CN117209621A - 一种沃柑多糖及其提取方法和应用 - Google Patents
一种沃柑多糖及其提取方法和应用 Download PDFInfo
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Abstract
本发明从沃柑果皮多糖中提取分离得到沃柑多糖,其分子量其分子量为50‑70kDa。其具有促进NK‑92MI细胞增殖、上调基因表达和提高细胞毒性方面的作用,可以用于制备促进NK‑92MI细胞增殖、上调基因表达和提高细胞毒性方面的药物。本发明制备得到的沃柑多糖还对Calu‑1肺癌细胞具有一定的抑制作用,可以用于制备抑制Calu‑1肺癌细胞的药物,大大提高沃柑果皮的利用价值。
Description
技术领域
本发明涉及食品资源化利用技术领域,具体涉及一种沃柑多糖及其提取方法和应用。
背景技术
柑橘是世界上最受欢迎的水果之一,仅橘子类的全球销售量每年就超过2900万吨。随着沃柑加工业的发展,产生出了越来越多的果皮。但它们大多被直接丢弃,从而给环境带来巨大压力,也浪费了资源。如何解决这一问题应该引起更多的关注和研究。
柑橘果皮是制备果胶等多糖的主要原料之一。由于其不同的特性,它们在食品、制药和化妆品行业得到了广泛的应用。目前对不同品种柑橘多糖的提取分离方法、结构鉴定、流变学性质分析、抗氧化活性、免疫功能等已经得到了大量研究,如专利申请号为CN201611238361.5公开的发明名称为“一种从柑橘皮中提取及纯化多糖的方法”,其主要通过如下步骤提取和纯化多糖:1)、柑橘皮多糖粗品的制备;2)、柑橘皮多糖粗品的提纯A、活性炭脱色、B、Sevag法脱蛋白。其制得的多糖声称具有一定的抗氧化活性,且产品纯度高、能耗低,高纯度的多糖在医药、保健品、食品等领域具有巨大的应用价值。但却很少有人研究沃柑中的多糖。而具有特定结构的多糖可以提高动物的免疫力,在开发功能性保健品方面具有巨大的优势和潜力。例如,一些多糖可以通过激活自然杀伤细胞(NK)来增强身体对入侵病毒的杀伤作用。当NK细胞被激活时,会释放出许多杀伤介质,如穿孔素和颗粒酶B。首先,穿孔素会在靶细胞膜上的形成孔道,然后颗粒酶B通过孔道进入,激活相关蛋白质,使其凋亡。本发明主要目的是研究沃柑果皮中的多糖是否具有激活NK细胞的特性,并对其结构进行鉴定和保护。在此基础上,进一步探讨多糖与NK细胞活性的量效关系,为提高沃柑果皮的高价值利用和产品开发提供参考数据。
发明内容
本发明的目的在于提供一种沃柑多糖及其提取方法和应用。
一种沃柑多糖,其从沃柑果皮中分离得到,其分子量为50-70kDa。
进一步的,所述沃柑多糖由如下方法制备得到:
S1:将沃柑果皮洗净后烘干,粉碎,用蒸馏水萃取,收集上清液;
S2:S1获得的上清液经浓缩后获得粘性液体,向粘性液体中加入无水乙醇进行醇沉收集沉淀物即为沃柑果皮粗多糖;
S3:将沃柑果皮粗多糖溶解于蒸馏水中配置成沃柑果皮粗多糖溶液,将沃柑果皮粗多糖溶液通过MinimatePall超滤系统,经过1000、500、300、100、70、50、30、10和5kDa的超滤膜后,将超滤得到的50-70kDa的多糖浓缩,再醇沉,将沉淀物冷冻干燥即获得所述沃柑多糖。
本发明的另一目的还在于保护上述一种沃柑多糖在制备促进NK-92MI细胞增殖、上调基因表达和提高细胞毒性方面的药物的应用。
本发明的另一目的还在于保护上述一种促进NK-92MI细胞增殖、上调基因表达和提高细胞毒性方面的药物,所述药物包含如上述的沃柑多糖。
本发明的另一目的还在于保护上述沃柑多糖在制备抑制Calu-1肺癌细胞药物中的应用。
本发明的另一目的还在于保护上述一种抑制Calu-1肺癌细胞药物,其包含上述沃柑多糖。
本发明与现有技术相比较具有以下有益效果:
本发明从沃柑果皮多糖中提取分离得到不同分子量的沃柑多糖,其中50-70kDa分子量的沃柑多糖具有促进NK-92MI细胞增殖、上调基因表达和提高细胞毒性方面的作用,可以用于制备促进NK-92MI细胞增殖、上调基因表达和提高细胞毒性方面的药物。本发明制备得到的沃柑多糖对Calu-1肺癌细胞具有一定的抑制作用,可以用于制备抑制Calu-1肺癌细胞的药物,提高沃柑果皮的利用价值。
附图说明
图1为实施例2中不同分子量沃柑多糖(OP1-OP10)对NK-92MI细胞增殖的影响效果图;
图2为实施例2中不同分子量沃柑多糖(OP1-OP10)对NK-92MI细胞中穿孔素表达的影响图;
图3为实施例2中不同分子量沃柑多糖(OP1-OP10)对NK-92MI细胞中颗粒酶B的表达效果图;
图4为实施例2中不同分子量沃柑多糖(OP1-OP10)对NK-92MI细胞中IFN-γ的表达效果图;
图5为实施例2中不同分子量沃柑多糖(OP1-OP10)对NK-92MI细胞毒性的影响结果图;
图6为实施例2中OP5的单糖组成图;
图7为实施例2中OP5的红外分析图谱;
图8为实施例2中OP5的氢谱图;
图9为实施例2中OP5的碳谱图。
具体实施方式
下面结合实施例和试验对本发明作进一步说明。
实施例1
一种沃柑多糖,其从沃柑果皮中分离得到,其分子量为50-70kDa。
实施例2
实施例1的沃柑多糖由如下方法制备得到:
S1:用蒸馏水冲洗沃柑果皮,然后在40℃的烘箱中干燥,用粉碎机将干燥的沃柑果皮粉碎成粉末,将粉末(100g)用蒸馏水(1900g)在100℃下萃取3h,收集上清液;
S2:使用旋转蒸发器将S1获得的上清液浓缩获得成粘性液体,将粘性液体(100毫升)加入无水乙醇(900毫升)中搅拌2分钟,然后在4℃下静置24小时。收集沉淀物并命名为沃柑果皮粗多糖(OP)。
S3:将沃柑果皮粗多糖溶解于100℃的蒸馏水中配置成沃柑果皮粗多糖溶液,将沃柑果皮粗多糖溶液通过MinimatePall超滤系统,该系统具有1000,500,300,100,70,50,30,10,5kDa的超滤膜,沃柑果皮粗多糖溶液通过该MinimatePall超滤系统后获得10份不同分子量的多糖溶液,将10份不同多糖溶液浓缩后醇沉,再将所有沉淀物冷冻干燥,制成不同分子量的沃柑多糖。
本实施例获得的10份不同分子量的多糖组分如下表1所示,10个具有不同分子量的多糖分别命名为(OP1-OP10)。其中,OP10的占比最高,为48.41%,其次是OP8(10.25%)、OP6(9.64%)和OP4(8.82%)。其他的占比小于7%。
其中,50-70kDa(OP5)的沃柑多糖即为实施例1的沃柑多糖。
表1
将上述10份不同分子量的多糖进行如下实验:
(一)细胞培养
1、NK-92MI细胞(自然杀伤细胞)
培养条件:MEMα+0.2mM Inositol+0.1mMβ-mercaptoethanol+0.02mM Folic Acid+12.5%HS+12.5%FBS+1%P/S 5%CO237℃培养。
2、Calu-1细胞(肺癌细胞)
培养条件:McCoy's 5A+10%FBS+1%P/S 5%CO237℃培养。
(二)NK-92MI细胞活性测定
1、不同分子量沃柑多糖(OP1-OP10)对NK-92MI细胞增殖的影响
将NK-92MI细胞接种在96孔板中。传代4小时后,将浓度分别为62.5、125、250、500和1000μg/mL的不同分子量沃柑多糖溶液加入样品组,而将等体积的PBS加入对照组。共培养16h后,用CCK8法检测NK-92MI细胞的增殖率。
其结果参见图1。由图1可以看出,OP1-OP10都能明显促进NK-92MI细胞的增殖。在某些浓度下,NK-92MI细胞的增殖率甚至超过150%。例如,500μg/mL的OP1和250μg/mL的OP9使NK-92MI细胞的增殖率分别达到156.45%和160.40%。总的来说,随着浓度的增加,各OP对NK-92MI细胞增殖的影响呈先增加后减少的趋势。在250μg/mL~500μg/mL的浓度范围内,各OP的促进作用较高。因此,在随后的实验中,缩小了范围以筛选出最佳浓度。
2、不同分子量沃柑多糖(OP1-OP10)对NK-92MI基因表达的影响
用TRIzol试剂提取处理好的NK-92MI细胞的总RNA。使用HiScript IIQRTSuperMix for qPCR(+gDNAwiper)构建cDNA。ChamQ Universal SYBR qPCR Master Mix用于定量实时PCR(qRT-PCR)扩增。荧光定量PCR的条件如下:在95℃下预变性3min。95℃15s、57℃15s和72℃20s的40个循环。通过倍数变化[2(-ΔΔCt)]法测定NK-92MI细胞中穿孔素(PFP)、颗粒酶B(GZMB)、干扰素-γ(IFN-γ)的靶基因表达。对于qRT-PCR分析,GAPDH被选择作为内部参考基因。
实验结果参见图2-图4所示,由图2可知,不同OP对NK-92MI细胞中穿孔素表达的影响显著不同。在实验浓度下,NK-92MI细胞中的穿孔素表达被4种沃柑多糖(OP7、OP8、OP9和OP10)下调,而被3种沃柑多糖(OP2、OP4和OP5)显著上调。在150μg/mL和250μg/mL之间,上调水平相对较高。由图3可知,在相同浓度下,OP2和OP5可以显著增加NK-92MI细胞中颗粒酶B的表达。在250~350μg/mL范围内,其增幅较大。提高1.29-1.50倍(OP2),1.32-1.41倍(OP5)。由图4可知,6种沃柑多糖增强了NK-92MI细胞中IFN-γ的表达(从OP1到OP6)。除OP3外,所有沃柑多糖在150μg/mL至250μg/mL的浓度范围内都能达到最大的上调水平。与对照组相比,OP5使IFN-γ的表达水平增长最高,达到5.39-7.40倍。
3、不同分子量沃柑多糖(OP1-OP10)对NK-92MI细胞毒性的影响
将NK-92MI细胞接种到6孔板中,在传代4小时后分为样品组和对照组。样品组具有不同分子量的沃柑多糖溶液即OP1-OP10(250μg/mL),对照组具有相同体积的PBS(250μg/mL)。两组必须全部培养16h。
另一方面,将在96孔板中培养的Calu-1细胞分为样品组、对照组和空白组。每组有8个复孔。在Calu-1细胞(靶细胞)粘附后,收集以上处理的两组NK-92MI细胞(效应细胞),并以10∶1的有效靶比相应地添加以一起培养。共培养4小时后,通过用CCK-8测定法计算Calu-1细胞活力来评估沃柑多糖对NK-92MI细胞毒性的影响。
经OP1-OP10处理后的NK-92MI细胞的细胞毒性发生了变化,结果参见图5所示,由图5可知,分别与OP7、OP8、OP9和OP10共培养的NK-92MI细胞的细胞毒性不同程度地降低。尽管其他6种沃柑多糖都能增强NK-92MI细胞的细胞毒性,但只有OP5达到了显著水平。因此,将对OP5的结构进行鉴定,以分析其构效关系。
(三)OP5的结构鉴定
(1)鉴定方法:
1、单糖组成检测
将OP5水解。将甘露糖、核糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖、木糖、阿拉伯糖和岩藻糖溶解在蒸馏水中作为混合标准溶液。通过液相检测OP5的单糖组成。柱温为30℃。流速为1.0mL/min。检测波长为250nm。进样量为20μL。流动相为0.05M磷酸二氢钾和乙腈的混合溶液(体积比为83:17)。
2、红外检测
将多糖研磨和压制后与KBr混合。通过红外光谱仪在400cm-1到4000cm-1范围内进行分析。
3、核磁共振检测
通过核磁共振仪器检测OP5在13C NMR(150MHz)和1H NMR(600MHz)光谱。氧化氘(D2O)作为溶剂。
(2)结果与分析
1、单糖组成
OP5的单糖组成如图6(图6中,1:甘露糖,2:核糖,3:鼠李糖,4:葡萄糖醛酸,5:半乳糖醛酸,6:N-乙酰葡糖胺,7:葡萄糖,8:N-乙酰半乳糖胺,9:半乳糖,10:木糖,11:阿拉伯糖,12:岩藻糖)所示,经与标准品的HPLC色谱图比较,发现OP5由10个单糖组成。它们的比例分别为甘露糖7.67%、核糖0.27%、鼠李糖2.39%、葡萄糖醛酸0.42%、半乳糖醛酸22.35%、葡萄糖15.95%、半乳糖16.72%、木糖1.30%、阿拉伯糖31.52%和岩藻糖1.41%。半乳糖醛酸含量高提示其为酸性多糖。
2、红外分析
OP5的红外分析如图7所示。由图7可知,在3416cm-1处的强吸收峰是由O-H键的拉伸振动引起的。2936cm-1处的吸收峰归因于C-H键的拉伸振动。在1751cm-1和1609cm-1处的两个吸收峰应分别是羧基(-COOH)C=O键的对称和不对称拉伸振动峰,这表明OP5中存在糖醛酸。1443cm-1处的吸收峰是由于C-H键的弯曲振动引起的。1234cm-1和1105cm-1处的吸收峰可能是由C-O键的拉伸振动引起的。在1018cm-1处观察到强吸收峰,表明OP5中存在吡喃环和C-O-C键的拉伸振动。633cm-1处的吸收峰可能是由O-H键的平面外弯曲振动引起的。
3、核磁共振
OP5的氢谱图如图8所示,碳谱图如图9所示。由图8可知,异头质子区内(δ4.3-5.9),在δ4.70处有一个明显的信号且小于5.0,表明糖环主要是β构型。在δ1.26处的信号应归因于鼠李糖中甲基的质子。从图9中可以看出,糖醛酸的特征信号区内(δ170-190),δ170.66处的清晰信号表明OP5具有糖醛酸。与单糖组成检测结果一致。在异头碳区(δ90-112)内,许多信号出现在低场(δ102-112),几个信号出现在高场(δ90-102),表明糖环主要为β构型,但仍存在α构型。δ82.33和δ83.88处的两个信号表明OP5中可能存在呋喃糖结构。此外,位于δ76至δ85区域内的4个信号(δ81.25、80.66、79.01和76.53)和位于δ70至δ75区域内的3个信号(δ74.42、74.12和71.11)表明吡喃糖的C2、C3和C4碳已被部分取代。δ69.65、δ69.18和δ67.53的信号提示某些吡喃糖上的C6被取代。在δ60至δ64之间出现了4个信号(δ63.86、63.34、61.07和60.73),表明某些吡喃糖的C6没有被取代。δ52.86处的信号应归因于N-Ac(CH3CON-)中N-取代基的碳。δ23.85处信号的存在表明,它可能是由N-Ac中甲基的碳引起的。δ19.89处的信号可能是由O-Ac(CH3COO-)中甲基的碳引起的。
多糖的免疫功效来源于其结构。许多因素会影响其活性,如分子量、单糖组成、溶解度、侧链和糖苷键。许多研究表明,多糖不能直接攻击肿瘤细胞和癌症细胞等靶细胞。它必须通过其作为全身反应的免疫增强或免疫调节作用来发挥生物学功能。具体而言,多糖必须首先与效应细胞的受体结合,才能产生宿主反应。因此,多糖的分子量尤其重要,因为只有适当的分子量才能使其更有利于与受体偶联。在发明中,分子量为50-70kDa的OP5在所有OP中表现出最高的生物活性。这可能是因为它的分子量不是太大也不是太小,所以它很容易与受体结合。然后刺激NK-92MI细胞分泌穿孔素、颗粒酶B、IFN-γ,以杀死Calu-1细胞。单糖组成是影响NK-92MI细胞活性的另一个因素。众所周知NK-92MI细胞的补体受体3型(CR3)是多糖识别的靶受体,它可以更容易地被含有葡萄糖、甘露糖和N-乙酰-d-葡糖胺等特殊单糖成分的多糖识别和结合。在OP5中,检测到大量的葡萄糖(15.95%)和一些甘露糖(7.67%)。这可能是支持其增强NK-92MI细胞活性的原因之一。此外,高占比的阿拉伯糖(31.52%)可能有助于提高OP5的免疫活性。良好水溶性是多糖发挥作用的必要基础,而糖醛酸含量会影响到多糖的水溶性。在OP5中存在大量具有高度亲水性羧基的半乳糖醛酸(22.35%),这对其在水中快速溶解并发挥随后的免疫作用是非常有益的。
基于以上的实验和分析,本实施例的OP1-OP10中,OP5在促进NK-92MI细胞增殖、上调基因表达和提高细胞毒性方面的综合作用最好。OP5的分子量不是太大也不是太小,在50和70kDa之间。它由阿拉伯糖(31.52%)、半乳糖醛酸(22.35%)、半乳糖(16.72%)、葡萄糖(15.95%)、甘露糖(7.67%)、鼠李糖(2.39%)、岩藻糖(1.41%)、木糖(1.30%)、葡萄糖醛酸(0.42%)和核糖(0.27%)组成。OP5分子量适中,阿拉伯糖、半乳糖醛酸、葡萄糖和甘露糖含量高,可能是其具有免疫活性的原因。
实施例3
实施例1的沃柑多糖在制备促进NK-92MI细胞增殖、上调基因表达和提高细胞毒性方面的药物的应用。
实施例4
一种促进NK-92MI细胞增殖、上调基因表达和提高细胞毒性方面的药物,所述药物包含如上述实施例1的沃柑多糖。
实施例5
实施例1的沃柑多糖在制备抑制Calu-1肺癌细胞药物中的应用。
实施例6
一种抑制Calu-1肺癌细胞药物,其包含上述实施例1沃柑多糖。
上述说明是针对本发明较佳可行实施例的详细说明,但实施例并非用以限定本发明的专利申请范围,凡本发明所提示的技术精神下所完成的同等变化或修饰变更,均应属于本发明所涵盖专利范围。
Claims (6)
1.一种沃柑多糖,其特征在于,其从沃柑果皮中分离得到,所述沃柑多糖多糖的分子量为50-70kDa。
2.根据权利要求1所述的一种沃柑多糖的制备方法,其特征在于,其由如下方法制备得到:
S1:将沃柑果皮洗净后烘干,粉碎,用蒸馏水萃取,收集上清液;
S2:S1获得的上清液经浓缩后获得粘性液体,向粘性液体中加入无水乙醇进行醇沉收集沉淀物即为沃柑果皮粗多糖;
S3:将沃柑果皮粗多糖溶解于蒸馏水中配置成沃柑果皮粗多糖溶液,将沃柑果皮粗多糖溶液通过MinimatePall超滤系统,经过1000、500、300、100、70、50、30、10和5kDa的超滤膜后,将超滤得到的50-70kDa的多糖浓缩,再醇沉,将沉淀物冷冻干燥即获得所述沃柑多糖。
3.如权利要求1所述一种沃柑多糖或权利要求2所述一种沃柑多糖的制备方法制备得到的沃柑多糖在制备促进NK-92MI细胞增殖、上调基因表达和提高细胞毒性方面的药物的应用。
4.一种促进NK-92MI细胞增殖、上调基因表达和提高细胞毒性方面的药物,其特征在于,其包含如权利要求1所述一种沃柑多糖或权利要求2所述一种沃柑多糖的制备方法制备得到的沃柑多糖。
5.如权利要求1所述一种沃柑多糖或权利要求2所述一种沃柑多糖的制备方法制备得到的沃柑多糖在制备抑制Calu-1肺癌细胞药物中的应用。
6.一种抑制Calu-1肺癌细胞药物,其特征在于,其包含如权利要求1所述一种沃柑多糖或权利要求2所述一种沃柑多糖的制备方法制备得到的沃柑多糖。
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