CN110229244B - 一种枝瑚菌多糖及其制备方法和应用 - Google Patents
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- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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Abstract
本发明公开了一种枝瑚菌多糖,所述多糖是由葡萄糖和半乳糖组成的杂多糖,所述葡萄糖与半乳糖的残基摩尔比为2:1,优选的,所述葡萄糖选自α‑D‑葡萄糖或β‑D‑葡萄糖,所述半乳糖选自α‑D‑半乳糖,更优选的,所述α‑D‑葡萄糖为α‑D‑吡喃型葡萄糖。
Description
技术领域
本发明属于真菌多糖应用技术领域,具体地说,涉及一种天然产物枝瑚菌多糖RF-1及其制备方法和应用。
背景技术
多糖(polysaccharides)是生物体普遍存在的一类生物大分子。多糖的发展较晚,结构又复杂多样,因此对研究其结构、活性等都较困难。多糖生物活性的研究报道主要集中在免疫调节、抗肿瘤、抗病毒、抗氧化和降血糖等方面。
枝瑚菌,拉丁学名Ramaria flaccida(Fr.)Quél.,又被称为扫帚菌、鸡爪菌、刷把菌、则梭校等,子实体小,分枝多且稠密,柄短,通常从柄基部分枝。菌肉柔软,担子细长呈棍棒状。夏秋季群生在阔叶林或针叶林中地上的落叶层或枯枝,落果上,往往密集在一起。
目前,对枝瑚菌多糖的精细结构及其抗肿瘤活性应用尚未见任何报道。
发明内容
本发明的目的在于克服上述技术存在的缺陷,提供一种枝瑚菌多糖及其制备方法和应用。
本发明的目的是通过以下及技术方案实现的:
本发明提供一种枝瑚菌多糖,所述多糖是由葡萄糖和半乳糖组成的杂多糖,所述葡萄糖与半乳糖的残基摩尔比为2:1。
优选的,所述葡萄糖选自α-D-葡萄糖或β-D-葡萄糖,所述半乳糖选自α-D-半乳糖。
更优选的,所述α-D-葡萄糖为α-D-吡喃型葡萄糖。
在本发明的一个优选实施方式中,所述枝瑚菌多糖主链由(1→6,2)-α-D-半乳糖和(1→6,4)-α-D-吡喃型葡萄糖组成,支链通过(1→6)-α-D-吡喃型葡萄糖与主链葡萄糖的4-O相连,在支链的1-O上和主链半乳糖的2-O上连接4-β-D-葡萄糖作为端基糖。
优选的,所述枝瑚菌多糖的重均分子量为12000-25000Da(如12000、15000、17000、20000、23000、25000Da);在本发明的一个优选实施方式中,所述枝瑚菌多糖的重均分子量为17093Da。
在本发明的优选实施方式中,所述枝瑚菌多糖的结构式如下:
其中,n为10-200的整数(如10、40、50、100、120、180、190、200)。
本发明提供一种枝瑚菌多糖的制备方法,包括如下步骤:
(1)取枝瑚菌子实体粉末,热水浸提,将所得水提物经浓缩、醇沉、除蛋白得到粗多糖;
(2)将步骤(1)所得粗多糖通过离子交换柱层析,洗脱,收集洗脱液;
(3)将步骤(2)所得洗脱液进行透析、浓缩。
(4)将步骤(3)透析袋内液体冷冻干燥,得到枝瑚菌多糖。
优选的,所述热水浸提步骤中,浸提温度为80-100℃(如80、85、90、95、100℃),在本发明的一个实施例中,该浸提温度为100℃。
优选的,热水浸提步骤中,枝瑚菌子实体粉末与水的体积比为1:1-10(如1:1、1:2、1:3、1:5、1:8、1:10);在本发明的一个实施例中,该体积比为1:3。
优选的,浸提次数为1次或多次(如2、3、4、5次);在本发明的一个实施例中,该浸提次数为3次。
优选的,每次浸提时间为1-10小时(如1、3、5、8、10小时);在本发明的一个实施例中,每次浸提时间为3小时。
在本发明的一个实施例中,热水浸提步骤包括:取枝瑚菌子实体粉末,与水混合,并置于水浴中煮沸。
优选的,醇沉步骤中,醇与水提物的浓缩液的体积比为1-10:1(如1:1、3:1、4:1、5:1、10:1);在本发明的一个实施例中,该体积比为3:1。
在本发明的一个实施例中,醇沉步骤中,醇为乙醇。
在本发明的一个实施例中,除蛋白采用Sevage法。
更优选的,步骤(1)包括:取枝瑚菌子实体粉末,热水浸提,收集上清液,浓缩,加入无水乙醇,收集沉淀,烘干,除去其中的蛋白质,得到粗多糖。
离子交换柱为纤维素柱,该纤维素柱的填料为DEAE-纤维素。
洗脱所用洗脱剂为蒸馏水或NaCl溶液。
优选的,洗脱液为NaCl溶液,该洗脱为梯度洗脱,洗脱剂浓度为0.01-1.0mol/L(如0.01、0.1、0.2、0.3、0.4、0.5、0.6、0.8、1.0)mol/L。
更优选的,步骤(2)包括:将步骤(1)所得粗多糖的水溶液通过纤维素柱,梯度洗脱,收集洗脱液,浓缩。
更优选的,步骤(3)包括:将步骤(2)所得洗脱液置于透析袋中进行透析,持续3天。
透析袋的截留分子量为5000-10000Da(如5000、7000、8000、10000Da);在本发明的一个实施例中,该截留分子量为7000Da。
本发明提供一种枝瑚菌多糖在制备预防和/或治疗肿瘤药物、保健品或食品中的应用。
与现有技术相比,本发明的有益效果为:
本发明一定剂量的枝瑚菌多糖RF-1具有显著的抗肿瘤活性,RF-1在浓度为20mg/kg时,可以显著抑制小鼠内S180肿瘤细胞的增殖,且效果优于甘露聚糖肽。
附图说明
图1枝瑚菌多糖RF-1HPGPC谱图;
图2枝瑚菌多糖RF-1的红外谱图;
图3枝瑚菌多糖RF-1的HPLC图谱;
图4枝瑚菌多糖RF-1的1H NMR图谱;
图5枝瑚菌多糖RF-1的13C NMR图谱;
图6枝瑚菌多糖RF-1的HH-COSY图谱;
图7枝瑚菌多糖RF-1的HMQC图谱;
图8枝瑚菌多糖RF-1的HMBC图谱;
图9枝瑚菌多糖RF-1的结构;
图10枝瑚菌多糖RF-1体内抑制S180肿瘤白光图。
具体实施方式
除非另有定义,本发明中所使用的所有科学和技术术语具有与本发明涉及技术领域的技术人员通常理解的相同的含义。
下面结合附图和具体实施例对本发明的技术方案作进一步详细地说明。
实施例1枝瑚菌多糖RF-1的分离提取
枝瑚菌多糖RF-1的分离提取
S1:通过热水提取法提取枝瑚菌粗多糖
将粉碎后的枝瑚菌子实体粉末和蒸馏水以1:3的体积比在100℃的水中煮沸3小时,上清液加入三倍体积的无水乙醇形成絮状沉淀物,收集沉淀并干燥,除蛋白,获得枝瑚菌粗多糖;
S2:DEAE-纤维素柱层析法分离纯化枝瑚菌粗多糖
称取DEAE-纤维素50.00g,活化,将粗多糖稀释后的上清液(3ml)加入DEAE纤维素柱中,加入蒸馏水洗脱,用透析袋(Mw=7kDa)透析,除去小分子化合物,冻干,得到枝瑚菌多糖,命名为RF-1。
实施例2枝瑚菌多糖RF-1的结构鉴定
运用水解、甲基化分析、气相色谱和质谱联用技术、红外谱技术、核磁共振技术、高效液相色谱,对枝瑚菌多糖RF-1进行结构解析。
1,分子量的测定
将实施例1制备的5mg枝瑚菌多糖RF-1样品用1ml ddH2O溶解,超声5min,进行HPGPC分析,结果如图1所示,枝瑚菌纯多糖重均分子量为17093Da。
2,枝瑚菌多糖RF-1的红外谱分析
将实施例1制备的枝瑚菌多糖RF-1样品2mg与KBr混合压片,用红外分光光度计扫描4000cm-1-400cm-1范围,结果如图2所示,通过傅里叶变换红外光谱进行RF-1的红外分析如下,3427.93cm-1为糖类分子内或分子间氢键O-H的伸缩振动峰,2922.96cm-1是饱和C-H的伸缩振动峰,多糖的特征吸收峰中,1634.83cm-1为C=O,C=C振动峰,1402.49cm-1为C-H的弯曲振动峰,1076.87cm-1为C-O-H伸缩振动和吡喃环中醚键C-O-C伸缩振动,573.00cm-1为C-H的摇摆振动峰。
3,枝瑚菌多糖RF-1的高效液相色谱分析
单糖标准品和经TFA酸水解后的枝瑚菌多糖RF-1样品,以75%乙腈作为流动相进行高效液相色谱(HPLC)分析,通过HPLC分析RF-1的酸性水解产物,结果如图3所示,其中峰1为葡萄糖,保留时间为14.184,峰面积为786490,含量为0.3424(单位为g/L),峰2为半乳糖,保留时间为16.690,峰面积为435197,含量为0.1744(单位为g/L),本发明制备的枝瑚菌多糖RF-1主要由葡萄糖和半乳糖组成,组成比为2:1。
4,枝瑚菌多糖RF-1的核磁共振分析
取实施例1制备的枝瑚菌多糖RF-1样品10mg溶于0.5mL重水(D2O)中,装入核磁管中,使用核磁共振仪检测RF-1的氢谱(1H NMR)和碳谱(13C NMR)。
1H NMR谱图如图4所示,枝瑚菌多糖RF-1具有六个异头氢信号(δ4.99,δ4.95,δ4.90,δ4.88,δ4.42和δ4.39),表明RF-1多糖由六种单糖构成。
13C NMR谱图如图5所示,枝瑚菌多糖RF-1的13CNMR光谱中δ103.01,δ102.92,δ102.44,δ101.35,δ97.95和δ97.89的信号为异质子信号峰。
1H-1H COSY谱图如图6所示,表明H2的化学位移分别为3.78,3.71,3.75,3.71,3.24和3.22ppm。我们将枝瑚菌多糖RF-1中的单糖的氢信号在1H NMR谱中进行了归属并列于表1中,通过HMQC光谱(图7)归属C1-C6的相关信号并列于表1中。根据RF-1的HMBC谱(图8),可以判定碳氢远程耦合情况。
表1 RF-1在1H NMR和13C-NMR中的化学位移
5,枝瑚菌多糖RF-1的甲基化与硅烷化衍生后进行GC-MS分析
使用硫酸二甲酯对枝瑚菌多糖RF-1进行甲基化,用氯仿萃取产物,干燥后得甲基化的枝瑚菌多糖RF-1,所述甲基化多糖经TFA完全酸水解后得甲基化完全酸水解产物,备用。将上述制备的样品1mg依次加入无水吡啶(0.2ml),六甲基二硅氮烷(0.2ml)和三甲基氯硅烷(0.1ml),50℃条件下反应20min,离心(10000rpm,20min),取上层溶液用于GC-MS分析。
枝瑚菌多糖RF-1的甲基化结果如下表2所示,吡喃型葡萄糖残基有1,6-和1,4,6-两种方式连接,以及4-端基的形式;而吡喃型半乳糖残基为1,2,6-方式连接。进一步根据气相色谱与质谱法检测得出的各单糖峰面积可算出1,6-Glc:1,4,6-Glc:4-Glc:1,2,6-Gal=1:1:3:2。综上可初步推断枝瑚菌多糖的结构如图9所示。
表2枝瑚菌多糖RF-1的GC-MS结果
实施例3枝瑚菌多糖RF-1的抗肿瘤活性研究
运用体内建立S180肿瘤模型法测定枝瑚菌多糖RF-1的抗肿瘤活性。
实验材料
S180小鼠肿瘤细胞,昆明(KM)雌性小鼠,甘露聚糖肽片等。
实验方法
选取15只生长状况良好的雌性昆明小鼠,分别在左腋皮下接种100μL浓度为3×106CFU/mL的S180肿瘤细胞,待肿瘤呈黄豆大小开始实验。
S180肿瘤模型组:随机挑选5只S180肿瘤的小鼠,每天自由进食饮水;
阳性对照组:随机挑选5只S180肿瘤的小鼠,在自由进食饮水4天后,每天灌胃给药(20mg/kg甘露聚糖肽,按照甘露聚糖肽说明书进行给药),自由进食饮水;
RF-1药物组:随机挑选5只S180肿瘤的小鼠,在自由进食饮水4天后,每天灌胃给药(20mg/kg RF-1溶液),自由进食饮水。
灌胃给药七天后,将小鼠处死,摘除肿瘤,肝脏,胸腺和脾进行称重,计算抑瘤率。抑瘤率(%)=[(A-B)/A]×100%,注:A:S180肿瘤模型组肿瘤的平均值;B:实验组(阳性对照组和RF-S药物组)肿瘤的平均值。
实验结果
实验结果数据如表3所示,具体肿瘤白光图如图10所示,接种前各组小鼠体质量为18-20g,灌胃给药七天后,S180肿瘤模型组小鼠的体重增长最快,表明模型组小鼠体重增长最快可能因肿瘤增殖迅速而导致。在整个实验中,RF-1组的小鼠在皮毛光泽度、食欲及活动方面几乎与甘露聚糖肽组相同。并且,这两组小鼠的平均肝重也没有差异,这说明RF-1对肝脏无损伤。当RF-1浓度为20mg/kg时,小鼠肿瘤平均重量为0.58g,对小鼠S180肿瘤的抑制率为40%,而甘露聚糖肽组对小鼠S180肿瘤的抑瘤率为53.81%。说明RF-1具有较强的体内抗肿瘤作用。
表3 RF-1对小鼠体内S180肿瘤的抑制作用
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (6)
2.一种权利要求1所述的枝瑚菌多糖的制备方法,包括如下步骤:
(1)取枝瑚菌子实体粉末,热水浸提,将所得水提物经浓缩、醇沉、除蛋白得到粗多糖;
(2)将步骤(1)所得粗多糖通过离子交换柱层析,洗脱,收集洗脱液;
(3)将步骤(2)所得洗脱液进行透析、浓缩;
(4)将步骤(3)透析袋内液体冷冻干燥,得到枝瑚菌多糖;
其中,所述热水浸提步骤中,浸提温度为100℃;
枝瑚菌子实体粉末与水的体积比为1:1-10。
3.根据权利要求2所述的制备方法,其特征在于,
浸提时间为1-10小时;
醇沉步骤中,醇与水提物的浓缩液的体积比为1-10:1。
4.根据权利要求3所述的制备方法,其特征在于,枝瑚菌子实体粉末与水的体积比为1:3;
醇沉步骤中,醇与水提物的浓缩液的体积比为3:1;
醇沉步骤中,醇为乙醇;
除蛋白采用Sevage法。
5.根据权利要求4所述的制备方法,其特征在于,离子交换柱为纤维素柱,该纤维素柱的填料为DEAE-纤维素;洗脱所用洗脱剂为蒸馏水或NaCl溶液;
透析袋的截留分子量为7000Da。
6.一种权利要求1所述的枝瑚菌多糖在制备预防和/或治疗肿瘤药物、保健品或食品中的应用。
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