CN112341549A - 一种罗汉松实多糖的组成及其用途 - Google Patents
一种罗汉松实多糖的组成及其用途 Download PDFInfo
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Abstract
本发明涉及一种罗汉松实多糖的组成及其在免疫调节功能方面的用途。罗汉松实多糖是从罗汉松或短叶罗汉松的花托中分离纯化获得的多糖。该多糖重均分子量为6018Da，数均分子量为6690Da；属于杂多糖，由甘露糖、葡萄糖和半乳糖组成(摩尔比为21.26∶73.96∶4.78)；连接方式为α‑和β‑构型的糖苷键。本发明所述的罗汉松实多糖能促进小鼠脾细胞中T、B淋巴细胞的增殖，从而促进机体的细胞免疫和体液免疫反应，提高机体免疫力；可通过调节血清细胞因子IL‑2、IL‑6、TNF‑α以及INF‑γ的表达，改善机体免疫功能。罗汉松实多糖可用于制备食品或制备增强免疫力的保健食品或药品。
Description
技术领域
本发明属于医药技术和保健食品领域，具体涉及罗汉松实多糖的组成及其增强免疫力的用途。
背景技术
罗汉松实多糖是一种从罗汉松Podocarpus macrophyllus(Thunb.)D.Don或短叶罗汉松Podocarpus macrophyllus(Thunb.)D.Don var.maki(Sieb.)Endl.的花托中提取分离纯化得到的天然活性多糖。有文献报道，罗汉松实多糖在罗汉松花托鲜品中的含量约为0.5％。来源于植物的多糖大多具有较好的生物活性，已报道的生物活性包括：免疫调节、抗肿瘤、降血脂、抗氧化、治疗动脉粥样硬化、治疗阿尔茨海默病等。如，有文献报道，香菇多糖具有抗肿瘤作用；金樱子多糖具有抗病毒作用；悬钩子木多糖具有免疫调节活性；连钱草多糖具有抗补体活性。尽管植物多糖的研究报道较多，但关于罗汉松实多糖的报道却极少。在发明专利方面，国内已公开的罗汉松实多糖的发明专利只有发明者黄增琼(ZL201910050934.9)公开了罗汉松实多糖的制备方法及其在抗肝癌方面的用途。此外，还有三件与罗汉松实相关的发明专利：
黄增琼(CN201610460262)公开了一种从罗汉松种子高效提取分离高纯度川芎嗪的方法；
张明洞(CN201110095608)公开了一种罗汉松实酒的制备方法；
张明洞(CN201110095609)公开了一种罗汉松实提取物及制品的生产方法，以及提取浓缩液在食品领域的应用；
以上发明专利都没有公开罗汉松实多糖的组成及其增强免疫力的用途。
除此之外，还有一些以罗汉松实药材作为中药复方组分，用于治疗各种疾病的中药复方专利申请。这些报道，都没有涉及罗汉松实多糖的组成，也没有公开罗汉松实多糖的免疫增强或免疫调节活性，及其在增强免疫力方面的用途。
虽然已有的报道证实了来源于植物的多糖具有多方面的生物活性，然而，由于多糖的来源不同，提取分离纯化方法不同，所获得多糖的分子量、单糖组成、单糖连接方式等也不相同。这些差异决定了多糖不一定都具有生物活性；也不一定都具有相似的生物活性；不同来源的多糖，即使具有相似的生物活性，其作用强度也可能不同；不同的多糖对生物体的毒性作用也是不相同的。因此，对于罗汉松实多糖而言，其是否具有免疫调节功能和增强免疫力的作用，作用效果如何，本领域专业人员是无法预知的。
发明内容
本发明的目的在于提供罗汉松实多糖的组成及其在调节免疫功能方面的用途。
本发明提供了一种罗汉松实多糖在制备食品或增强免疫力的保健食品或药品中的用途。
本发明所述的罗汉松实多糖是从罗汉松科罗汉松属植物罗汉松Podocarpusmacrophyllus (Thunb.)D.Don或短叶罗汉松Podocarpus macrophyllus(Thunb.)D.Donvar.maki(Sieb.)Endl.的花托中提取分离纯化获得。
所述的罗汉松实多糖具有如下特征：
(1)重均分子量为6018Da，数均分子量为6690Da；
(2)属于杂多糖，由甘露糖、葡萄糖和半乳糖组成，这三种单糖的摩尔比为21.26∶73.96∶4.78；连接方式为α-和β-构型的糖苷键。
根据本发明的另一方面，本发明提供了罗汉松实多糖在饮料、方便食品、糖果制品或特殊膳食食品方面的用途；以及在制备增强免疫力的保健食品或药品方面的用途。
本发明罗汉松实多糖，经动物实验证实，其能促进脾细胞中T、B淋巴细胞的增殖，从而促进机体的细胞免疫和体液免疫反应，提高机体免疫力；可通过调节血清细胞因子IL-2、IL-6、TNF-α以及INF-γ的表达，从而改善机体的免疫功能。
下面将结合具体实施例对本发明的上述内容再作进一步的详细说明。但不应该将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1罗汉松实多糖凝胶渗透色谱图
图2混合单糖标准品高效液相色谱图
图3罗汉松实多糖单糖组成分析高效液相色谱图
图4罗汉松实多糖1H-核磁共振谱图
图5罗汉松实多糖13C-核磁共振谱图
具体实施方式
实施例1
罗汉松实多糖分子量测定
采用凝胶渗透色谱(GPC)法测定罗汉松实多糖分子量。
将不同相对分子质量的葡聚糖标准品和罗汉松实多糖用磷酸二氢钾(KH2PO4)流动相配制成2.0mg/ml的标准品溶液和样品待测液，用0.45μm滤膜过滤，分别进样检测。色谱条件：色谱柱：TSK G-5000 PWXL柱(7.8×300mm)和TSK G-3000 PWXL柱(7.8×300mm)串联；流动相：0.02mol/L KH2PO4缓冲溶液；流速：0.6mL/min；进样量：10μL；Waters2414示差检测器；柱温35℃；测定时间：35min。记录图谱。用Breeze GPC软件分析结果，以葡聚糖标准品的相对分子质量分数(logMw)为纵坐标，洗脱体积(V)为横坐标，制作标准曲线。根据标准曲线计算多糖样品等分子量。
结果，葡聚糖标准品的相对分子质量分数(Log MW)对洗脱体积(V)回归处理的标准曲线方程为：Log MW＝11.23e-0.98eV+6.23e-1V27.67e-2V3+8.67e-3V4
图1为罗汉松实多糖的GPC图。经Breeze GPC软件分析，罗汉松实多糖的重均分子量为6018Da及数均分子量为6690Da。
实施例2
罗汉松实多糖组成分析
单糖组成测定：采用PMP柱前衍生化-HPLC法进行测定。
精密称取罗汉松实多糖10mg于20mL的钳口瓶中，加入5mL的2mol/L三氟乙酸(TFA)，充入氮气封管(10L/min，1min)。于100℃烘箱中水解反应120min；冷却后加入1mL甲醇，置于70℃水浴中，通入氮气吹干，重复两次以上操作，使完全除去TFA；用1mL 0.3mol/LNaOH溶液溶解上一步产物，得多糖水解液。
分别精密量取400μL的混合单糖标准液和多糖水解液于5mL的具塞试管，再加入400μLPMP甲醇，充分混合，在70℃水浴条件下反应120min，后取出降至室温；后加入400μL0.3mol/L的HCl进行中和，调节pH至6～7；随后加入1200μL纯水和1200μL氯仿，充分振摇混匀，静置待分层，弃去氯仿相，重复以上步骤两次。水相用0.45μm滤膜过滤用于HPLC进样分析。色谱条件：色谱柱C18柱(250mm×4.6mm×5μm)；流动相A：100mM磷酸钠缓冲液(pH＝6.4)；流动相B：乙腈；检测波长：250nm；柱温30℃；流速1ml/min；进样量20μL。
结果如图2和3所示，图2显示了13种混合单糖标准品的高效液相色谱图；图3显示了3种单糖的色谱峰，分别为甘露糖、葡萄糖和半乳糖。根据色谱峰的峰面积，及各单糖相对分子量，三种单糖的摩尔比为：21.26∶73.96∶4.78，组分中不含糖醛酸，说明罗汉松实多糖是中性杂多糖。
实施例3
罗汉松实多糖核磁共振波谱分析
取真空干燥后的罗汉松实多糖40mg，用氘代二甲亚砜(DMSO)溶解，转移到核磁管中，用核磁共振波谱仪检测，得到罗汉松实多糖的1H-核磁共振谱图和13C-核磁共振谱图。
图4和5分别为1H-核磁共振谱图和13C-核磁共振谱图。图4显示在5.36ppm有一个质子信号峰，在小于5.0ppm处有丰富的质子信号峰，表明罗汉松实多糖主要含有β-型糖苷键，少量α-型糖苷键。质子共振峰出现在3.2ppm-3.65ppm非常小的区域。其中，位于δ5.36信号峰为α-D-甘露糖的异头氢，δ4.76归属为β-D-葡萄糖异头氢，δ4.67处，β-D-半乳糖异头氢信号峰最强。图5显示，在δ90-100ppm有极弱的β-构型异头碳信号，在δ100-110ppm有极弱的α-构型异头碳信号，表明罗汉松实多糖存在α-构型和β-构型糖苷键。此外，在δ82-84ppm的范围内无信号，结合单糖组成分析结果，表明罗汉松实多糖不含呋喃糖，但含有吡喃糖。在δ62-63ppm范围内的信号峰，结合单糖组成分析结果，表明含有半乳糖，在δ18ppm处无信号，结合单糖组成分析结果，表明不含鼠李糖。
实施例4
罗汉松实多糖对荷瘤小鼠脾细胞中T、B淋巴细胞增殖的影响
采用MTT法测定罗汉松实多糖对荷瘤小鼠脾组织中T、B淋巴细胞增殖能力。
将荷瘤小鼠随机分为：模型组、环磷酰胺组(CTX)、香菇多糖组(LNT)、罗汉松实多糖(LHSP)高(H)、中(M)、低(L)剂量组；另设正常对照组。正常组和模型组灌胃给予生理盐水，10ml/kg；CTX组20mg/kg；LNT组100mg/kg；LHSP高、中、低剂量组分别为480，240，120mg/kg/d；各组给药容量均为10ml/kg，每天1次，连续给药14天。给药结束后，各组小鼠按以下方法操作。
小鼠脾细胞悬浮液制备：无菌坏境下摘取小鼠脾脏，浸没在PBS中的200目纱布上，将脾脏磨碎，将滤液转移至15mL离心管中。1000rpm离心5min，弃去上清液，加入2mL红细胞裂解液，吹打1min后加入4mLPBS平衡，混匀后离心，得到脾细胞沉淀。用RPMI-1640培养基(含10％胎牛血清)吹打悬浮，使脾细胞完全分散，将脾细胞浓度调整至1.0×107个/ml，待用。
小鼠脾细胞增殖：将脾细胞悬浮液分别加到两块96孔板中，每孔装入100μL脾细胞悬浮液。其中，一板加入浓度为5μg/mL的刀豆蛋白A(ConA)溶液，测定T细胞增殖能力；另一板加入浓度为10μg/mL的细菌脂多糖(LPS)溶液，测定B细胞增殖能力。将细胞板放在温度为37℃、饱和湿度为5％的CO2恒温培养箱中培养3天。后加入20μL 5mg/mLMTT溶液，继续避光培养4h，最后加入MTT Formazan溶解液终止反应，酶标仪570nm处测定光密度值(OD)。
结果如表1所示，与正常组相比，罗汉松实多糖各剂量组T、B淋巴细胞增殖能力显著升高(P＜0.05或P＜0.01)；模型组相比，罗汉松实多糖各剂量组B淋巴细胞增殖能力显著升高(P＜0.05)，T细胞增殖能力无明显差异(P＞0.05)；与环磷酰胺组比较，香菇多糖组、罗汉松实多糖各剂量组T、B淋巴细胞增殖能力均显著升高(P＜0.05或P＜0.01)；与香菇多糖组比较，各指标均无显著性差异(P＞0.05)。
表明，罗汉松实多糖各剂量组均能促进脾细胞中T、B淋巴细胞的增殖，从而促进机体的细胞免疫和体液免疫反应。
表1 罗汉松实多糖对荷瘤小鼠脾细胞中T、B淋巴细胞增殖能力的影响
注：与空白组比较，*P＜0.05，**P＜0.01；与模型组相比较，#P＜0.05，##P＜0.01；
与CTX组比较，aP＜0.05，aaP＜0.01；与LNT组比较，bP＜0.05，bbP＜0.01。
实施例5
罗汉松实多糖对荷瘤小鼠血清细胞因子IL-2、IL-6、TNF-α、INF-γ的影响
将荷瘤小鼠随机分为：模型组、环磷酰胺组(CTX)、香菇多糖组(LNT)、罗汉松实多糖(LHSP)高(H)、中(M)、低(L)剂量组；另设正常对照组。正常组和模型组灌胃给予生理盐水，10ml/kg；CTX组20mg/kg；LNT组100mg/kg；LHSP高、中、低剂量组分别为480，240，120mg/kg/d；各组给药容量均为10ml/kg，每天1次，连续给药14天。给药结束后，各组小鼠摘眼球取血，收集血液，静置3～4h后于4℃、3000rpm/离心10min，取上层血清备用。采用ELISA法，测定各组小鼠血清中IL-2、IL-6、TNF-α、INF-γ的含量。
结果如表2所示。与空白组比较，模型组小鼠血清中IL-6含量显著提高(P＜0.05)，IL-2、TNF-α、INF-γ含量显著降低(P＜0.05)；与模型组比较，罗汉松实多糖各剂量组血清IL-2、TNF-α和INF-γ均明显升高(P＜0.01)，且呈剂量依赖性；罗汉松实多糖各剂量组IL-6无显著差异(P＞0.05)；与环磷酰胺组比较，罗汉松实多糖各剂量组IL-2、TNF-α和INF-γ均明显升高(P＜0.05，P＜0.01)，且呈剂量依赖性；IL-6水平无显著差异(P＞0.05)；与香茹多糖组相比，罗汉松实多糖各剂量组IL-2明显升高(P＜0.05)，IL-6、TNF-α和INF-γ均无显著差异(P＞0.05)。
以上结果表明，罗汉松实多糖各剂量组、香菇多糖组均明显升高荷瘤小鼠血清中IL-2、TNF-α及INF-γ细胞因子的水平，降低IL-6细胞因子的表达。证实罗汉松实多糖可通过调节血清细胞因子IL-2、IL-6、TNF-α以及INF-γ的表达，从而改善机体的免疫功能。
表2 罗汉松实多糖对荷瘤小鼠血清IL-2、IL-6、TNF-α、INF-γ影响
注：与空白组比较，*P＜0.05，**P＜0.01；与模型组相比较，#P＜0.05，##P＜001；
与CTX组比较，aP＜0.05，aaP＜0.01；与LNT组比较，bP＜0.05，bbP＜0.01。
实施例6
罗汉松实多糖饮料的制备
取罗汉松实多糖50g，蜂蜜10g，柠檬酸3g，维生素C2g，橙味香精3g，由梨酸钾0.1g，加纯净水200mL溶解，混匀，加纯净水至500mL，巴氏杀菌，灌装。
实施例7
罗汉松实多糖黑米糊的制备
取黑米粉200g，无糖奶粉100g，混合均匀；另取罗汉松实多糖100g，加500mL纯净水溶解，得罗汉松实多糖溶液；将罗汉松实多糖溶液加入已混合均匀的黑米粉和无糖奶粉中，混合均匀，烘干，粉碎，过200目筛，110℃瞬间杀菌，包装，得罗汉松实多糖黑米糊。
Claims (4)
1.一种具有免疫调节功能的罗汉松实多糖，其特征在于：
(1)所述的多糖重均分子量为6018Da，数均分子量为6690Da；
(2)所述的多糖属于杂多糖，由甘露糖、葡萄糖和半乳糖组成，这三种单糖的摩尔比为21.26∶73.96∶4.78；连接方式为α-和β-构型的糖苷键。
2.一种权利要求1所述的具有免疫调节功能的罗汉松实多糖的用途，其特征在于：可用于制备食品。
3.一种权利要求1所述的具有免疫调节功能的罗汉松实多糖的用途，其特征在于：可用于制备增强免疫力的保健食品或药品。
4.根据权利要求2所述的用途，其特征在于：所述的食品为：饮料、方便食品、糖果制品或特殊膳食食品。
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| CN102199509A (zh) * | 2011-04-15 | 2011-09-28 | 张明洞 | 一种罗汉松实酒的制备方法 |
| CN102217755A (zh) * | 2011-04-15 | 2011-10-19 | 张明洞 | 罗汉松实提取物及制品的生产方法 |
| CN109602759A (zh) * | 2019-01-17 | 2019-04-12 | 广西医科大学 | 罗汉松实多糖的用途 |
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| CN102199509A (zh) * | 2011-04-15 | 2011-09-28 | 张明洞 | 一种罗汉松实酒的制备方法 |
| CN102217755A (zh) * | 2011-04-15 | 2011-10-19 | 张明洞 | 罗汉松实提取物及制品的生产方法 |
| CN109602759A (zh) * | 2019-01-17 | 2019-04-12 | 广西医科大学 | 罗汉松实多糖的用途 |
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