improve the yield and hypoglycemic activity of Cordyceps militaris polysaccharides, and is significantly superior to existing technologies such as hot water extraction.

②本发明提供的蚕蛹虫草纯化多糖对α-葡萄糖苷酶具有极高的抑制率,5mg/mL时显著高于同浓度的阿卡波糖,表现出优良的降糖功能,可用于制备降糖相关疾病的产品。② The purified polysaccharide of silkworm chrysalis and Cordyceps militaris provided by the present invention has an extremely high inhibition rate on α-glucosidase, which is significantly higher than acarbose at the same concentration at 5 mg/mL, showing excellent hypoglycemic function, and can be used to prepare products for hypoglycemic-related diseases.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1.不同处理电压对蚕蛹虫草多糖得率的影响(不同的上标小写字母代表存在统计学差异);Figure 1. Effects of different treatment voltages on the yield of Cordyceps militaris polysaccharides (different superscript lowercase letters represent statistical differences);

图2.不同处理电压对蚕蛹虫草多糖抑制α-葡萄糖苷酶活性的影响(不同的上标小写字母代表存在统计学差异);Figure 2. Effects of different treatment voltages on the inhibition of α-glucosidase activity by Cordyceps militaris polysaccharides (different superscript lowercase letters represent statistical differences);

图3.蚕蛹虫草纯化多糖的单糖组成色谱图(标准品中的峰:1-岩藻糖;2-鼠李糖;3-阿拉伯糖;4-半乳糖;5-葡萄糖;6-木糖;7-甘露糖;8-果糖;9-半乳糖醛酸;10-葡萄糖醛酸);Figure 3. Chromatogram of monosaccharide composition of purified polysaccharide of Cordyceps militaris (peaks in standard: 1-fucose; 2-rhamnose; 3-arabinose; 4-galactose; 5-glucose; 6-xylose; 7-mannose; 8-fructose; 9-galacturonic acid; 10-glucuronic acid);

图4.蚕蛹虫草纯化多糖的分子量分布图;Figure 4. Molecular weight distribution of purified polysaccharides from silkworm chrysalis and Cordyceps militaris;

图5.蚕蛹虫草纯化多糖的红外光谱图;Figure 5. Infrared spectrum of purified polysaccharide from silkworm chrysalis and Cordyceps militaris;

图6.蚕蛹虫草纯化多糖的扫描电镜图;Figure 6. Scanning electron micrograph of purified polysaccharide from silkworm chrysalis;

图7.蚕蛹虫草纯化多糖对α-葡萄糖苷酶的抑制作用(不同的上标小写字母代表组内存在统计学差异;不同的上标大写字母代表相同浓度下组间存在统计学差异)。Figure 7. Inhibitory effect of purified polysaccharides from Cordyceps militaris on α-glucosidase (different superscript lowercase letters represent statistical differences within the group; different superscript uppercase letters represent statistical differences between groups at the same concentration).

具体实施方式Detailed ways

蚕蛹虫草由苏州吴江家和蚕业专业合作社提供;闪式提取器由西安太康生物科技有限公司提供,型号为JHBE-20A;分子量采用Waters的高效液相色谱仪测定,型号为1525;单糖组成采用戴安的离子色谱仪测定,型号为ICS-5000;红外光谱由PerkinElmer的傅里叶变换红外显微镜系统测定,型号为Spotlight200i;扫描电子显微镜(SEM)由HITACHI超高分辨场发射扫描电子显微镜测定,型号为Regulus8100;所有实验均为三平行,数据表达为均值±SD,数据的统计分析采用t-检验或ANOVA分析,P<0.05认为存在统计学差异。Silkworm pupa and Cordyceps were provided by Suzhou Wujiang Jiahe Sericulture Professional Cooperative; the flash extractor was provided by Xi'an Taikang Biotechnology Co., Ltd., model JHBE-20A; the molecular weight was determined by Waters high performance liquid chromatograph, model 1525; the monosaccharide composition was determined by Dionex ion chromatograph, model ICS-5000; the infrared spectrum was determined by PerkinElmer Fourier transform infrared microscope system, model Spotlight200i; the scanning electron microscopy (SEM) was determined by HITACHI ultra-high resolution field emission scanning electron microscope, model Regulus8100; all experiments were performed in triplicate, and the data were expressed as mean ± SD. The statistical analysis of the data was performed by t-test or ANOVA analysis, and P < 0.05 was considered statistically significant.

实施例1蚕蛹虫草多糖的闪式提取及处理电压筛选Example 1 Flash Extraction of Silkworm Cordyceps Polysaccharide and Screening of Treatment Voltage

将新鲜蚕蛹虫草于60℃烘干至恒重,粉碎后过40目筛制成蚕蛹虫草粉备用;加入石油醚避光脱脂12h,减压抽滤得蚕蛹虫草脱脂粉。将蚕蛹虫草脱脂粉以1:40g/mL的料液比在水中浸泡2h,然后置于闪式提取器中,在一定电压下(70~150V)处理1min。结束后离心取上清液,将上清液浓缩至原体积1/4,加入3倍体积无水乙醇于4℃沉淀12h,沉淀物经冷冻干燥得蚕蛹虫草多糖。Fresh silkworm pupa cordyceps is dried at 60℃ to constant weight, crushed and sieved through a 40-mesh sieve to make silkworm pupa cordyceps powder for later use; petroleum ether is added to degrease in the dark for 12 hours, and the silkworm pupa cordyceps defatted powder is obtained by vacuum filtration. The silkworm pupa cordyceps defatted powder is soaked in water at a solid-liquid ratio of 1:40g/mL for 2 hours, then placed in a flash extractor and treated at a certain voltage (70-150V) for 1 minute. After the end, the supernatant is centrifuged and concentrated to 1/4 of the original volume, and 3 times the volume of anhydrous ethanol is added to precipitate at 4℃ for 12 hours. The precipitate is freeze-dried to obtain silkworm pupa cordyceps polysaccharide.

多糖得率(%)=m/M×100,其中:m为冷冻干燥后的蚕蛹虫草多糖质量(g);M为蚕蛹虫草脱脂粉质量(g)。多糖含量测定采用苯酚-硫酸法,葡萄糖标准方程为:Y=9.541X+0.0075,R2=0.9997。Polysaccharide yield (%) = m/M×100, where: m is the mass of freeze-dried Cordyceps militaris polysaccharide (g); M is the mass of defatted powder of Cordyceps militaris (g). The polysaccharide content was determined using the phenol-sulfuric acid method, and the glucose standard equation was: Y = 9.541X + 0.0075, R 2 = 0.9997.

将不同电压下提取的蚕蛹虫草多糖分别溶于蒸馏水,制备成5mg/mL的溶液,用于测定对α-葡萄糖苷酶的抑制活性。具体测定方法如下:The Cordyceps militaris polysaccharides extracted at different voltages were dissolved in distilled water to prepare 5 mg/mL solutions for the determination of the inhibitory activity against α-glucosidase. The specific determination method is as follows:

精确移取100μL蚕蛹虫草多糖溶液与300μL 0.24U/mL的α-葡萄糖苷酶溶液混合,然后再加入600μL磷酸盐缓冲盐(PBS)缓冲液(pH=6.8);振荡均匀后,于37℃孵育15min;加入2.74mg/mL 4-硝基苯基-β-D-吡喃半乳糖苷(PNPG)溶液并充分混匀,继续在37℃孵育20min;最后加入4mL的碳酸钠以终止反应,于400nm处测定吸光度值。α-葡萄糖苷酶抑制率按下式计算:α-葡萄糖苷酶抑制率(%)=[1-(A样品-A背景)/(A阴性-A空白)]×100;表1为α-葡萄糖苷酶抑制活性实验反应体系。Accurately pipette 100 μL of silkworm chrysalis Cordyceps polysaccharide solution and mix with 300 μL of 0.24U/mL α-glucosidase solution, then add 600 μL of phosphate buffered saline (PBS) buffer (pH=6.8); after oscillation, incubate at 37°C for 15 min; add 2.74 mg/mL 4-nitrophenyl-β-D-pyranogalactoside (PNPG) solution and mix thoroughly, continue to incubate at 37°C for 20 min; finally, add 4 mL of sodium carbonate to terminate the reaction, and measure the absorbance at 400 nm. The α-glucosidase inhibition rate is calculated as follows: α-glucosidase inhibition rate (%) = [1-(A sample -A background )/(A negative -A blank )] × 100; Table 1 is the α-glucosidase inhibition activity experimental reaction system.

表1.α-葡萄糖苷酶抑制活性实验反应体系Table 1. α-glucosidase inhibitory activity test reaction system

1.1不同处理电压对蚕蛹虫草多糖得率的影响1.1 Effect of different treatment voltages on the yield of polysaccharides from silkworm chrysalis

固定料液比为1:40g/mL、闪式提取时间为1min,考察不同处理电压(70~150V)对蚕蛹虫草多糖得率的影响,结果如图1所示。The solid-liquid ratio was fixed at 1:40 g/mL and the flash extraction time was 1 min. The effect of different treatment voltages (70-150 V) on the yield of Cordyceps militaris polysaccharides was investigated. The results are shown in Figure 1.

由图1可知,在70~110V范围内,多糖得率随电压值升高而提高,当电压值达到110V时,多糖得率为18.3±1.82%,显著高于70V和90V时的多糖得率(P<0.01);当电压值达到130V时,多糖得率最高,为20.4±2.35%,虽然高于110V时的多糖得率,但没有统计学差异(P>0.05);当电压值升高至150V时,多糖得率显著下降(P<0.05)。As shown in Figure 1, in the range of 70-110V, the polysaccharide yield increases with the increase of voltage value. When the voltage value reaches 110V, the polysaccharide yield is 18.3±1.82%, which is significantly higher than the polysaccharide yields at 70V and 90V (P<0.01); when the voltage value reaches 130V, the polysaccharide yield is the highest, which is 20.4±2.35%. Although it is higher than the polysaccharide yield at 110V, there is no statistical difference (P>0.05); when the voltage value increases to 150V, the polysaccharide yield decreases significantly (P<0.05).

1.2不同处理电压对蚕蛹虫草多糖抑制α-葡萄糖苷酶活性的影响1.2 Effects of different treatment voltages on the inhibitory activity of α-glucosidase by Cordyceps militaris polysaccharides

固定料液比为1:40g/mL、闪式提取时间为1min、蚕蛹虫草多糖浓度为5mg/mL,考察不同处理电压(70~150V)对蚕蛹虫草多糖抑制α-葡萄糖苷酶活性的影响,结果如图2所示。The solid-liquid ratio was fixed at 1:40 g/mL, the flash extraction time was 1 min, and the concentration of Cordyceps militaris polysaccharide was 5 mg/mL. The effect of different treatment voltages (70-150 V) on the inhibition of α-glucosidase activity by Cordyceps militaris polysaccharide was investigated. The results are shown in Figure 2.

由图2可知,在70~110V范围内,α-葡萄糖苷酶抑制率随电压值升高而显著提高(P<0.01),当电压值达到110V时,抑制率达到了83.2±7.64%,显著高于70V和90V时的抑制率(P<0.01);当电压值升高至130V时,抑制率开始显著下降(P<0.01);当电压值继续升高至150V时,抑制率降至53.5±6.69%。As shown in Figure 2, in the range of 70 to 110 V, the inhibition rate of α-glucosidase increased significantly with the increase of voltage value (P<0.01). When the voltage value reached 110 V, the inhibition rate reached 83.2±7.64%, which was significantly higher than the inhibition rates at 70 V and 90 V (P<0.01). When the voltage value increased to 130 V, the inhibition rate began to decrease significantly (P<0.01). When the voltage value continued to increase to 150 V, the inhibition rate dropped to 53.5±6.69%.

从提取效果、α-葡萄糖苷酶抑制活性、能耗等方面综合考虑,选择处理电压110V左右为宜,此时的蚕蛹虫草多糖得率为18.3±1.82%、对α-葡萄糖苷酶的抑制率为83.2±7.64%。Taking extraction effect, α-glucosidase inhibitory activity, energy consumption and other aspects into consideration, a processing voltage of about 110 V was appropriate, at which the yield of Cordyceps militaris polysaccharide was 18.3±1.82% and the inhibition rate of α-glucosidase was 83.2±7.64%.

对比例Comparative Example

在实施例1基础上,为了进一步证明本发明所提供的闪式提取技术能高效制备高降糖活性的蚕蛹虫草多糖,本发明实施了如下对比试验:On the basis of Example 1, in order to further prove that the flash extraction technology provided by the present invention can efficiently prepare Cordyceps militaris polysaccharides with high hypoglycemic activity, the present invention implemented the following comparative test:

方案A:热水提取法Option A: Hot water extraction

将蚕蛹虫草脱脂粉以1:40g/mL的料液比在水中浸泡2h,然后在80℃下提取2h;冷却后离心取上清液,将上清液浓缩至原体积1/4,加入3倍体积无水乙醇于4℃沉淀12h,沉淀物经冷冻干燥得蚕蛹虫草多糖;计算多糖得率并测定对α-葡萄糖苷酶的抑制活性,方法同实施例1。The defatted powder of silkworm pupa Cordyceps militaris was soaked in water at a solid-liquid ratio of 1:40 g/mL for 2 hours, and then extracted at 80° C. for 2 hours; after cooling, the supernatant was centrifuged and concentrated to 1/4 of the original volume, 3 times the volume of anhydrous ethanol was added and precipitated at 4° C. for 12 hours, and the precipitate was freeze-dried to obtain silkworm pupa Cordyceps militaris polysaccharide; the polysaccharide yield was calculated and the inhibitory activity against α-glucosidase was determined, using the same method as in Example 1.

结果表明:采用热水提取法获得的蚕蛹虫草多糖得率为7.52±1.63%(比本发明技术方案低2.4倍);5mg/mL时对α-葡萄糖苷酶的抑制率为31.5±4.81%(比本发明技术方案低2.6倍)。The results showed that the yield of Cordyceps militaris polysaccharide obtained by hot water extraction was 7.52±1.63% (2.4 times lower than the technical solution of the present invention); the inhibition rate of α-glucosidase at 5 mg/mL was 31.5±4.81% (2.6 times lower than the technical solution of the present invention).

方案B:酶辅助提取法Option B: Enzyme-assisted extraction

将蚕蛹虫草脱脂粉以1:40g/mL液料比在水中浸泡2h,然后将pH值调节至5.0左右,加入虫草脱脂粉质量2.0%的纤维素酶(酶活为10U/mg),置于摇床中于50℃左右孵育2h,结束后将酶解液置于沸水浴中灭酶15min;冷却后离心取上清液,将上清液浓缩至原体积1/4,加入3倍体积无水乙醇于4℃沉淀12h,沉淀物经冷冻干燥得蚕蛹虫草多糖;计算多糖得率并测定对α-葡萄糖苷酶的抑制活性,方法同实施例1。The silkworm pupa cordyceps defatted powder is soaked in water at a liquid-to-solid ratio of 1:40 g/mL for 2 hours, and then the pH value is adjusted to about 5.0, and 2.0% of the mass of the cordyceps defatted powder is added with cellulase (enzyme activity is 10 U/mg), and the mixture is placed in a shaker at about 50° C. for incubation for 2 hours. After the incubation, the enzymolysis solution is placed in a boiling water bath for 15 minutes to inactivate the enzyme; after cooling, the supernatant is centrifuged to obtain the supernatant, and the supernatant is concentrated to 1/4 of the original volume, 3 times the volume of anhydrous ethanol is added to precipitate at 4° C. for 12 hours, and the precipitate is freeze-dried to obtain silkworm pupa cordyceps polysaccharide; the polysaccharide yield is calculated and the inhibitory activity to α-glucosidase is determined, and the method is the same as that in Example 1.

结果表明:采用酶辅助提取法获得的蚕蛹虫草多糖得率为10.7±0.93%(比本发明技术方案低1.7倍);5mg/mL时对α-葡萄糖苷酶的抑制率为35.4±2.95%(比本发明技术方案低2.4倍)。The results showed that the yield of Cordyceps militaris polysaccharide obtained by enzyme-assisted extraction was 10.7±0.93% (1.7 times lower than the technical solution of the present invention); the inhibition rate of α-glucosidase at 5 mg/mL was 35.4±2.95% (2.4 times lower than the technical solution of the present invention).

方案C:微波辅助提取法Scheme C: Microwave-assisted extraction

将蚕蛹虫草脱脂粉以1:40g/mL液料比在水中浸泡2h,然后转移至微波超声波组合合成萃取仪(北京祥鹄科技发展有限公司,型号为XH-300B);开启微波模式,在500W功率、70℃条件下提取8min;结束后离心取上清液,将上清液浓缩至原体积1/4,加入3倍体积无水乙醇于4℃沉淀12h,沉淀物经冷冻干燥得蚕蛹虫草多糖;计算多糖得率并测定对α-葡萄糖苷酶的抑制活性,方法同实施例1。The defatted powder of silkworm chrysalis and Cordyceps militaris was soaked in water at a liquid-to-solid ratio of 1:40 g/mL for 2 h, and then transferred to a microwave-ultrasonic combined synthetic extractor (Beijing Xianghu Technology Development Co., Ltd., model XH-300B); the microwave mode was turned on, and the extraction was carried out at 500 W power and 70° C. for 8 min; after the extraction, the supernatant was centrifuged and concentrated to 1/4 of the original volume, 3 times the volume of anhydrous ethanol was added and precipitated at 4° C. for 12 h, and the precipitate was freeze-dried to obtain silkworm chrysalis and Cordyceps militaris polysaccharide; the polysaccharide yield was calculated and the inhibitory activity against α-glucosidase was determined, using the same method as in Example 1.

结果表明:采用微波辅助提取法获得的蚕蛹虫草多糖得率为14.4±2.03%(比本发明技术方案低1.3倍);5mg/mL时对α-葡萄糖苷酶的抑制率为48.6±5.11%(比本发明技术方案低1.7倍)。The results showed that the yield of Cordyceps militaris polysaccharides obtained by microwave-assisted extraction was 14.4±2.03% (1.3 times lower than the technical solution of the present invention); the inhibition rate of α-glucosidase at 5 mg/mL was 48.6±5.11% (1.7 times lower than the technical solution of the present invention).

方案D:超声辅助提取法Scheme D: Ultrasound-assisted extraction

将蚕蛹虫草脱脂粉以1:40g/mL液料比在水中浸泡2h,然后转移至微波超声波组合合成萃取仪(北京祥鹄科技发展有限公司,型号为XH-300B);开启超声模式,在300W功率、60℃条件下提取30min;结束后离心取上清液,将上清液浓缩至原体积1/4,加入3倍体积无水乙醇于4℃沉淀12h,沉淀物经冷冻干燥得蚕蛹虫草多糖;计算多糖得率并测定对α-葡萄糖苷酶的抑制活性,方法同实施例1。The defatted powder of silkworm chrysalis and Cordyceps militaris was soaked in water at a liquid-to-solid ratio of 1:40 g/mL for 2 h, and then transferred to a microwave-ultrasonic combined synthetic extractor (Beijing Xianghu Technology Development Co., Ltd., model XH-300B); the ultrasonic mode was turned on, and the extraction was carried out at 300 W power and 60° C. for 30 min; after the extraction, the supernatant was centrifuged and concentrated to 1/4 of the original volume, 3 times the volume of anhydrous ethanol was added and precipitated at 4° C. for 12 h, and the precipitate was freeze-dried to obtain silkworm chrysalis and Cordyceps militaris polysaccharide; the polysaccharide yield was calculated and the inhibitory activity against α-glucosidase was determined, using the same method as in Example 1.

结果表明:采用超声辅助提取法获得的蚕蛹虫草多糖得率为13.6±1.53%(比本发明技术方案低1.3倍);5mg/mL时对α-葡萄糖苷酶的抑制率为50.7±4.69%(比本发明技术方案低1.6倍)。The results showed that the yield of Cordyceps militaris polysaccharides obtained by ultrasound-assisted extraction was 13.6±1.53% (1.3 times lower than the technical solution of the present invention); the inhibition rate of α-glucosidase at 5 mg/mL was 50.7±4.69% (1.6 times lower than the technical solution of the present invention).

综上,采用本发明提供的闪式提取技术获得的蚕蛹虫草多糖得率高于传统热水提取法、酶辅助提取法、微波辅助提取法及超声辅助提取法,具体能耗低、效率极高的优势(仅需提取1min);对α-葡萄糖苷酶的抑制率也同样高于上述方法,表明:110V的闪式处理不仅能够提高蚕蛹虫草多糖得率,还能优化多糖结构,提高降糖活性。In summary, the yield of silkworm Cordyceps militaris polysaccharides obtained by the flash extraction technology provided by the present invention is higher than that of traditional hot water extraction, enzyme-assisted extraction, microwave-assisted extraction and ultrasound-assisted extraction, and has the advantages of low energy consumption and extremely high efficiency (only 1 minute of extraction is required); the inhibition rate of α-glucosidase is also higher than that of the above methods, indicating that the 110V flash treatment can not only increase the yield of silkworm Cordyceps militaris polysaccharides, but also optimize the polysaccharide structure and improve the hypoglycemic activity.

实施例2蚕蛹虫草多糖的纯化Example 2 Purification of Silkworm Cordyceps Polysaccharide

采用Sevag试剂(氯仿:正丁醇=4:1,体积比)脱蛋白3次;通过离心除去变性的蛋白和残留的Sevag试剂后在蒸馏水中透析3次(透析袋截留分子量为7000Da、间隔12h更换一次蒸馏水);透析液冷冻干燥后得脱蛋白多糖。称取100mg脱蛋白多糖溶于20mL去离子水中,上样至二乙氨基乙基(DEAE)纤维素色谱柱(2.6cm×60cm);用0.5mol/L的NaCl溶液进行缓慢洗脱,流速0.5mL/min,每管2mL;采用苯酚-硫酸法测定每管洗脱液的吸光度,当多糖全部洗脱下来后合并洗脱液,浓缩后透析3次(方法同上),冷冻干燥得蚕蛹虫草纯化多糖。Deproteinization was performed 3 times using Sevag reagent (chloroform: n-butanol = 4:1, volume ratio); denatured proteins and residual Sevag reagent were removed by centrifugation and dialyzed 3 times in distilled water (dialysis bag molecular weight cutoff was 7000Da, distilled water was replaced every 12 hours); the dialyzate was freeze-dried to obtain deproteinized polysaccharide. 100 mg of deproteinized polysaccharide was weighed and dissolved in 20 mL of deionized water, and loaded onto a diethylaminoethyl (DEAE) cellulose chromatographic column (2.6 cm × 60 cm); 0.5 mol/L NaCl solution was used for slow elution, with a flow rate of 0.5 mL/min, and 2 mL per tube; the absorbance of each tube of eluate was determined by the phenol-sulfuric acid method, and the eluate was combined when all the polysaccharides were eluted, concentrated, and dialyzed 3 times (the method is the same as above), and freeze-dried to obtain the purified polysaccharide of silkworm chrysalis.

实施例3蚕蛹虫草纯化多糖的成分分析与表征Example 3 Composition Analysis and Characterization of Purified Polysaccharides from Silkworm Cordyceps

苯酚-硫酸法测定蚕蛹虫草纯化多糖中总糖含量为83.5±1.46%;考马斯亮蓝法测定蛋白含量为7.26±2.05%;间羟基联苯法测定糖醛酸含量为4.16±0.68%;氯化钡明胶比浊法测定硫酸基含量为3.71±0.69%。The total sugar content in the purified polysaccharide from silkworm pupa Cordyceps militaris was determined by the phenol-sulfuric acid method and was 83.5±1.46%; the protein content was determined by the Coomassie Brilliant Blue method and was 7.26±2.05%; the uronic acid content was determined by the m-hydroxybiphenyl method and was 4.16±0.68%; and the sulfate content was determined by the barium chloride gelatin turbidimetry method and was 3.71±0.69%.

单糖组成分析:称取5mg蚕蛹虫草纯化多糖于5mL的具塞刻度试管中,加入1mL2mol/L三氟乙酸,121℃烘箱水解2h后,蒸馏水定容至50mL,用0.45微米微孔滤膜过滤后供进样分析,进样体积为20μL。仪器及色谱条件:美国戴安ICS-5000离子色谱仪,配备脉冲安培检测器,色谱柱为CarboPac PA20(6.5μm,3mm×150mm)。采用梯度洗脱,流动相为:98%水+2%250mM NaOH(0-21min);93%水+2%250mM NaOH+5%1M NaAc(21.1-30min);20%水+80%250mM NaOH(30.1-50min)。流速为0.5mL/min。Monosaccharide composition analysis: Weigh 5 mg of purified polysaccharide from Cordyceps militaris into a 5 mL stoppered test tube, add 1 mL of 2 mol/L trifluoroacetic acid, and hydrolyze in an oven at 121°C for 2 h. Then distilled water is diluted to 50 mL, filtered with a 0.45 μm microporous filter membrane for injection analysis, and the injection volume is 20 μL. Instrument and chromatographic conditions: American Dionex ICS-5000 ion chromatograph, equipped with a pulsed amperometric detector, and the chromatographic column is CarboPac PA20 (6.5 μm, 3 mm×150 mm). Gradient elution is used, and the mobile phase is: 98% water + 2% 250 mM NaOH (0-21 min); 93% water + 2% 250 mM NaOH + 5% 1 M NaAc (21.1-30 min); 20% water + 80% 250 mM NaOH (30.1-50 min). The flow rate is 0.5 mL/min.

分子量分析:将蚕蛹虫草纯化多糖配制成2mg/mL的溶液,进样前用0.45μm微孔滤膜过滤样品,进样品体积为20μL。分析仪器为Waters1525高效液相色谱仪,2414示差折光检测器和Empower 3工作站,色谱柱为UltrahydrogelTMLinear(2μm,7.8mm×300mm)。以不同分子量的葡聚糖作为标准糖,制备系列不同分子量的标准糖溶液,根据保留时间和分子量值建立多糖分子量测定的三阶校正曲线。色谱条件为流动相:0.1M NaNO3;流速:0.9mL/min;柱温:45℃。Molecular weight analysis: The purified polysaccharide of Cordyceps militaris was prepared into a 2 mg/mL solution. The sample was filtered with a 0.45 μm microporous filter membrane before injection. The injection volume was 20 μL. The analytical instrument was a Waters 1525 high performance liquid chromatograph, a 2414 differential refractometer and an Empower 3 workstation. The chromatographic column was an Ultrahydrogel TM Linear (2 μm, 7.8 mm × 300 mm). Dextran with different molecular weights was used as a standard sugar to prepare a series of standard sugar solutions with different molecular weights. The third-order calibration curve for the determination of polysaccharide molecular weight was established based on the retention time and molecular weight value. The chromatographic conditions were as follows: mobile phase: 0.1 M NaNO 3 ; flow rate: 0.9 mL/min; column temperature: 45 °C.

红外光谱分析:取2mg干燥的蚕蛹虫草纯化多糖与100mg干燥的溴化钾混合、压片后,放入红外光谱仪中进行测定,扫描范围为4000cm-1~400cm-1Infrared spectrum analysis: 2 mg of dried purified polysaccharide of Cordyceps militaris was mixed with 100 mg of dried potassium bromide, pressed into tablets, and then placed in an infrared spectrometer for measurement in a scanning range of 4000 cm -1 to 400 cm -1 .

扫描电镜分析:取干燥的蚕蛹虫草纯化多糖,用棉签将其粘贴在双面导电胶的载物台上,用洗耳球吹去没有粘住的多糖粉末。将其放置在离子溅射仪中进行喷箔处理,再将处理好的样品按顺序放入扫描电子显微镜下,待仪器设置稳定之后观察其形貌。Scanning electron microscope analysis: Take the dried silkworm pupa Cordyceps purified polysaccharide, stick it on the double-sided conductive adhesive stage with a cotton swab, and use an ear-cleaning bulb to blow away the unsticky polysaccharide powder. Place it in an ion sputtering instrument for foil spraying, and then put the treated samples in order under the scanning electron microscope, and observe their morphology after the instrument settings are stable.

如图3所示,通过与标准糖进行比对确定蚕蛹虫草纯化多糖由阿拉伯糖、半乳糖、葡萄糖、木糖、半乳糖醛酸、葡萄糖醛酸以31.6:21.6:274:23.6:1.00:3.56的摩尔比构成。As shown in FIG3 , by comparing with standard sugars, it was determined that the purified polysaccharide of silkworm chrysalis consisted of arabinose, galactose, glucose, xylose, galacturonic acid, and glucuronic acid in a molar ratio of 31.6:21.6:274:23.6:1.00:3.56.

如图4所示,蚕蛹虫草纯化多糖的数均分子量(Mn)为1.85kDa、重均分子量(Mw)为5.09kDa。As shown in FIG4 , the number average molecular weight (Mn) of the purified polysaccharide of Cordyceps militaris was 1.85 kDa, and the weight average molecular weight (Mw) was 5.09 kDa.

如图5所示,从蚕蛹虫草纯化多糖的红外光谱图中,可以观察到3422cm-1附近存在一个强吸收峰,这是由-OH的伸缩振动引起的;2931cm-1为甲基CH不对称伸缩振动;1645cm-1为羰基CO的伸缩振动;1404cm-1为CO伸缩振动;1063cm-1附近的吸收峰为C-O-C糖苷键的骨架振动;863cm-1为α-型葡萄糖苷键的特征峰;598cm-1为吡喃糖环的吸收峰。红外光谱分析进一步证实蚕蛹虫草纯化多糖符合多糖的结构特征。As shown in Figure 5, from the infrared spectrum of the purified polysaccharide from silkworm chrysalis, it can be observed that there is a strong absorption peak near 3422cm -1 , which is caused by the stretching vibration of -OH; 2931cm -1 is the asymmetric stretching vibration of methyl CH; 1645cm -1 is the stretching vibration of carbonyl CO; 1404cm -1 is the stretching vibration of CO; the absorption peak near 1063cm -1 is the skeleton vibration of COC glycosidic bond; 863cm -1 is the characteristic peak of α-glucosidic bond; 598cm -1 is the absorption peak of pyranose ring. Infrared spectrum analysis further confirmed that the purified polysaccharide from silkworm chrysalis conforms to the structural characteristics of polysaccharides.

如图6所示,在×1k的放大倍数下,蚕蛹虫草纯化多糖的表面粗糙,呈现出不规则的孔洞,其中还散在分布一些大小不一的颗粒。As shown in Figure 6, at a magnification of ×1k, the surface of the purified polysaccharide from silkworm chrysalis and Cordyceps militaris is rough, showing irregular holes, with some particles of different sizes scattered in it.

实施例4蚕蛹虫草纯化多糖抑制α-葡萄糖苷酶活性的评价Example 4 Evaluation of the Inhibition of α-glucosidase Activity by Purified Polysaccharides from Silkworm Cordyceps

为了考察纯化对蚕蛹虫草多糖降糖活性的影响,本发明进一步评价了蚕蛹虫草纯化多糖对α-葡萄糖苷酶的抑制作用,并以同浓度的阿卡波糖(已上市的α-葡萄糖苷酶抑制剂)为阳性对照,方法同实施例1。In order to investigate the effect of purification on the hypoglycemic activity of Cordyceps militaris polysaccharides, the present invention further evaluated the inhibitory effect of purified Cordyceps militaris polysaccharides on α-glucosidase, and used acarbose (an α-glucosidase inhibitor already on the market) at the same concentration as the positive control, using the same method as Example 1.

如图7所示,在1~5mg/mL浓度范围内,蚕蛹虫草纯化多糖对α-葡萄糖苷酶的抑制作用随浓度升高而显著提高(P<0.01),当浓度达到5mg/mL时,对α-葡萄糖苷酶的抑制率达到了99.4±1.25%,显著高于同浓度的阿卡波糖(92.5±2.11%,P<0.05);也显著高于110V处理电压下的α-葡萄糖苷酶的抑制率(83.2±7.64%,P<0.01,见实施例1),表明:纯化能进一步提高蚕蛹虫草多糖的降糖活性;采用本发明技术方案提取纯化的蚕蛹虫草多糖对α-葡萄糖苷酶的半数抑制浓度为1.48±0.21mg/mL,比文献报道的4.22mg/mL(现代食品科技,2014,30(12):50-60)低了2.85倍,表明:采用本发明技术方案提取纯化的蚕蛹虫草多糖具有极高的α-葡萄糖苷酶抑制作用,呈现出优良的降糖功能。As shown in FIG. 7 , within the concentration range of 1 to 5 mg/mL, the inhibitory effect of the purified polysaccharide of silkworm chrysalis on α-glucosidase increased significantly with increasing concentration (P<0.01). When the concentration reached 5 mg/mL, the inhibition rate of α-glucosidase reached 99.4±1.25%, which was significantly higher than that of acarbose at the same concentration (92.5±2.11%, P<0.05); it was also significantly higher than the inhibition rate of α-glucosidase under the treatment voltage of 110 V (83.2±7.64%, P<0.01, see actual results). Example 1) shows that purification can further improve the hypoglycemic activity of silkworm Cordyceps polysaccharides; the half inhibition concentration of silkworm Cordyceps polysaccharides extracted and purified by the technical solution of the present invention on α-glucosidase is 1.48±0.21 mg/mL, which is 2.85 times lower than the 4.22 mg/mL reported in the literature (Modern Food Science and Technology, 2014, 30(12):50-60), indicating that the silkworm Cordyceps polysaccharides extracted and purified by the technical solution of the present invention have extremely high α-glucosidase inhibition and exhibit excellent hypoglycemic function.

实施例5蚕蛹虫草纯化多糖口服液的制备Example 5 Preparation of Purified Silkworm Cordyceps Polysaccharide Oral Liquid

取2.0g蚕蛹虫草纯化多糖加入100mL纯化水中,室温搅拌至溶解,加入0.5g赤藓糖醇,0.2g山梨酸钾,搅拌均匀后,罐装瞬时灭菌,装瓶,封口,即得蚕蛹虫草纯化多糖口服液。Take 2.0g of purified polysaccharide from silkworm chrysalis and Cordyceps militaris, add it into 100mL of purified water, stir it at room temperature until dissolved, add 0.5g of erythritol and 0.2g of potassium sorbate, stir evenly, sterilize it in cans instantly, bottle it, and seal it to obtain purified polysaccharide oral liquid from silkworm chrysalis and Cordyceps militaris.

实施例6蚕蛹虫草纯化多糖胶囊剂的制备Example 6 Preparation of Purified Polysaccharide Capsules from Silkworm Chrysalis and Cordyceps

取5.0g过40目筛的蚕蛹虫草纯化多糖,按1:1比例喷入90%乙醇,混合均匀,加10%淀粉制软材,过20目筛制粒,置于60℃烘箱中干燥1h,再过20目筛整粒,在相对湿度低于65%的环境下装填于3号空胶囊中,即得蚕蛹虫草纯化多糖胶囊剂。Take 5.0g of purified polysaccharide from silkworm chrysalis and Cordyceps militaris passed through a 40-mesh sieve, spray it with 90% ethanol at a ratio of 1:1, mix evenly, add 10% starch to make a soft material, pass through a 20-mesh sieve to granulate, place it in a 60°C oven to dry for 1h, pass through a 20-mesh sieve to granulate, and fill it into No. 3 empty capsules in an environment with a relative humidity of less than 65% to obtain purified polysaccharide capsules from silkworm chrysalis and Cordyceps militaris.

实施例7蚕蛹虫草纯化多糖片剂的制备Example 7 Preparation of Purified Polysaccharide Tablets from Silkworm Chrysalis and Cordyceps

取5.0g蚕蛹虫草纯化多糖与300mg聚乙烯吡咯烷酮、4.0mg磷酸氢钙混合,研细,过100目筛;搅拌时加入5%聚维酮K30的95%乙醇溶液制软材,湿法制粒(过16目筛);60℃干燥,过筛,整粒,加入1%的硬脂酸镁和2%的微粉硅胶,混合均匀,压片即得蚕蛹虫草纯化多糖片剂。5.0 g of purified polysaccharide from silkworm chrysalis and Cordyceps militaris was mixed with 300 mg of polyvinyl pyrrolidone and 4.0 mg of calcium hydrogen phosphate, ground into powder, and passed through a 100-mesh sieve; 5% of a 95% ethanol solution of povidone K30 was added during stirring to prepare a soft material, and wet granulation was performed (passed through a 16-mesh sieve); the mixture was dried at 60° C., sieved, and granulated; 1% of magnesium stearate and 2% of micro-powdered silica gel were added, the mixture was evenly mixed, and tableting was performed to obtain purified polysaccharide tablets from silkworm chrysalis and Cordyceps militaris.

上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围。The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims (9)

1.一种利用闪式提取高效制备高降糖活性蚕蛹虫草多糖的方法,以蚕蛹虫草脱脂粉为原料,在水中浸泡,然后置于闪式提取器中,在一定电压下处理;经离心、浓缩上清液、乙醇沉淀、冷冻干燥得蚕蛹虫草多糖。1. A method for efficiently preparing silkworm pupa cordyceps polysaccharide with high hypoglycemic activity by flash extraction, which comprises taking silkworm pupa cordyceps defatted powder as raw material, soaking it in water, and then placing it in a flash extractor and treating it under a certain voltage; centrifuging, concentrating the supernatant, ethanol precipitation, and freeze-drying to obtain silkworm pupa cordyceps polysaccharide. 2.根据权利要求1所述的方法,所述一定的处理电压为70~150V。2. The method according to claim 1, wherein the certain processing voltage is 70 to 150V. 3.根据权利要求1所述的方法,所述蚕蛹虫草脱脂粉由石油醚脱脂所得。3. The method according to claim 1, wherein the silkworm chrysalis and Cordyceps defatted powder is obtained by defatting with petroleum ether. 4.根据权利要求1或2所述的方法,所述一定的处理电压为110V,采用西安太康生物科技有限公司型号为JHBE-20A的闪式提取器。4. The method according to claim 1 or 2, wherein the certain processing voltage is 110V, and a flash extractor model JHBE-20A of Xi'an Taikang Biotechnology Co., Ltd. is used. 5.根据权利要求1或2所述的方法,具体步骤为:5. The method according to claim 1 or 2, wherein the specific steps are: 将新鲜蚕蛹虫草于烘干至恒重,粉碎后过筛制成蚕蛹虫草粉备用;加入石油醚避光脱脂,减压抽滤得蚕蛹虫草脱脂粉;Dry fresh silkworm pupa cordyceps to constant weight, crush and sieve to make silkworm pupa cordyceps powder for later use; add petroleum ether to defat in dark, and filter under reduced pressure to obtain silkworm pupa cordyceps defatted powder; 将蚕蛹虫草脱脂粉在水中浸泡,然后置于闪式提取器中,在电压110V下处理;结束后离心取上清液,将上清液浓缩得浓缩液,加入无水乙醇沉淀,沉淀物经冷冻干燥得蚕蛹虫草多糖;The defatted powder of silkworm pupa and Cordyceps militaris is soaked in water, and then placed in a flash extractor and processed at a voltage of 110V; after the extraction, the supernatant is centrifuged and concentrated to obtain a concentrated solution, anhydrous ethanol is added for precipitation, and the precipitate is freeze-dried to obtain silkworm pupa and Cordyceps militaris polysaccharide; 所述蚕蛹虫草多糖得率为18.3±1.82%、5mg/mL时对α-葡萄糖苷酶的抑制率为83.2±7.64%。The yield of the silkworm chrysalis cordyceps polysaccharide is 18.3±1.82%, and the inhibition rate of α-glucosidase at 5 mg/mL is 83.2±7.64%. 6.根据权利要求1所述的方法,所述的蚕蛹虫草多糖的制备方法中,还包括对制备出的蚕蛹虫草多糖进行纯化,采用Sevag试剂脱蛋白,除去残留的Sevag试剂后在蒸馏水中透析,透析液冷冻干燥后得脱蛋白多糖,经二乙氨基乙基(DEAE)纤维素色谱柱纯化得蚕蛹虫草纯化多糖。6. The method according to claim 1, wherein the preparation method of the silkworm chrysalis Cordyceps polysaccharide further comprises purifying the prepared silkworm chrysalis Cordyceps polysaccharide, deproteinizing with Sevag reagent, dialyzing in distilled water after removing residual Sevag reagent, freeze-drying the dialysate to obtain deproteinized polysaccharide, and purifying with a diethylaminoethyl (DEAE) cellulose chromatographic column to obtain purified silkworm chrysalis Cordyceps polysaccharide. 7.根据权利要求6所述的方法,所述纯化的具体方法为:采用Sevag试剂脱蛋白,除去残留的Sevag试剂后在蒸馏水中透析;透析液冷冻干燥后得脱蛋白多糖;取脱蛋白多糖溶于去离子水中,上样至二乙氨基乙基(DEAE)纤维素色谱柱;用NaCl溶液进行缓慢洗脱,采用苯酚-硫酸法测定每管洗脱液的吸光度,当多糖全部洗脱下来后合并洗脱液,浓缩后透析,冷冻干燥得蚕蛹虫草纯化多糖;7. The method according to claim 6, wherein the specific method of the purification is: deproteinizing with Sevag reagent, dialyzing in distilled water after removing residual Sevag reagent; freeze-drying the dialyzate to obtain deproteinized polysaccharide; dissolving the deproteinized polysaccharide in deionized water, and loading it onto a diethylaminoethyl (DEAE) cellulose chromatographic column; slowly eluting with NaCl solution, measuring the absorbance of each tube of eluate with phenol-sulfuric acid method, combining the eluates after all the polysaccharides have been eluted, concentrating, dialyzing, and freeze-drying to obtain purified polysaccharide of Bombyx mori. 所述的蚕蛹虫草纯化多糖中满足下述条件中的一种或多种:1)总糖含量为83.5±1.46%;2)蛋白含量为7.26±2.05%;3)糖醛酸含量为4.16±0.68%;4)硫酸基含量为3.71±0.69%;5)所述的蚕蛹虫草纯化多糖由阿拉伯糖、半乳糖、葡萄糖、木糖、半乳糖醛酸、葡萄糖醛酸以31.6:21.6:274:23.6:1.00:3.56的摩尔比构成;6)所述的蚕蛹虫草纯化多糖的数均分子量(Mn)为1.85kDa;7)重均分子量(Mw)为5.09kDa。The purified polysaccharide from silkworm chrysalis and Cordyceps militaris satisfies one or more of the following conditions: 1) the total sugar content is 83.5±1.46%; 2) the protein content is 7.26±2.05%; 3) the uronic acid content is 4.16±0.68%; 4) the sulfate content is 3.71±0.69%; 5) the purified polysaccharide from silkworm chrysalis and Cordyceps militaris is composed of arabinose, galactose, glucose, xylose, galacturonic acid, and glucuronic acid in a molar ratio of 31.6:21.6:274:23.6:1.00:3.56; 6) the number average molecular weight (Mn) of the purified polysaccharide from silkworm chrysalis and Cordyceps militaris is 1.85 kDa; 7) the weight average molecular weight (Mw) is 5.09 kDa. 8.一种权利要求7所述方法得到的蚕蛹虫草纯化多糖的用途,用于制备降糖功能产品中的用途。8. Use of the purified polysaccharide of silkworm chrysalis and Cordyceps militaris obtained by the method of claim 7 for preparing a product with hypoglycemic function. 9.根据权利要求8所述的用途,蚕蛹虫草纯化多糖可用于制备口服液、胶囊或片剂。9. The use according to claim 8, wherein the purified polysaccharide from silkworm chrysalis and Cordyceps militaris can be used to prepare oral liquid, capsule or tablet.
CN202410158285.5A 2024-02-04 2024-02-04 Method for flash-type efficient extraction of Cordyceps militaris polysaccharide with high hypoglycemic activity and application thereof Pending CN118027236A (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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