CN113717294A - 一种菊粉型党参果聚糖cpw1及其制备方法与应用 - Google Patents
一种菊粉型党参果聚糖cpw1及其制备方法与应用 Download PDFInfo
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- CN113717294A CN113717294A CN202110959994.XA CN202110959994A CN113717294A CN 113717294 A CN113717294 A CN 113717294A CN 202110959994 A CN202110959994 A CN 202110959994A CN 113717294 A CN113717294 A CN 113717294A
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- cpw1
- codonopsis pilosula
- fructan
- polysaccharide
- selenium
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Abstract
本发明公开了一种菊粉型党参果聚糖CPW1,其特征在于:该多糖是菊粉型果聚糖,分子量为5623,是β‑D‑呋喃果糖残基以β‑(2→1)‑糖苷键连接而成、端基是α‑D‑葡萄糖‑1键接的直链均一多糖,该结构清晰单一;其产品可以进一步用于稳定和分散硒纳米粒子,增加其生物活性,具有很高的临床应用价值。
Description
技术领域
本发明涉及中药学领域,尤其是一种菊粉型党参果聚糖CPW1及其制备方法与应用。
背景技术
党参为桔梗科植物党参[Codonopsis pilosula(Franch.)Nannf.]、素花党参[Codonopsis pilosula Nannf.var.modesta(Nannf.)L.T.Shen]或川党参(Codonopsistangshen Oliv.)的干燥根,是我国常用的传统中药,具有健脾益肺、补中益气以及调理胃肠运动的效用。而党参中主要的活性成分为多糖。目前,已有许多研究者从党参中提取到党参多糖,并证实了其具有多种生物活性,但党参多糖提取技术主要是水提,所得党参多糖所含杂质较多,结构不明或结构不均一,无法重复,十分不利于实际生产应用。
党参多糖目前的研究多集中于药理作用,对其在生物医用材料领域的应用报道较少,因此,拓宽中药材在各领域的应用,有利于更加充分发挥中药材的作用。硒(Se)是人体健康所必需的微量元素。近年来,零价硒(如硒纳米粒子、纳米硒)逐渐出现,可在靶向给药中实现抗癌作用。因此,硒纳米粒子已作为抗癌、抗糖尿病和抗氧化剂,或作为营养补充剂,也作为抗艾滋病的药物补充剂或治疗各种自身免疫性疾病。通常,硒纳米粒子是通过使用还原剂来还原亚硒酸盐、亚硒酸盐或二氧化硒来制备的。然而,纳米颗粒易于聚集,从而导致低生物利用度和高毒性。因此,有效分散硒纳米粒子至关重要。
发明内容
本发明的目的是提供一种菊粉型党参果聚糖CPW1及其制备方法与应用,以至少克服上述一项技术缺陷。
为实现上述目的,本发明采取的技术方案如下:
第一方面,提供一种菊粉型党参果聚糖CPW1,该多糖是菊粉型果聚糖,分子量为5623,分子量为5623,是β-D-呋喃果糖残基以β-(2→1)-糖苷键连接而成、端基是α-D-葡萄糖-1键接的直链均一多糖。
本发明第二方面保护第一方面所述的一种菊粉型党参果聚糖CPW1的制备方法,其特征在于:包括以下步骤:
S1、脱脂脱色:将党参洗净粉粹后放入索氏提取器,回流收集样品备用;
S2、提取:将经步骤S1获得的产品置入盐水内搅拌、收集滤液备用;
S3、脱蛋白:将步骤S2所得滤液浓缩,用SEVERAGE法除蛋白后脱色后获得党参粗多糖;
S4、极性分离:将步骤S3获得的党参粗多糖用DEAE Sepharose Fast Flow色谱柱进行极性分离,其中采用四组溶剂洗脱以获得四组洗脱组分,命名为CPW、CPS0.2、CPS0.5、CPS1;
S5、色谱柱纯化:将步骤S4中得到的水洗组分CPW通过色谱柱进一步纯化获得均一多糖,命名为CPW1。
优选地,步骤S1中,需将粉粹后的党参用100木纱布的样品袋装载后放入索氏提取器,且使用乙酸乙酯和丙酮分别回流2~3小时后收集。
优选地,步骤S2中,盐水为0.9%氯化钠配置的盐水,且于80~90℃搅拌4h后收集滤液,并将剩余残渣重复步骤S2前述步骤2-3次后将所得滤液收集。
优选地,步骤S3中,除蛋白的具体操作是
将滤液:氯仿:正丁醇按体积比25:5:1比例混匀后,快速搅拌30分钟,离心,收集上清液备用;
步骤S3中,脱色的具体步骤是:
在除蛋白后的上清液中滴加30%双氧水,保持pH=8~9,直到溶液变为浅黄色,将溶液对水透析7-10天后,浓缩,冻干,得党参粗多糖。
优选地,步骤S4中,极性分离的具体步骤是:
S41、DEAE Sepharose Fast Flow填料先用0.5mol/L的盐酸浸泡1h,然后洗去填料中的杂质物,用4-5倍体积的蒸馏水洗脱至中性;
S42、调整流速为5ml/min,蒸馏水平衡2h后用蒸馏水溶解粗多糖、加热、斡旋,12000rpm离心,取上清液上样;调整流速为15ml/min的蒸馏水进行洗脱;
S43、四组溶剂洗脱:三倍柱体积的水、0.2M NaCl、0.5M NaCl、1.0M NaCl洗脱;采用苯酚硫酸法进行追踪检测,酶标仪490nm进行检测,并根据绘制散点图峰形,分别收集,浓缩,透析,冷冻干燥。以得到四组洗脱组分,命名为CPW、CPS0.2、CPS0.5、CPS1。
本发明第三方面保护一种硒纳米粒子制剂CPW1-Se,是由第一方面所述的CPW1和硒纳米粒子SeNPs复合而成的制剂,为一种无定型态非晶体。
优选地,CPW1分子链上的羟基和SeNPs上的硒原子以Se-O键螯合,使得硒纳米粒子制剂CPW1-Se不会聚集,可稳定分布于水中至少15天。
优选地,硒纳米粒子制剂CPW1-Se的尺寸,可以通过控制亚硒酸钠的浓度控制其粒径大小在20-110nm内。
本发明第四方面保护第三方面所述一种硒纳米粒子制剂CPW1-Se在抗肝癌领域中的应用。
本发明通过热盐水提取、色谱柱分离纯化的方法从党参中提取一种产率较高、纯度较高的水溶性多糖,获得途径方便,工艺简单、科学合理,只需要经过葡聚糖凝胶柱就可以快速得到高纯度的产品。该产品是一种菊粉型党参果聚糖CPW1,其结构简单,可用于增加纳米粒子的生物相容性,更好的发挥生物活性,并可进一步用于稳定和分散硒纳米粒子,增加其生物相容性,具有很高的临床应用价值;本发明提供的一种硒纳米粒子制剂CPW1-Se对肝癌细胞(HepG2、Huh7)有明显的促凋亡作用。
附图说明
图1CPW1的高效液相色谱曲线;
图2CPW1的二维核磁HMBC图谱;
图3SeNPs的TEM图像;
图4CPW1-Se的TEM图像;
图5CPW1-Se的粒径稳定性;
图6CPW1-Se的Zeta电位的稳定性;
图7CPW1和CPW1-Se的C1s的XPS图谱;
图8CPW1和CPW1-Se的红外图谱;
图9SeNPs的细胞毒性;
图10CPW1-Se的细胞毒性;
具体实施方式
以下结合附图对本发明的优选实施例进行说明,应当理解,此处所描述的优选实施例仅用于说明和解释本发明,并不用于限定本发明。
实施例1:
本发明提供一种杂质少且结构均一的菊粉型党参果聚糖CPW1,具体为菊粉型果聚糖,分子量为5623,分子量为5623,是β-D-呋喃果糖残基以β-(2→1)-糖苷键连接而成、端基是α-D-葡萄糖-1键接的直链均一多糖;
其化学结构式为α-D-Glcp-1→(2-β-D-Fruf-1)n→2-β-D-Fruf,其中n=35-36。
获得一种菊粉型党参果聚糖CPW1的具体制备方法,包括以下步骤:
S1、脱脂脱色:将党参药材洗净烘干后,粉碎,装入100木纱布缝制的样品袋中,放入索氏提取器,乙酸乙酯和丙酮分别回流2h,收集样品备用;
S2、提取:将步骤S1获得的样品放入0.9%氯化钠配置的盐水中,85℃搅拌4小时,收集滤液,剩余残渣重复本步骤2次,将三次所得滤液收集备用;
S3、脱蛋白:将步骤S2所得滤液浓缩,用SEVERAGE法除蛋白,具体是药材滤液:氯仿:正丁醇按体积比25:5:1混匀后,快速搅拌30分钟,离心,收集上清液;
然后将上清液缓慢滴加加入30%双氧水,保持pH=8.5,直到溶液变为浅黄色,将溶液对水透析7天后,浓缩,冻干,得党参粗多糖;
S4、极性分离:将党参粗多糖用DEAE Sepharose Fast Flow色谱柱进行极性分离。DEAE Sepharose Fast Flow填料先用0.5mol/L的盐酸浸泡1小时,然后洗去填料中的杂质物,用4倍体积的蒸馏水洗脱至中性。调整流速为5mL/min,蒸馏水平衡2h。用蒸馏水溶解粗多糖,加热,斡旋,12000rpm离心,取上清液上样。调整流速为15mL/min的蒸馏水进行洗脱;
四组溶剂洗脱:三倍柱体积的水、0.2M NaCl、0.5M NaCl、1.0M NaCl洗脱。采用苯酚硫酸法进行追踪检测,酶标仪490nm进行检测。根据检测所得峰形,分别收集,浓缩,透析,冷冻干燥。得到四组洗脱组分,命名为CPW、CPS0.2、CPS0.5、CPS1;
S5、色谱柱纯化:将水洗组分CPW通过色谱柱进一步纯化。凝胶纯化色谱柱为填料Sephacryl S-100,尺寸:2.6×70cm,流速1.0ml/min,柱温30℃,流动相为0.2M氯化钠溶液。根据凝胶纯化色谱图,只收集主峰的对称部分,通过旋转蒸发仪进行浓缩,冷冻干燥,得到均一多糖,命名为CPW1。
实施例2:
对实施例1获得的菊粉型党参果聚糖CPW1的结构进行鉴定,包括以下步骤:
(1)纯度鉴定:对本发明提取方法获得的CPW1采用高效液相色谱法进行纯度和分子量鉴定,证明已是单一组成(如图1所示)。色谱柱:BRT105-104-102串联凝胶柱(8×300mm);流动相:0.05M NaCl溶液;流速:0.6mL/min,柱温:40℃;进样量:20μl;检测器:示差检测器RI-10A。
结果表明,CPW1是一种纯多糖,分子量为5623。
(2)单糖组成:采用离子色谱对CPW1的单糖组成进行分析,证明是由果糖和少量葡聚糖组成。精密称量10mg的CPW1置于安瓿瓶中,加入3M TFA 10ml,80℃水解3h。准确吸取酸水解溶液转移至管中氮吹吹干,加入10ml水涡旋混匀,吸取100uL加入900uL去离子水,12000rpm离心5min。取上清进离子色谱(ThermoFisher,ICS5000)分析。
其中16种单糖标准品(岩藻糖、鼠李糖、阿拉伯糖、半乳糖、葡萄糖、木糖、甘露糖、果糖、核糖、半乳糖醛酸、葡萄糖醛酸、氨基半乳糖盐酸盐、盐酸氨基葡萄糖、N-乙酰-D氨基葡萄糖、古罗糖醛酸、甘露糖醛酸)的标准母液(1mg/ml),按照下表1的标准品序列配置混标,进行离子色谱分析。
表1混标中16种单糖标准品浓度及出峰时间
色谱柱:DionexCarbopacTMPA20(3*150);流动相:A:H2O;B:15mMNaOHC:15mMNaOH&100mM NaOAC;流速:0.3ml/min;进样量:5μL;柱温:30℃;检测器:电化学检测器。
结果表明,CPW1单糖由果糖和少量葡萄糖组成,摩尔比0.879:0.121(如下表2所示)。
表2 CPW1的单糖组成
(3)键接方式:CPW1经完全水解和甲基化后经气相色谱-质谱联用分析(如下表3所示),以及一维和二维核磁共振分析(如下表4所示),可知CPW1是β-D-呋喃果糖残基以β-(2→1)-糖苷键连接而成、端基是α-D-Glcp-1键接的直链均一多糖。
表3 CPW1甲基化糖醇乙酰酯(PMAA)结果分析
表4 CPW1的氢谱和碳谱化学位移归属
具体操作如下:
样品经甲基化、水解、乙酰化后,经GC-MS测定并与标准质谱图库进行比对。称量多糖样品(5-15mg)置于玻璃反应瓶中,加入1mL无水DMSO,快速加入甲基化试剂A液,封闭,在超声作用下溶解,再加入甲基化试剂B液。在磁力搅拌水浴30℃反应60min反应。最后将2mL超纯水加入到上述混合物中终止甲基化反应。
取甲基化后的多糖,加入1ml的2M三氟乙酸(TFA)水解90min,旋转蒸发仪蒸干。残基加入2ml双蒸水,60mg硼氢化钠还原8小时,加入冰醋酸中和,旋蒸,101度烘箱烘干,然后加入1ml乙酸酐乙酰化100℃反应1h,冷却。然后加入3mL甲苯,减压浓缩蒸干,重复4次,以除去多余的醋酐。
将乙酰化后的产物用3mL CH2Cl2溶解后转移至分液漏斗,加入少量蒸馏水充分震荡后,除去上层水溶液,如此重复4次。CH2Cl2层以适量的无水硫酸钠干燥,定容10mL,放入液相小瓶。分析采用Shimadzu GCMS-QP 2010气相色谱-质谱联用仪测定乙酰化产物样品;
GC-MS条件:RXI-5SIL MS色谱柱30m*0.25mm*0.25um;程序升温条件为:起始温度120℃,以3℃/min升温至250℃/min;保持5min;进样口温度为250℃,检测器温度为250℃/min,载气为氦气,流速为1mL/min。
单糖组成结果表明该多糖为果糖和葡萄糖组成;而甲基化结果表明为呋喃甘露糖和呋喃葡萄糖。由于果糖为酮糖,在还原过程中异构化成甘露糖和葡萄糖。得到的甲基糖苷异构化成呋喃环的甘露糖苷和葡萄糖苷。因此,整合后的甲基化结果如上表3所示,链接方式主要为→1)-Fruf-(2→。
NMR实验步骤:称取多糖样品50mg,将其溶于0.5ml的重水中并冷冻干燥。随后将冻干粉再溶于0.5ml的重水中,并继续冷冻干燥,重复以上过程,以充分交换活泼氢。然后将样品溶于0.5ml的重水中,在室温25℃下置于以600MHz的核磁共振仪测定1H NMR谱、13C NMR谱、DEPT135一维图谱和二维图谱。
CPW1的1H和13C的化学位移归属如上表4所示。结合单糖组成和甲基化分析的结果可知CPW1可能是一种菊粉型果聚糖,而菊粉型果聚糖的一个特征就是在其端位存在葡萄糖残基。在CPW1的二维HMBC图谱(如图2)中的异头质子共振区存在一个强烈的交叉峰H1(α-D-Glcp-1)-C2(β-D-Fruf-2,1),表明存在α-D-Glcp-1→2-β-D-Fruf-1→。检测到H1a,1b(β-D-Fruf-2,1)-C2(β-D-Fruf-2,1)的交叉峰,这表明果糖残基是以2,1-linkage方式连接成链的。
最终确定:CPW1分子量为5623,是β-D-呋喃果糖残基以β-(2→1)-糖苷键连接而成、端基是α-D-葡萄糖-1键接的菊粉型果聚糖;
其化学结构式为α-D-Glcp-1→(2-β-D-Fruf-1)n→2-β-D-Fruf,其中n=35-36。
实施例3:
制备一种硒纳米粒子制剂CPW1-Se,具体步骤如下:
制备:将配置好的CPW1(1,5,10mg/ml,10ml)水溶液与亚硒酸钠水溶液(0.1M,0.2M,0.4M,0.4ml)混合,室温下连续搅拌4小时,将抗坏血酸(0.2M,0.4M,0.8M,0.8ml)水溶液逐滴加到溶液中,继续室温下搅拌24小时,透析,冻干。最优反应条件为党参多糖(5mg/ml,10ml)/亚硒酸钠(0.2M,0.4ml)/抗坏血酸(0.2M,0.8ml)。
表征和稳定性评价:透射电镜用来表征CPW1-Se的形貌表征(日本东京,Jeol,JEM-2010HR)。傅里叶变换红外(FT-IR)光谱(Nicolet 5700,Perkineller,Waltham,MA,USA)、X射线光电子能谱(XPS)(Kratos,XSAM800 X)用来表征CPW1和CPW1-Se粉末的基团变化。采用马尔文粒径仪(Malvern Instruments,Malvern,UK)测量CPW1-Se的Zeta电位和尺寸分布。采用电感耦合等离子体质谱(ICP-MS)分析法测定硒含量。
结果表明,在不添加CPW1的情况下,合成的硒纳米粒子(SeNPs)易于相互作用,然后聚集成大直径(300~500nm)的纳米颗粒,并且溶液呈红色且浑浊(图3)。然而,添加CPW1后,获得橙红色透明的溶液,并且随着CPW1浓度的增加,纳米粒子趋向于更加分散,这表明CPW1可以用来分散硒纳米粒子。随着CPW1浓度的变化,硒纳米粒子的平均粒径之间没有显著差异。然而,硒纳米粒子的平均尺寸随着亚硒酸钠浓度的增加而增加,从平均54nm增加到平均79nm,这表明可以通过调整亚硒酸钠的浓度来控制产生的硒纳米粒子的尺寸(如图4所示)。
对放置不同天数(0~15天)的CPW1-Se的大小和zeta电位结果(如图5、6所示)进行了评估,结果表明CPW1-Se在15天内保持相对稳定。此外,利用X射线光电子能谱(XPS)研究了CPW1和CPW1-Se的化合物价态。结果发现两者C1s峰类型几乎相同,而CPW1-Se的O1s结合能比CPW1增加,表明O原子参与了反应(如图7所示)。同时,在CPW1和CPW1-Se的傅里叶红外光谱中观察到的吸收带几乎相同,表明CPW1的骨架结构在SeNPs形成后保持不变。值得注意的是,O-H的拉伸振动从CPW1的3403cm-1降低到CPW1-Se的3378cm-1,表明硒纳米粒子优先通过Se-O键与OH相互作用(如图8所示)。
实施例4:
一种硒纳米粒子制剂CPW1-Se在抗肝癌活性中的应用。具体实施步骤如下:
取96孔板,向每孔接种5000个细胞,将细胞分为空白组、对照组和不同浓度给药组。空白组中加入培养基。细胞培养24h后,PBS清洗2次。空白组和对照组中加入培养基,给药组中分别加入含药培养基(CPW1-Se终浓度为5、10、20、40、60、80、100、200mg/mL;CPW1终浓度为5、10、20、40、60、80、100、200mg/mL;SeNPs终浓度为0.5、1、2、4、6、8、10、20mg/mL),每组设置4个复孔。分别培养24、48h后,吸取PBS到各孔中,充分清洗细胞,操作重复3次。每孔中加入含10μL CCK-8试剂的培养基,培养2h。打开酶标仪,设置测定波长为450nm,测定各孔吸光度值(A),计算各组细胞存活率[细胞存活率=(A给药组-A空白组)/(A对照组-A空白组)×100%]。
结果表明:在没有CPW1的情况下,SeNPs会聚集成大颗粒,从而导致对癌细胞和正常细胞的高毒性。而CPW1-Se以剂量依赖性方式显著抑制癌细胞增殖,尤其是对HepG2细胞(如图9、10所示)。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (10)
1.一种菊粉型党参果聚糖CPW1,其特征在于:该多糖是菊粉型果聚糖,分子量为5623,分子量为5623,是β-D-呋喃果糖残基以β-(2→1)-糖苷键连接而成、端基是α-D-葡萄糖-1键接的直链均一多糖。
2.根据权利要求1所述的一种菊粉型党参果聚糖CPW1的制备方法,其特征在于:包括以下步骤:
S1、脱脂脱色:将党参洗净粉粹后放入索氏提取器,回流收集样品备用;
S2、提取:将经步骤S1获得的产品置入盐水内搅拌、收集滤液备用;
S3、脱蛋白:将步骤S2所得滤液浓缩,用SEVERAGE法除蛋白后脱色后获得党参粗多糖;
S4、极性分离:将步骤S3获得的党参粗多糖用DEAE Sepharose Fast Flow色谱柱进行极性分离,其中采用四组溶剂洗脱以获得四组洗脱组分,命名为CPW、CPS0.2、CPS0.5、CPS1;
S5、色谱柱纯化:将步骤S4中得到的水洗组分CPW通过色谱柱进一步纯化获得均一多糖,命名为CPW1。
3.根据权利要求2所述的一种菊粉型党参果聚糖CPW1的制备方法,其特征在于:步骤S1中,需将粉粹后的党参用100-200目纱布的样品袋装载后放入索氏提取器,且使用乙酸乙酯和丙酮分别回流2~3小时后收集。
4.根据权利要求2所述的一种菊粉型党参果聚糖CPW1的制备方法,其特征在于:步骤S2中,盐水为0.9%氯化钠配置的盐水,且于80-90℃搅拌4h后收集滤液,并将剩余残渣重复步骤S2前述步骤2-3次后将所得滤液收集。
5.根据权利要求2所述的一种菊粉型党参果聚糖CPW1的制备方法,其特征在于:步骤S3中,除蛋白的具体操作是
将滤液:氯仿:正丁醇按体积比25:5:1混匀后,快速搅拌30分钟,离心,收集上清液备用;
步骤S3中,脱色的具体步骤是:
在除蛋白后的上清液中滴加30%双氧水,保持pH=8-9之间,直到溶液变为浅黄色,将溶液对水透析7-10天后,浓缩,冻干,得党参粗多糖。
6.根据权利要求2所述的一种菊粉型党参果聚糖CPW1的制备方法,其特征在于:步骤S4中,极性分离的具体步骤是:
S41、DEAE Sepharose Fast Flow填料先用0.5mol/L的盐酸浸泡1h,然后洗去填料中的杂质物,用4-5倍体积的蒸馏水洗脱至中性;
S42、调整流速为5mL/min,蒸馏水平衡2h后用蒸馏水溶解粗多糖、加热、斡旋,12000rpm离心,取上清液上样;调整流速为15mL/min的蒸馏水进行洗脱;
S43、四组溶剂洗脱:三倍柱体积的水、0.2M NaCl、0.5M NaCl、1.0M NaCl洗脱;采用苯酚硫酸法进行追踪检测,酶标仪490nm进行检测,并根据绘制散点图峰形,分别收集,浓缩,透析,冷冻干燥。以得到四组洗脱组分,命名为CPW、CPS0.2、CPS0.5、CPS1。
7.一种硒纳米粒子制剂CPW1-Se,其特征在于:是由如权利要求1所述的CPW1和硒纳米粒子SeNPs复合而成的制剂,为一种无定型态非晶体。
8.根据权利要求7所述一种硒纳米粒子制剂CPW1-Se,其特征在于:CPW1分子链上的羟基和SeNPs上的硒原子以Se-O键螯合,使得硒纳米粒子制剂CPW1-Se不会聚集,可稳定分布于水中至少15天。
9.根据权利要求7所述一种硒纳米粒子制剂CPW1-Se,其特征在于:硒纳米粒子制剂CPW1-Se的尺寸,可以通过控制亚硒酸钠的浓度控制其粒径大小在20-110nm内。
10.根据权利要求7-9任一所述一种硒纳米粒子制剂CPW1-Se在抗肝癌领域中的应用。
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