CN112759661B - 金樱子多糖制备方法、鉴定方法和应用 - Google Patents
金樱子多糖制备方法、鉴定方法和应用 Download PDFInfo
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Abstract
本发明属于医药及保健食品技术领域，具体涉及金樱子多糖及其制备方法、鉴定方法和应用。本发明提供的金樱子多糖的相对分子量1.26×104Da。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金樱子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，本发明得到的金樱子多糖纯品为金樱子多糖药物、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，具体涉及金樱子多糖及其制备方法、鉴定方法和应用。
背景技术
免疫应答是针对入侵病原体的非特异性防御的第一线，这是生物体中的一种自我保护功能，可以调节免疫应答以治疗疾病。具有免疫刺激活性的多糖可以直接或间接与免疫系统相互作用，从而激活免疫系统以表现出生物学活性。肿瘤是世界上高发病率和高死亡率的疾病之一，它是由受损白细胞的高增殖引起的。免疫活性和抗肿瘤活性之间存在一定的关系。大多数多糖在体外和体内均具有免疫调节活性。天然多糖具有抗肿瘤活性和低毒性，可以克服耐药性，为开发抗肿瘤药物提供了丰富的资源。因此，寻找具有免疫活性和抗肿瘤作用的天然多糖具有重要意义。
多糖又称多聚糖，是一类广泛存在于动植物与微生物体内的生物大分子。对多糖的相关研究已有近70年的历史，尤其是近些年，糖生物学成为了继基因组学、蛋白质组学之后的又一大研究热点与难点，已有研究表明，多糖具有免疫调节、抗炎、抗肿瘤、降血糖、降血脂等多种生物活性，其中免疫调节作用是多糖类物质最重要的作用之一。我国在中药多糖的研究及开发利用方面近年来也取得很多成果，灵芝多糖、黄芪多糖和人参多糖等中药多糖产品已经通过分析鉴定投放市场，经过深加工的多糖保健品也相继出现。虽然中药多糖免疫调节作用的研究较多，但却不够深入。
金樱子属于蔷薇科，主要分布在中国南部。金樱子的根，花，果实和叶子均可入药。特别是，其果实被列为中国卫生部批准的新食品资源。金樱子果实具有多种药理作用，如抗氧化，降血脂，抗炎和镇痛作用等，其化学成分主要包括三萜类化合物，类固醇，酚类，单宁酸，有机酸和木聚糖。多糖是其主要活性成分之一。因此，期望将金樱子多糖开发为具有免疫调节和抗肿瘤活性的药物。
斑马鱼肿瘤移植模型已成为人类癌症转化研究的新模型，该模型可以显示出癌症生物学的重要迹象，例如肿瘤细胞增殖，转移和肿瘤抑制血管生成。斑马鱼体积小，产卵量大，胚胎透明，与人类基因的遗传相似性为87％。因此，该模型被广泛用于抗肿瘤药物和疗法的早期筛选，并可以准确地测试肿瘤特异性免疫疗法的效果，从而改善了新的抗癌疗法的发展。在天然多糖抗肿瘤活性筛选实验中，斑马鱼肿瘤移植实验所需的多糖含量较少，筛选周期短，能准确反映生物活性，为今后的肿瘤活性筛选提供了新的模式。
发明内容
为了解决现有技术存在的问题，本发明提供了金樱子多糖及其制备方法、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金樱子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，本发明得到的金樱子多糖纯品为金樱子多糖药物、质量控制及深入研究其构效关系和作用机制奠定基础。
本发明提供了一种金樱子多糖RL1-2。发明人通过对金樱子多糖粗品提纯分离得到金樱子多糖RL1-2，且经结构分析得到其绝对分子量为1.26×104g/mol。
进一步地，所述金樱子多糖RL1-2的结构如图9所示.
同时，本发明也提供了金樱子多糖的制备方法，包括以下步骤：
S1水提：将金樱子果实加入水中，加热提取，过滤，得提取液和药渣；
S2分级醇沉：
将步骤S1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RL1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RL2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RL3；
其中10≤a＜b＜c＜100；
S3纯化：
一次纯化：
将步骤S2所得粗多糖RL1进行除蛋白，透析，冻干，得金樱子多糖；
二次纯化：
将一次纯化后的多糖RL1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RL1-2。
本发明采用水提与分级醇沉法相结合，乙醇浓度由低到高进行分级醇沉，对金樱子多糖进行初步分离，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S1中水的添加量为所述金樱子果实重量的5～15倍，加热温度为60～100℃，提取时间1～10h。
进一步地，所述步骤S2中减压浓缩温度为40～70℃，静置的时间为10～28h。
另外，本发明还提供了两种金樱子多糖的鉴定方法，包括以下步骤：
(1)取金樱子多糖样品，完全酸水解，水解产物液相色谱检测；
(2)取金樱子多糖样品，完全酸水解，水解产物衍生化进行D/L构型分析；
(3)取金樱子多糖样品，干燥，压片，红外光谱检测；
(4)取金樱子多糖样品，甲基化，水解、还原，乙酰化，进行GC-MS检测；
(5)取金樱子多糖样品溶于D2O，进行核磁共振分析。
本发明人为了进一步开发金樱子这一宝贵资源，发掘新药源，通过大量实验研究得到本发明：以金樱子果实为原料，采用水提醇沉法分离得到粗多糖，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金樱子粗多糖，首次制备出金樱子多糖纯品，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，成功得出了金樱子多糖的结构信息，其中金樱子多糖RL1-2为水溶性杂多糖，主链由(1→5)-α-L-Araf,(1→2,5)-α-L-Araf,(1→3,5)-α-L-Araf,(1→4)-α-D-Glcp,(1→6)-α-D-Glcp,(1→3,6)-β-D-Glcp,(1→4)-α-D-Galp,(1→6)-α-L-Galp,(1→4)-α-L-Galp,(1→6)-β-D-Galp，和(1→2)-β-D-Xylp组成，支链由(1→5)-α-L-Araf,(1→6)-β-D-Galp,末端由α-L-arabinose和β-D-mannose组成。本发明还涉及了金樱子多糖在制备免疫调节药物或抗肿瘤中的应用。并通过细胞实验和斑马鱼实验研究对其免疫调节活性和抗肿瘤进行评价。
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，对金樱子多糖进行初步分离，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金樱子粗多糖进行二次分离纯化，效果显著，首次制备出金樱子多糖纯品；
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.本发明得到的金樱子多糖纯品，组分保存完好，结构明确，质量可控，可增强RAW264.7巨噬细胞的吞噬能力，促进一氧化氮(NO)，白介素(IL)-6，IL-1β，肿瘤坏死因子(TNF)-α等分子及炎症因子的释放，同时能提高斑马鱼胚胎中NO和活性氧(ROS)等分子的释放，为金樱子多糖在医药、保健品等领域的应用提供依据。此外，在斑马鱼异种移植模型中通过抑制肿瘤细胞的增殖和迁移而表现出体内抗肿瘤作用，这可能成为开发新抗肿瘤药物的潜在资源。
5.同时，本发明为金樱子多糖药物、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RL1-2单糖组成的高效液相色谱图；
图2为RL1-2单糖D/L构的高效液相色谱图；
图3为RL1-2的红外图谱；
图4为RL1-2的1H NMR图谱；
图5为RL1-2的13C NMR图谱；
图6为RL1-2的HSQC图谱；
图7为RL1-2的HMBC图谱；
图8为RL1-2的1H-1H COSY图谱；
图9为RL1-2的一级结构；
图10为RL1-2对RAW264.7细胞的免疫调节作用；
图11为RL1-2对斑马鱼胚胎中ROS、NO释放的影响；
图12为RL1-2对斑马鱼肿瘤种植模型中的体内抗肿瘤作用；
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金樱子多糖的制备方法
所述金樱子多糖的制备方法包括以下步骤：
S1水提：向金樱子果实中加入其重量10倍的水，加热至80℃提取，提取3h，收集提取液，药渣晾干，得提取液和药渣；
S2分级醇沉：
将步骤S1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RL1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24h后，离心，收集沉淀和上清液，得粗多糖RL2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24h后，离心，收集沉淀和上清液，得粗多糖RL3；
S4纯化：利用Sevag法分别将步骤S2所得RL1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3000Da)进行透析、冻干，得金樱子多糖RL1。
实施例2金樱子多糖RL1
所述金樱子多糖RL1-2是由实施例1所得金樱子多糖RL1二次纯化所得，具体方法如下：
1)离子交换柱层析：取200mg实施例1所得金樱子多糖RL1，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一个峰，其中洗脱峰为0.05M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RL1；
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，得金樱子多糖RL1-2
实施例3金樱子多糖RL1-2的结构分析(一)试验材料：金樱子多糖RL1-2。
(二)试验方法：
1.单糖组成分析
样品处理：
精确称取金樱子多糖样品各4.0mg试验材料于具塞试管中，加入2M三氟乙酸(TFA)2.0mL，置于油浴锅中在120℃油浴水解6h。冷却至室温，重复加甲醇旋干，去除TFA，用去离子水溶解至1mL，离心，各吸取100μL样品溶液，加入100μL 0.3M的NaOH溶液，再加入100μL0.5M PMP甲醇溶液，混匀，70℃水浴锅上反应30min，冷却，加入105μL 0.3M的HCL溶液中和，加去离子水至1mL，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0.45μm滤膜过滤后供HPLC进样分析。
色谱条件：
色谱柱：Kromasil 100-5-C18，4.6×250mm，5μm；流动相：0.1M磷酸盐(pH＝6.9)缓冲液-乙腈(v/v为83：17)；检测波长：250nm；流速：1mL/min；进样体积：20μL。
2.单糖D/L构型分析
金樱子多糖加入三氟乙酸(2M，2mL)，120℃下油浴反应4h。反应结束后，加等体积氯仿萃取两遍，弃去氯仿层，水层旋干。
样品旋干后，加入左旋半胱氨酸甲酯盐酸盐(L-cysteine methyl ester,2.5mg)、无水吡啶(1mL)后，60℃水浴反应1h。再加入邻苯基异硫代氰酸酯(O-tolylisothiocyanate,5μL)，继续60℃水浴反应1h。反应完毕，样品过0.45μm微孔滤膜后，进液相分析。
色谱条件：
流动相：25％CH3CN-H2O(0.1％乙酸)；流速：0.8mL/min；进样量：10μL；检测波长：UV250nm；检测时间：60min。
3.红外光谱检测
将干燥的试验材料2.0mg与KBr研磨后，压片，用Perkin EImer FT/IR-100在4000-400cm-1范围内进行扫描。
4.甲基化/GC-MS分析
称取干燥的试验材料8.0mg于反应瓶中，加入无水DMSO 8mL，再加入干燥的氢氧化钠800mg，超声30min，冰浴条件下，避光加入碘甲烷3.0mL，分三次加入，每次冰浴超声30min，反应结束后加入2mL的蒸馏水分解残留的碘甲烷，并加入1mL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2mol/L的TFA在120℃恒温油浴水解6h，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20mg的NaBH4在40℃下反应30min还原水解产物。再使用100μL的冰醋酸终止反应，样品在低压下旋干，然后加入2mL的乙酰酐和吡啶用来乙酰化。保持95℃磁力搅拌反应2h。然后反复加甲醇3次，旋干，用1mL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。
5.核磁共振分析
取金樱子多糖样品RL1-2反复多次冻干后，取60mg溶于0.6mLD2O中，置于核磁管中，用400MHz核磁共振仪BrukerAV-400记录其1HNMR，13C NMR，HSQC，HMBC等图谱。
(三)试验结果：
1.金樱子多糖RL1-2结构分析
(1)单糖组成分析
如图1所示，由HPLC图谱可知，RL1-2有甘露糖，葡萄糖，半乳糖，木糖和阿拉伯糖组成。(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)单糖D/L构型分析
如图2所示，由HPLC图谱可知，RL1-2中的甘露糖，葡萄糖，半乳糖，木糖构型为D构型，阿拉伯糖的构型为L构型。
(2)红外光谱分析
如图3所示，由RL1-2红外光谱图可知，金樱子RL1-2含有多糖的红外特征吸收峰。
(3)甲基化/GC-MS分析
RL1-2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，RL1-2含有t-L-Araf，t-D-Manp，(1→2,5)-L-Araf,(1→3,5)-L-Araf，(1→3,6)-D-Glcp，(1→5)-L-Araf，(1→4)-D-Glcp，(1→6)-D-Glcp，(1→4)-D-Galp，(1→6)-D-Galp和(1→4)-D-Xylp等糖残基。
(4)核磁共振分析
本试验通过1H NMR、13C NMR、HSQC对RL1-2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4-7为RL1-2的1HNMR、13C NMR、HSQC和HMBC图谱。
根据图4-7的核磁图谱，RL1-2碳氢归属如下表1所示。
表1 RL1-2核磁共振分析结果
aUnresolved from other signals，nd：no detected.
综上所述：RL1-2有甘露糖，葡萄糖，半乳糖，木糖和阿拉伯糖组成，从甲基化分析说明其含有t-L-Araf，t-D-Manp，(1→2,5)-L-Araf,(1→3,5)-L-Araf，(1→3,6)-D-Glcp，(1→5)-L-Araf，(1→4)-D-Glcp，(1→6)-D-Glcp，(1→4)-D-Galp，(1→6)-D-Galp和(1→4)-D-Xylp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RL1-2的结构如图8所示。
实施例4金樱子多糖纯品对巨噬细胞的免疫调节作用研究
(一)试验材料：金樱子多糖RL1-2。
(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RAW 264.7模型，设置空白组,阳性药组及金樱子多糖给药组，其中金樱子多糖RL1-2的浓度为50μg/mL，100μg/mL和200μg/mL，阳性药为脂多糖(LPS)，浓度为1μg/mL。
2.母液的配置及细胞计数
取RL1-2样品20mg，溶于1ml的PBS缓冲盐中，将配制完的母液过0.22μm的滤膜并且紫外照射30min灭菌，然后置于-20℃冰箱中待用。
将培养瓶置于超净工作台中，移除旧培养基，加入5mL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600μL胰蛋白酶，消化RAW 264.7细胞1min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20μL上述单细胞悬液加入180μLDMEM培养基中，反复吹打后取10μL于细胞计数板中计数，将细胞稀释为5×104个/mL，铺板。
3.给药
将RAW264.7细胞接种到96孔板，培养24h后加入待测样品，RL1-2给药浓度分别为50μg/mL，100μg/mL和200μg/mL,给药后的RAW264.7细胞置于CO2培养箱中培养24h。
4.MTT实验
移除96孔板中的培养液，然后每孔加入5mg/mL的噻唑蓝(MTT)溶液20μL，于CO2培养箱中培养4h。4h后移除96孔板中的MTT溶液，每孔加入150μL二甲基亚砜(分析纯)溶解甲瓒结晶，用酶标仪检测492nm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。
5.中性红吞噬实验
RAW264.7细胞经不同浓度的RL1-2给药处理后经PBS洗涤三次后加入100μL的中性红溶液(0.1％)孵育1h，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)150μL，室温下孵育1h，用酶标仪于540nm处测定OD值，实验重复三次。
6.NO释放测试
RAW264.7细胞接种于96孔板，4h后用不同浓度的RL1-2(50μg/mL，100μg/mL和200μg/mL)及1μg/mL LPS处理24h后，收集上清液并与Griess试剂反应，于550nm处用酶标仪测定OD值。
7.RL1-2对RAW264.7细胞炎症因子的影响
RAW264.7细胞接种于24孔板孵育4h,(50μg/mL，100μg/mL和200μg/mL)RL1-2及1μg/mL LPS处理24h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
如图9所示，经不同浓度的金樱子多糖RL1-2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),表明金樱子多糖RL1-2在50μg/mL-200μg/mL安全且无细胞毒性。其次，RAW264.7细胞经不同浓度的RL1-2处理后，其吞噬活性显著性增加(P<0.05或P<0.01)及NO,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)，表明金樱子多糖RL1-2能通过提高机体巨噬细胞的吞噬能力，增加NO释放及促进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。
实施例5金樱子多糖纯品RL1-2对斑马鱼胚胎的免疫调节作用研究(一)实验方法
1.斑马鱼胚胎收集与培养
控制斑马鱼成鱼日夜节奏，昼夜时间控制在14h:10h，收集斑马鱼受精卵，置于培养皿中，加入培养基(0.2％的速溶海盐溶于去离子水中)，置于恒温培养箱中培养，培养温度问28.5℃。
2.实验设计与分组
本发明采用斑马鱼胚胎模型，建立一种快速，高效的免疫活性筛选模型。将受精后7-8小时后的斑马鱼胚胎置于12孔板中，每孔6条斑马鱼幼鱼，用不同浓度的RL1-2(50μg/mL,100μg/mL,200μg/mL)于28.5℃下培养24h。24h后，移去含有RL1-2的培养基，并更换定期新鲜的培养基，继续培养至斑马鱼幼鱼受精后72h，培养过程中斑马鱼禁食。
3.斑马鱼在体荧光进行成像观察
(1)活性氧(ROS)释放测定
斑马鱼幼鱼按上述方法培养至受精后72h，于培养基中加入ROS荧光探针DCF-DA(20μg/mL)并在黑暗下继续孵育1h。孵育结束后，养鱼用水反复冲洗斑马鱼幼鱼三次，随后用0.02％的三卡因溶液麻醉斑马鱼幼鱼并用3％的甲基纤维素进行固定。激光共聚焦显微镜下观察斑马鱼幼鱼体内的相对荧光强度并拍照。使用Image J软件进行斑马鱼幼鱼体内相对荧光强度进行定量分析，以此来检测RL1-2对斑马鱼体内ROS释放量的影响。
(2)一氧化氮(NO)释放测定
斑马鱼幼鱼按上述方法培养至受精后72h，于培养基中加入NO荧光探针DAF-FMDA(5μM)并在黑暗下继续孵育2h。孵育结束后，养鱼用水反复冲洗斑马鱼幼鱼三次，随后用0.02％的三卡因溶液麻醉斑马鱼幼鱼并用3％的甲基纤维素进行固定。激光共聚焦显微镜下观察斑马鱼幼鱼体内的相对荧光强度并拍照。使用Image J软件进行斑马鱼幼鱼体内相对荧光强度进行定量分析，以此来检测RL1-2对斑马鱼体内NO释放量的影响。
(二)实验结果
与对照组相比，RLP50-2处理组中ROS和NO的产生显着增加。具体而言，在不同浓度的RLP50-2下，ROS的产量分别比对照高64％，146％和283％。同时，RLP50-2可以显着促进斑马鱼体内NO的产生，分别是对照组的2.0倍，2.9倍和4.1倍。以上结果表明RLP50-2可以通过增加体内ROS和NO的产生来实现其免疫调节作用。
实施例6金樱子多糖纯品RL1-2对斑马鱼胚胎肿瘤移植模型抗肿瘤作用研究(一)实验方法
使用斑马鱼模型的体内抗肿瘤测定
具体而言，斑马鱼受精(dpf)后两天，胚胎用于建立异种移植肿瘤模型。微量注射之前，在室温下跟踪K562细胞并用细胞跟踪仪CM-Dil标记至终浓度2μM/L。然后将细胞悬浮于无FBS的培养基中，密度为1×107cell/mL。然后将标记的K562细胞微注射到麻醉后的胚胎的卵黄囊中。将胚胎在28.5℃下浸入含有不同浓度RLP50-2的培养基中48小时。受精后五天(dpf)，通过激光共聚焦显微镜和ImageJ软件观察到斑马鱼中K562细胞的增殖和迁移。红色荧光的密度和焦点的数量分别代表了体细胞K562细胞的增殖和迁移。所有涉及动物的过程均已获得南开大学动物保护委员会的许可。
(二)实验结果
使用斑马鱼异种移植模型，通过注射人慢性髓细胞白血病K562，检查了RLP50-2在体内阻断肿瘤细胞的增殖和迁移。用RLP50-2处理显着降低了红色荧光的强度和焦点，结果表明RLP50-2可以剂量依赖的方式抑制斑马鱼中肿瘤细胞的增殖和迁移。此外，与阳性对照(依托泊苷)相比，RLP50-2在100和300μg/mL时更有效。
综上所述，本发明制备的金樱子多糖纯品RL1-2均能通过增加RAW246.7细胞的吞噬能力和促进相关炎症因子的释放，能增加斑马鱼幼鱼体内ROS和NO的释放，从而增强机体的免疫调节能力。此外通过抑制斑马鱼肿瘤移植模型中的肿瘤细胞增殖和迁移而显示出体内抗肿瘤作用，这可能成为开发新的抗肿瘤药物的潜在资源。
Claims (5)
1.一种金樱子多糖RL1-2，其特征在于，所述金樱子多糖RL1-2的结构如下所示：
2.如权利要求1所述的金樱子多糖RL1-2的制备方法，其特征在于，包括以下步骤：
S1水提：将金樱子果实加入水中，加热提取，过滤，得提取液和药渣；所述加热温度为60～100℃；
S2醇沉：将步骤S1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50％，静置，收集沉淀和上清液，得粗多糖RL1和上清液1；S3纯化：
一次纯化：
将步骤S2所得粗多糖RL1进行除蛋白，透析，冻干，得金樱子多糖；
二次纯化：
将一次纯化后的多糖RL1进行DEAE-Sepharose FF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，将洗脱峰为0.05M的NaCl洗脱部分浓缩、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Sephadex G-75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RL1-2。
3.如权利要求2所述的金樱子多糖RL1-2的制备方法，其特征在于，所述步骤S1中水的添加量为所述金樱子果实重量的5～15倍，提取时间1～10h。
4.如权利要求2所述的金樱子多糖RL1-2的制备方法，其特征在于，所述步骤S2中减压浓缩温度为40～70℃，静置的时间为10～28h。
5.如权利要求1所述的金樱子多糖RL1-2以及权利要求2-4所述的金樱子多糖RL1-2的制备方法制备得到的金樱子多糖RL1-2在制备增强免疫力药物或抗白血病药物中的应用。
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