CN110540603A - 知母多糖及其制备方法、鉴定方法和应用 - Google Patents
知母多糖及其制备方法、鉴定方法和应用 Download PDFInfo
- Publication number
- CN110540603A CN110540603A CN201910812081.8A CN201910812081A CN110540603A CN 110540603 A CN110540603 A CN 110540603A CN 201910812081 A CN201910812081 A CN 201910812081A CN 110540603 A CN110540603 A CN 110540603A
- Authority
- CN
- China
- Prior art keywords
- polysaccharide
- rhizoma anemarrhenae
- supernatant
- anemarrhenae polysaccharide
- water
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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Classifications
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- A—HUMAN NECESSITIES
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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- A61K31/733—Fructosans, e.g. inulin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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Abstract
本发明属于医药及保健食品技术领域,具体涉及一种知母多糖及其制备方法、鉴定方法和应用。本发明提供的知母多糖的分子量范围为1000‑100000 Da。本发明制备方法简单,反应条件温和,可大规模生产;且本发明对得到的高纯度知母多糖化学结构进行了鉴定,明确了其结构,为探究其药理活性机制提供结构依据。同时,本发明得到的知母多糖纯品为知母多糖药物、保健食品、功能食品,及其质量控制和深入研究其构效关系、作用机制奠定基础。
Description
技术领域
本发明属于医药及保健食品技术领域,具体涉及一种知母多糖及其制备方法、鉴定方法和应用。
背景技术
神经退行性疾病(Neurodegenerative diseases)diseases)包括阿尔茨海默病(Alzheimer's disease,AD)、亨廷顿病(Huntington's disease,HD)、帕金森病(Parkinson'sdisease,PD)等,是由于神经元变性、凋亡所导致的进行性发展的致死性复杂疾病。世界卫生组织预测,到2030年,全球范围内罹患神经退行性疾病的人数将高达近1亿人,而到2040年,神经退行性疾病将取代癌症成为人类第二大致死疾病。神经退行性疾病病因复杂,目前其发病机制尚不完全清楚, 临床上缺乏特异性的治疗方案。相关研究表明神经细胞的凋亡是神经退行性疾病的主要机制,因此寻找能一种高效、低毒且能减少脑神经细胞凋亡的药物可能成为治疗神经退行性疾病的新思路。
知母(Anemarrhena asphodeloides Bunge)是百合科植物知母的干燥根茎,习称为“毛知母”。其味苦、性寒,归肺、胃、肾经,具有清热泻火、生津润燥的功效。临床上用于治疗外感热病,高热,烦渴,肺热燥咳,骨蒸潮热,内热消渴,肠燥便秘。知母的化学成分主要有皂苷类、多糖、黄酮类、微量元素及氨基酸等多种成分。同时现代药理研究表明,多糖作为知母的主要活性成分之一,其药理作用较为广泛,具有降血糖、神经保护、增强免疫、抗氧化、抗肿瘤、促进体液免疫和细胞免疫等多种活性。目前关于知母多糖的研究主要集中于对其降血糖活性方面的研究,对知母多糖结构方面的研究不够深入,且在其在免疫调节和神经退行性疾病方面的报道较少,因此,有必要提供一种提取纯化、结构鉴定的方法,为知母多糖药物、质量控制及深入研究其构效关系和作用机制奠定基础。
发明内容
为了解决现有技术存在的问题,本发明提供了一种知母多糖及其制备方法、鉴定方法和应用。本发明制备方法简单,反应条件温和,可大规模生产;且本发明对得到的高纯度知母多糖化学结构进行了鉴定,明确了其成分,为探究其药理活性机制提供结构依据。同时,本发明得到的知母多糖纯品为知母多糖药物、质量控制及深入研究其构效关系和作用机制奠定基础。
本发明提供了一种知母多糖AA2-1。发明人通过对知母多糖粗品提纯分离得到知母多糖AA2-1,且经结构分析得到其分子量范围为1000-100000 Da。
进一步地,所述知母多糖AA2-1的结构如图7所示,其中n为1~14。
同时,本发明也提供了一种知母多糖的制备方法,包括以下步骤:
S1 剪切:将知母干燥根切成小段,用水清洗干净、晾干,得知母段;
S2 水提:将步骤S1所得知母段加入水中,加热提取,过滤,得提取液和药渣;
S3 分级醇沉:
将步骤S2所得提取液减压浓缩,得浓缩液1;向浓缩液1中加入乙醇,至乙醇体积浓度为a%,静置,收集沉淀和上清液,得粗多糖AA1和上清液1;
将上清液1减压浓缩,得浓缩液2;向浓缩液2中加入乙醇,至乙醇体积浓度为b%,静置,收集沉淀和上清液,得粗多糖AA2和上清液2;
将上清液2减压浓缩,得浓缩液3;向浓缩液3中加入乙醇,至乙醇体积浓度为c%,静置,收集沉淀,得粗多糖AA3;
其中10≤a<b<c<100;
S4 纯化:
一次纯化:
将步骤S3所得粗多糖AA2进行除蛋白,透析,冻干,得知母多糖;
二次纯化:
将一次纯化后的多糖AA2进行离子交换柱层析,用0~2 M的NaCl进行梯度洗脱,使用苯酚-硫酸法跟踪洗脱曲线,根据洗脱曲线分别收集糖部分,浓缩、冷冻干燥;再分别用水进行溶解,离心,取上清液;
将上清液再进行分子筛凝胶柱层析,用水进行洗脱,利用苯酚-硫酸法检测洗脱曲线,根据洗脱曲线收集糖部分,浓缩、冷冻干燥,得AA-2。
本发明人通过研究发现知母段的优选长度为1~3 cm;同时,本发明采用水提分级醇沉法的结合,乙醇浓度由低到高进行分级醇沉,对知母多糖进行初步分离,且高浓度乙醇能将极性大、水溶性好的多糖与极性小、水溶性差的多糖分离,解决了传统水煮法提取多糖导致其后期分离繁杂、困难的问题。
进一步地,所述步骤S2中水的添加量为所述知母段重量的5~15倍,加热温度为60~100℃,提取时间1~10 h。
进一步地,所述步骤S3中减压浓缩温度为40~70℃,静置的时间为10~28 h。
另外,本发明还提供了一种知母多糖的鉴定方法,包括以下步骤:
(1)取知母多糖样品,完全酸水解,水解产物液相色谱检测;
(2)取知母多糖样品,干燥,压片,红外光谱检测;
(3)取知母多糖样品,甲基化,水解、还原,乙酰化,进行GC-MS检测;
(4)取知母多糖样品溶于D2O,进行核磁共振分析。
本发明人为了进一步开发知母这一宝贵资源,发掘新药源,通过大量实验研究得到本发明:以知母干燥根为原料,采用水提醇沉法得到粗多糖,对提取的粗多糖进行脱蛋白,然后利用离子交换层析和凝胶分子筛柱层析方法纯化知母粗多糖,首次制备出一种知母多糖纯品,对此多糖纯品的理化性质、分子量、单糖组成等进行了系统的分析鉴定,成功得出了知母多糖的结构信息,其中知母多糖AA2-1由葡萄糖和果糖组成的果聚糖,主链由→2)-α-D-Fruf-(6→组成,支链由→2)-β-D-Fruf-(1→,→1, 2)-α-D-Fruf-(6→和→6)-α-D-Glcp-(1→组成,末端糖残基由→2)-β-D-Fruf组成;
本发明还涉及了知母多糖在制备治疗神经保护药物,免疫调节药物或保健品中的应用。并通过实验研究对其神经损伤保护活和免疫调节活性进行评价。
与现有技术相比,本发明具有以下优势:
1. 本发明采用水提醇沉法,对知母多糖进行初步分离,效果显著,且本制备方法简单,反应条件温和,可大规模生产;
2. 本发明通过柱层析法对知母粗多糖进行二次分离纯化,效果显著,首次制备出一种知母多糖纯品;
3. 本发明对纯化得到的多糖纯品的结构进行了鉴定,明确了各多糖组分的理化性质及结构,为探究其药理活性机制提供结构依据;
4. 本发明得到的知母多糖纯品,组分保存完好,结构明确,质量可控,在CoCl2诱导的SH-SY5Y神经细胞损伤模型中显示具有神经保护活性和增强RAW264.7巨噬细胞的免疫调节活性,为知母多糖在医药、保健品等领域的应用提供依据。
同时,本发明为知母多糖药物、质量控制及深入研究其构效关系和作用机制奠定基础。
附图说明
图1为AA2-1单糖组成的高效液相色谱图;
图2为AA2-1的红外图谱;
图3为AA2-1的1H NMR图谱;
图4为AA2-1的13C NMR图谱;
图5为AA2-1的HSQC图谱;
图6为AA2-1的HMBC图谱;
图7为AA2-1的一级结构;
图8为AA2-1对SH-SY5Y细胞增殖的促进作用;
图9为AA2-1对SH-SY5Y细胞凋亡的保护作用;
图10为AA2-1对RAW264.7细胞的免疫调节作用。
具体实施方式
下面通过具体实施例对本发明做进一步说明,需要指出的是,以下说明仅仅是对本发明要求保护的技术方案的举例说明,但并不因此而限制本发明。本发明的保护范围以所附权利要求书记载的内容为准。
实施例1 知母多糖的制备方法
所述知母多糖的制备方法包括以下步骤:
S1 剪切:将5 kg的知母干燥根用剪刀剪至1~3 cm,用冷水快速清洗,晾干,得知母段;
S2 水提:向步骤S1所得知母段中加入其重量10倍的水,加热至80℃提取,提取3 h,收集提取液,药渣晾干,得提取液和药渣;
S3 分级醇沉:
将步骤S2所得提取液于60℃减压浓缩后,加入乙醇至乙醇体积浓度为50%,室温静置24h后,离心,收集沉淀和上清液,得粗多糖AA1和上清液1;
上清液1于60℃减压浓缩后,加入乙醇至乙醇体积浓度为70%,室温静置24 h后,离心,收集沉淀和上清液,得粗多糖AA2和上清液2;
上清液2于60℃减压浓缩后,加入乙醇至乙醇体积浓度为90%,室温静置24 h后,离心,收集沉淀和上清液,得粗多糖AA3;
S4 纯化:利用Sevag法分别将步骤S3所得AA2粗多糖进行除蛋白,除蛋白后粗多糖用透析袋(截留分子量为1000 Da)进行透析、冻干,得知母多糖AA2。
实施例2 知母多糖AA2-1
所述知母多糖AA2-1是由实施例1所得知母多糖AA1二次纯化所得,具体方法如下:
1)离子交换柱层析:取200 mg实施例1所得知母多糖AA2,溶于5mL的去离子水中,上样于DEAE-Sepharose FF柱,在不同盐浓度的洗脱液条件下出现1个峰,其中洗脱峰为0.1M的NaCl洗脱部分(洗脱过程中使用苯酚-硫酸法跟踪洗脱曲线,根据洗脱曲线分别收集糖部分),分别将所得洗脱液浓缩、冷冻干燥后,得到一种多糖;
2)分子筛凝胶层析:将上述冻干后的多糖样品,用水进行溶解,离心,取上清液,上Sephadex G-75柱,用水进行洗脱,使用苯酚-硫酸法跟踪洗脱曲线,出现一个单一对称峰,收集主峰,浓缩,冷冻干燥,得知母多糖AA2-1。
实施例3 知母多糖AA2-1的结构分析
(一)试验材料:知母多糖AA2-1。
(二)试验方法:
1. 单糖组成分析(PMP柱前衍生法)
样品处理:
精确称取4.0 mg试验材料于具塞试管中,加入2 M三氟乙酸(TFA)2.0 mL,置于油浴锅中在120℃油浴水解6 h。冷却至室温,重复加甲醇旋干,去除TFA,用去离子水溶解至1 mL,离心,各吸取100 μL样品溶液,加入100 μL 0.3 M的NaOH溶液,再加入100 μL 0.5 M PMP甲醇溶液,混匀,70℃水浴锅上反应30 min,冷却,加入105 μL 0.3 M的HCL溶液中和,加去离子水至1 mL,再加入等体积的氯仿溶液,剧烈震摇,离心,去氯仿相,如此反复2次萃取,取水相用0.45 μm滤膜过滤后供HPLC进样分析。
色谱条件:
色谱柱:Kromasil 100-5-C18,4.6×250 mm,5 μm;流动相:0.1 M磷酸盐(pH = 6.9)缓冲液-乙腈(v/v为83:17);检测波长:250 nm;流速:1 mL/min;进样体积:20 μL。
红外光谱检测
将干燥的试验材料2.0 mg与KBr研磨后,压片,用Perkin EImer FT/IR-100在4000-400cm-1范围内进行扫描。
甲基化/GC-MS分析
称取干燥的试验材料8.0 mg于反应瓶中,加入无水DMSO 8 mL,再加入干燥的氢氧化钠800 mg,超声30 min,冰浴条件下,避光加入碘甲烷3.0 mL,分三次加入,每次冰浴超声30min,反应结束后加入2 mL的蒸馏水分解残留的碘甲烷,并加入1 mL的氯仿萃取,离心取氯仿层。
甲基化完全后样品置于具塞试管中用2 mol/L的TFA在120℃恒温油浴水解6 h,减压蒸发至干,再重复多次加入甲醇旋干至pH为中性,然后用20 mg的NaBH4在40℃下反应30min还原水解产物。再使用100 μL的冰醋酸终止反应,样品在低压下旋干,然后加入2 mL的乙酰酐和吡啶用来乙酰化。保持95℃磁力搅拌反应2 h。然后反复加甲醇3次,旋干,用1 mL氯仿溶解,再用等体积的蒸馏水洗涤3次,除去水层,最后用无水硫酸钠干燥氯仿层,再过滤除去硫酸钠固体,减压浓缩蒸干,供GC-MS分析。
核磁共振分析
取各知母多糖样品反复多次冻干后,取60 mg溶于0.6 mL D2O中,置于核磁管中,用400MHz核磁共振仪Bruker AV-400记录其1H NMR,13C NMR,HSQC,HMBC等图谱。
(三)试验结果:
1.知母多糖AA2-1结构分析
(1)单糖组成分析
如图1所示,由HPLC图谱可知,AA2-1仅含有葡萄糖。(色谱峰顺序:1:甘露糖,2:鼠李糖,3:葡萄糖醛酸,4:半乳糖醛酸,5:葡萄糖,6:半乳糖,7:木糖,8:阿拉伯糖,9:岩藻糖。)
(2)红外光谱分析
如图2所示,由AA2-1红外光谱图可知,AA2-1含有多糖的特征吸收峰:3389 cm-1为O-H伸缩振动,2934 cm-1为C-H伸缩振动,1647 cm-1为结合水吸收峰。1740cm-1区间未见吸收峰说明AA2-1不含有糖醛酸,923 cm-1和821 cm-1为呋喃环的特征吸收峰;601 cm-1处有吸收峰,说明AA2-1为主要由β型糖残基组成。
(3)甲基化/GC-MS分析
AA2-1甲基化分析,经过水解,还原乙酰化后,GC-MS检测,由GC-MS图谱可知,AA2-1含有→1)-β-D-Fruf-(2→, →2)-β-D-Fruf-(6→, →1, 2)-β-D-Fruf-(6→, →6)-α-D-Glcp-(1→和β-D-Fruf-(2→等糖残基。
(4)核磁共振分析
本试验通过1H NMR、13C NMR、HSQC对AA2-1的糖残基的碳原子和氢原子的化学位移进行归属,然后用HMBC确认其连接顺序。图3-6为AA2-1的1HNMR、13C NMR、HSQC和HMBC图谱。
根据图3-6的核磁图谱,AA2-1碳氢归属如下表1所示。
表1 AA2-1核磁共振分析结果
aUnresolved from other signals,nd:no detected.
综上所述:AA2-1是一种由葡萄糖和果糖组成的果聚糖,从甲基化分析说明其含有→1)-β-D-Fruf-(2→,→6)-β-D-Fruf-(2→,→1, 6)-β-D-Fruf-(2→, →6)-α-D –Glcp-(1→和β-D-Fruf-(2→等糖残基,不同糖残基之间的连接顺序由二维核磁HMBC谱图分析得出,由以上分析得出AA2-1的结构如图7所示,其中n为1~14。
实施例4 知母多糖纯品的神经保护作用研究
(一)试验材料:知母多糖AA2-1。
(二)试验对象:SH-SY5Y细胞(由中国科学院上海细胞库提供)。
(三)试验方法:
1. 实验设计与分组:
本发明采用人神经母细胞瘤细胞(SH-SY5Y)模型,以CoCl2为模型药,以生育酚为阳性药,建立一种快速,高效的神经损伤模型。设置空白组,模型组,阳性药组及给药组,其中知母多糖AA2-1的浓度为36.8 μM, 73.5 μM和147.1 μM, 生育酚的浓度为300 μM。
母液的配置:
取AA2-1 20 mg,溶于1 ml的PBS缓冲盐中,将配制完的母液过0.22 µm的滤膜并且紫外照射30 min灭菌,然后置于-20℃冰箱中待用。
细胞计数和铺板:
将培养瓶置于超净工作台中,移除旧培养基,加入5 mL的 PBS缓冲液清洗细胞,清洗完细胞后移除PBS缓冲液并向培养瓶中加入600 µL胰蛋白酶,消化SH-SY5Y细胞1 min后再加入DMEM培养基,反复吹打使其成为均匀的单细胞悬液。取20 µL上述单细胞悬液加入180 µLDMEM培养基中,反复吹打后取10 µL于细胞计数板中计数,将细胞稀释为5 × 104 个/mL,铺板。
给药:
将SH-SY5Y细胞接种到96孔板,培养24 h后加入待测样品,AA2-1给药浓度分别为36.8μM, 73.5 μM和147.1 μM,生育酚的给药浓度为50 μM,给药后的SH-SY5Y细胞置于CO2培养箱中培养24 h,随后再加入300 μM的CoCl2培养24 h。
MTT检测:
移除96孔板中的培养液,然后每孔加入5 mg/mL的噻唑蓝(MTT)溶液20 µL,于CO2培养箱中培养4 h。4 h后移除96孔板中的MTT溶液,每孔加入150 µL二甲基亚砜(分析纯)溶解甲瓒结晶,用酶标仪检测492 nm下吸光度值(OD值)。计算样品对各细胞增殖的抑制率,实验重复三次。
SH-SY5Y细胞凋亡实验:
SH-SY5Y 细胞用不同浓度的AA2-1 (36.8 μM, 73.5 μM和147.1 μM)和300 μM的CoCl2分别处理24 h后转移至离心管中,贴壁的细胞PBS洗涤三次后用0.25%的胰酶进行消化,收集底部沉淀并用PBS洗涤两次后用195 μL的染色缓冲液重悬,随后加入5 μL的膜联蛋白V-FITC溶液和10 μL的碘化丙啶溶液,室温避光条件下孵育20 min后使用流式细胞仪进行分析,实验重复三次。
(四) 试验结果
1. 知母多糖AA2-1的神经保护作用
知母多糖AA2-1对SH-SY5Y细胞的保护作用见图8及图9。如图8所示,经不同浓度的知母多糖AA2-1处理后,AA2-1对SH-SY5Y的增殖作用具有显著性的促进作用(P < 0.001)且具有剂量依赖性。如图9所示,不同浓度的AA2-1对SH-SY5Y细胞的凋亡具有显著的改善作用(P <0.01 或P < 0.001),表明知母多糖AA2-1可通过抑制SH-SY5Y细胞的凋亡从而改善CoCl2所引起的神经细胞损伤。
实施例5 知母多糖纯品的免疫调节作用研究
(一) 试验材料:知母多糖AA2-1。
(二) 试验对象:RAW264.7细胞(由中国科学院上海细胞库提供)。
(三) 试验方法:
1. 实验设计与分组:
本发明采用巨噬细胞RAW264.7模型,设置空白组, 阳性药组及知母多糖给药组,其中知母多糖AA2-1的浓度为18.4 μM,36.8 μM, 73.5 μM和147.1 μM,阳性药为脂多糖 (LPS),浓度为1 μg/mL。
母液的配置及细胞计数
母液配置及细胞计数方法与实施例二中相同。
给药
将RAW264.7细胞接种到96孔板,培养24 h后加入待测样品,AA2-1给药浓度分别为18.4μM, 36.8 μM, 73.5 μM和147.1 μM, 给药后的RAW264.7细胞置于CO2培养箱中培养24 h。
MTT实验
移除96孔板中的培养液,然后每孔加入5 mg/mL的噻唑蓝(MTT)溶液20 µL,于CO2培养箱中培养4 h。4 h后移除96孔板中的MTT溶液,每孔加入150 µL二甲基亚砜(分析纯)溶解甲瓒结晶,用酶标仪检测492 nm下吸光度值(OD值)。计算样品对RAW264.7细胞的增殖促进作用,实验重复三次。
中性红吞噬实验
RAW264.7 细胞经不同浓度的AA2-1给药处理后经PBS洗涤三次后加入100 μL的中性红溶液(0.1%)孵育1 h,弃去中性红溶液,PBS洗涤三次,吸干,加入细胞裂解液(冰醋酸:乙醇=1:1) 150 μL,室温下孵育1 h,用酶标仪于540 nm处测定OD值,实验重复三次。
NO释放测试
RAW264.7细胞接种于96孔板,4 h后用不同浓度的AA2-1(18.4 μM, 36.8 μM, 73.5 μM和147.1 μM)及1 μg/mL LPS 处理24 h后,收集上清液并与Griess试剂反应,于550 nm处用酶标仪测定OD值。
AA2-1对RAW264.7细胞炎症因子的影响
RAW264.7细胞接种于24孔板孵育4 h, (18.4 μM, 36.8 μM, 73.5 μM和147.1 μM)及1μg/mL LPS 处理24 h后,收集上清并用ELISA试剂盒进行TNF-α, IL-6 和IL-1β等炎症因子的检测。
(四)实验结果
1.知母多糖AA2-1的免疫调节作用
如图10所示,经不同浓度的知母多糖AA2-1处理后,RAW264.7细胞的活性与空白组相比无显著性差异(P > 0.05),表明知母多糖安全且无细胞毒性。其次,RAW264.7细胞经不同浓度的AA2-1处理后,其吞噬活性显著性增加(P < 0.01 或P < 0.001)及TNF-α, IL-6 和IL-1β等炎症因子的表达水平显著性增加(P < 0.05,P < 0.01 或P < 0.001),表明知母多糖AA2-1能通过提高机体巨噬细胞的吞噬能力及促进TNF-α, IL-6 和IL-1β等炎症因子的释放增强机体的免疫调节能力。
综上所述,本发明制备的知母多糖纯品AA2-1能通过减少SH-SY5Y细胞凋亡显著改善CoCl2所引起的神经损伤,且通过增加RAW246.7细胞的吞噬能力及促进相关炎症因子的释放从而增强机体的免疫调节能力。
Claims (7)
1.一种知母多糖,其特征在于,包括知母多糖AA2-1;
所述知母多糖AA2-1由葡萄糖和果糖组成,其分子量范围为1000-100000 Da。
2.如权利要求1所述的知母多糖,其特征在于,所述知母多糖AA2-1的结构如图7所示,其中n为1~14。
3.如权利要求1所述的知母多糖的制备方法,其特征在于,包括以下步骤:
S1 剪切:将知母干燥根切成小段,用水清洗干净、晾干,得知母段;
S2 水提:将步骤S1所得知母段加入水中,加热提取,过滤,得提取液和药渣;
S3 分级醇沉:
将步骤S2所得提取液减压浓缩,得浓缩液1;向浓缩液1中加入乙醇,至乙醇体积浓度为a%,静置,收集沉淀和上清液,得粗多糖AA1和上清液1;
将上清液1减压浓缩,得浓缩液2;向浓缩液2中加入乙醇,至乙醇体积浓度为b%,静置,收集沉淀和上清液,得粗多糖AA2和上清液2;
将上清液2减压浓缩,得浓缩液3;向浓缩液3中加入乙醇,至乙醇体积浓度为c%,静置,收集沉淀,得粗多糖AA3;
其中10≤a<b<c<100;
S4 纯化:
一次纯化:
将步骤S3所得粗多糖AA1、AA2、AA3进行除蛋白,透析,冻干,得知母多糖;
二次纯化:
将一次纯化后的多糖AA2进行离子交换柱层析,用0~2M的NaCl进行梯度洗脱,使用苯酚-硫酸法跟踪洗脱曲线,根据洗脱曲线分别收集糖部分,浓缩、冷冻干燥;再分别用水进行溶解,离心,取上清液;
将上清液再进行分子筛凝胶柱层析,用水进行洗脱,利用苯酚-硫酸法检测洗脱曲线,根据洗脱曲线收集糖部分,浓缩、冷冻干燥,得AA2-1。
4. 如权利要求3所述的知母多糖的制备方法,其特征在于,所述步骤S2中水的添加量为所述下知母段重量的5~15倍,加热温度为60~100℃,提取时间1~10 h。
5.如权利要求3所述的知母多糖的制备方法,其特征在于,所述步骤S3中减压浓缩温度为40~70℃,静置的时间为10~28 h。
6.如权利要求1-5所述的知母多糖的鉴定方法,其特征在于,包括以下步骤:
取知母多糖样品,完全酸水解,水解产物离子色谱/液相色谱检测;
取知母多糖样品,干燥,压片,红外光谱检测;
取知母多糖样品,甲基化,水解、还原,乙酰化,进行GC-MS检测;
取知母多糖样品溶于D2O,进行核磁共振分析。
7.如权利要求1-6所述的知母多糖在制备治疗神经损伤保护药物,免疫调节药物或保健品中的应用。
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