CN110204627B - 一种美网柄牛肝菌多糖及其制备方法和应用 - Google Patents
一种美网柄牛肝菌多糖及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种美网柄牛肝菌多糖及其制备方法和应用。该多糖是自美网柄牛肝菌子实体依次通过水提醇沉、除蛋白、离子交换柱层析和透析浓缩得到的,其为由α‑D‑半乳糖和β‑D‑葡萄糖组成的杂多糖,其中α‑D‑半乳糖和β‑D‑葡萄糖的残基的摩尔比为5:1。该多糖具有显著的免疫调节活性,且对多种肿瘤细胞具有显著的抑制活性。
Description
技术领域
本发明属于真菌多糖应用技术领域,具体地说,涉及一种美网柄牛肝菌多糖及其制备方法和应用。
背景技术
多糖(polysaccharide)是由10个以上单糖通过糖苷键连接起来的天然生物大分子。目前,研究已发现多糖具有多种生理作用,如免疫调节、抗肿瘤、抗病毒、抗氧化、降血糖等。而且,二维核磁共振谱(1H-1H NMR、HSQC、HMBC等)、气相色谱-质谱联用(GC-MS)、高效液相色谱(HLPC)、X光衍射和扫描电子显微镜等技术的快速发展,在很大程度上促进了多糖结构的研究。
美网柄牛肝菌(Boletus reticulates Schaeff.)属于伞菌目、牛肝菌科、牛肝菌属,可食用而且味道鲜美。美网柄牛肝菌的子实体呈中等至大型,菌盖厚呈半球形或扁半球形,后期渐呈扁平。菌肉白色,后呈乳白色,表皮下稍有褐色,伤痕处稍变褐色,干燥有愉快的香气。菌管白色或污白色带黄色,后期管孔带青褐绿色。菌柄粗壮,近梭形或棒状圆柱形。目前,有关美网柄牛肝菌子实体多糖的精细结构、免疫活性和抗肿瘤活性还未见报道。
发明内容
本发明的目的在于提供一种美网柄牛肝菌多糖及其制备方法和应用。
其具体技术方案为:
本发明提供一种美网柄牛肝菌多糖,其为由α-D-半乳糖和β-D-葡萄糖组成的杂多糖,其中α-D-半乳糖和β-D-葡萄糖的残基的摩尔比为5:1。
进一步地,该多糖的重均分子量为100000-200000Da(如100000、120000、150000、160000、180000、200000Da);在本发明的一个实施例中,该多糖的重均分子量为150935Da。
进一步地,该多糖的化学结构中包含1,6-连接的α-D-半乳糖残基和4-连接的β-D-葡萄糖残基。
再进一步地,该多糖的化学结构中包含由1,6-连接的α-D-半乳糖残基组成的主链和由4-连接的β-D-葡萄糖残基组成的侧链。
在本发明的一个实施例中,该多糖具有如下结构式:
其中,n为90-180的整数(如90、110、140、150、167、180)。
上述多糖是自美网柄牛肝菌子实体依次通过水提醇沉、除蛋白、离子交换柱层析和透析浓缩得到的。
本发明还提供一种上述美网柄牛肝菌多糖的制备方法,其包括如下步骤:
(1)取美网柄牛肝菌子实体粉末,热水浸提,所得水提物依次经浓缩、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)所得洗脱液用透析袋进行透析浓缩。
优选地,(4)将步骤(3)完成后的透析袋内液体离心,取上清液,冷冻干燥。
进一步地,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℃。
进一步地,热水浸提步骤中,水与美网柄牛肝菌子实体粉末的液料比为1-10:1(体积质量比,如1:1、2:1、3:1、5:1、6:1、8:1、10:1,mL/g);在本发明的一个实施例中,该液料比为3:1。
进一步地,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。
进一步地,每次浸提时间为4-10小时(如4、6、8、10小时);在本发明的一个实施例中,每次浸提时间为6小时。
进一步地,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。
进一步地,步骤(1)包括:取美网柄牛肝菌子实体粉末,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。
进一步地,离子交换柱为纤维素柱,该纤维素柱的填料如DEAE-52纤维素。
进一步地,洗脱所用洗脱剂为水。
进一步地,步骤(2)包括:将步骤(1)所得粗多糖的水溶液通过纤维素柱,洗脱,收集洗脱液,浓缩,离心,收集上清液。
进一步地,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。
进一步地,步骤(3)包括:将步骤(2)所得洗脱液置于透析袋,半天换一次水,透析两天。
本发明还提供一种上述美网柄牛肝菌多糖在制备增强免疫的药物、保健品和食品中的应用。
本发明还提供一种上述美网柄牛肝菌多糖在制备用于预防和/或治疗肿瘤的药物、保健品和食品中的应用。
上述应用中,美网柄牛肝菌多糖可单独使用,可也与其他活性成分联合使用。
本发明自美网柄牛肝菌分离纯化得到多糖BRS-X,其具有显著的免疫调节活性,例如,采用20μg/mL的BRS-X,对T细胞增殖率可达38.88%,对B细胞增殖率可达30.97%,采用10μg/mL的BRS-X,对RAW264.7细胞增殖率可达122.49%;且LDS-1对多种肿瘤细胞具有显著的抑制活性,例如,LDS-1的质量浓度为10μg/mL时,对S180细胞和MFC细胞的抑制率分别为37.45%和23.95%,LDS-1的质量浓度为5μg/mL时,对L929细胞的抑制率可达40.77%。
附图说明
图1所示为BRS-X的HPGPC谱图;
图2所示为BRS-X的红外光谱图;
图3所示为BRS-X的高效液相(HLPC)谱图;
图4所示为BRS-X的1H NMR谱图;
图5所示为BRS-X的13C NMR谱图;
图6所示为BRS-X的化学结构;
图7所示为BRS-X对T细胞增殖活性影响的实验结果;
图8所示为BRS-X对B细胞增殖活性影响的实验结果;
图9所示为BRS-X对RAW264.7细胞增殖活性影响的实验结果;
图10所示为BRS-X对RAW264.7细胞吞噬活性影响的实验结果;
图11所示为BRS-X对S180细胞生长的抑制率实验结果;
图12所示为BRS-X对MFC细胞生长的抑制率实验结果;
图13所示为BRS-X对L929细胞生长的抑制率实验结果。
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。
1.美网柄牛肝菌多糖BRS-X的结构解析
1.1美网柄牛肝菌多糖的提取与分离纯化
1.1.1通过热水提取法提取美网柄牛肝菌粗多糖
将烘干的美网柄牛肝菌子实体打粉,按照液料比为3:1的比例在100℃水浴6小时,重复3次,收集上清液并浓缩。利用三倍体积的无水乙醇沉淀粗多糖,粗多糖烘干后用Sevage法除去其中的蛋白质。
1.1.2 DEAE-52纤维素柱层析法分离纯化美网柄牛肝菌粗多糖
用蒸馏水将DEAE-52纤维素50.00g搅拌均匀后,加入0.5mol/L NaOH溶液浸泡2小时以上。反复用蒸馏水洗至中性,加入0.5mol/L HC1溶液浸泡2小时以上。反复用蒸馏水洗至中性,加入0.5mol/L NaOH溶液浸泡2小时以上,反复用蒸馏水洗至中性。
将中性的纤维素装入玻璃柱,利用蒸馏水作为流动相将纤维素压实。取适量的粗多糖溶解于蒸馏水,并加入至纤维素柱中。蒸馏水作为流动相,收集洗脱液并利用硫酸-苯酚法定性检测。浓缩洗脱液并离心,上清液转移至透析袋(7000Da),半天换一次水,透析两天。将透析袋中的液体离心,将上清液冷冻干燥得到美网柄牛肝菌多糖,并命名为BRS-X。
1.2美网柄牛肝菌多糖BRS-X的结构解析
1.2.1分子量的测定
将10mg BRS-X样品溶解于3mL蒸馏水,超声混匀5分钟并过滤,采用高效凝胶渗透色谱法(HPGPC)测定多糖的分子量。
1.2.2傅里叶红外光谱分析
将5mg BRS-X和KBr研磨混匀并压片,在傅里叶变换红外光谱仪中对混合物在4000cm-1到400cm-1范围内进行扫描。
1.2.3单糖的组成分析
单糖标准品和通过TFA酸水解后的20mg BRS-X,以75%乙腈作为流动相进行高效液相色谱(HPLC)分析。
1.2.4核磁共振法分析
称取20mg BRS-X并溶解于500μl D2O,使用核磁共振仪检测BRS-X的氢谱(1H NMR)、碳谱(13C NMR)。
1.2.5多糖连接位点分析
将碘甲烷加入20mg BRS-X中进行甲基化,甲基化的BRS-X用TFA进行酸水解,水解的BRS-X用无水吡啶(0.2mL)、六甲基二硅氮烷(0.2mL)和三甲基氯硅烷(0.1mL)进行硅烷化。将BRS-X硅烷化后的衍生物使用气相色谱与质谱联用仪(GC-MS)检测连接位点。1.3结果
1.3.1美网柄牛肝菌多糖BRS-X的分子量
BRS-X的HPGPC谱图如图1所示,其表明,BRS-X的重均分子量(Mw)为150,935Da。
1.3.2美网柄牛肝菌多糖BRS-X的傅里叶红外光谱分析
BRS-X的傅里叶红外光谱如图2所示。3,432cm-1处代表O-H的伸缩振动。2,929cm-1处是C-H的伸缩振动。1,637cm-1处代表C=O的伸缩振动;1,401cm-1处是C-H的弯曲振动;1,145cm-1和1,076cm-1处的吸收峰在1,200-1,000cm-1范围内,表明单糖具有吡喃环。800-300cm-1区域的峰值对应于C=C拉伸和C-OH弯曲模式。557cm-1处为C-H摇摆振动峰。1.3.3美网柄牛肝菌多糖BRS-X的单糖组成分析
BRS-X的高效液相色谱如图3所示,其中峰1为葡萄糖,保留时间为14.208,峰面积为130145,含量为0.0567(单位为g/L),峰2为半乳糖,保留时间为16.692,峰面积为643732,含量为0.2579(单位为g/L)。结果显示,BRS-X由α-D-半乳糖和β-D-葡萄糖以5:1的比例组成。
1.3.4美网柄牛肝菌多糖BRS-X的核磁共振法分析
BRS-X的1H NMR谱图如图4所示。在1H NMR谱中,信号δ4.93、δ4.93、δ4.85和δ4.64表示BRS-X有四个异头质子。δ4.93、δ4.93和δ4.85处的氢信号归属于α-吡喃糖残基,δ4.64处的氢信号归属于β-吡喃糖残基。δ3.32-δ4.07处的峰是由大量重叠的氢信号组成,这归属于每个单糖中H2-H6的信号。水的氢信号为δ4.70。
BRS-X的13C NMR谱图如图5所示。在BRS-X的13C NMR谱中,δ101.41、δ97.79、δ97.78和δ97.72处的峰为四个异头碳信号,表明BRS-X中单体存在α和β型。在δ80-δ55ppm之间有很强的信号,这些可归属于葡萄糖和半乳糖的C2-C6的碳信号。BRS-X中的单糖的碳信号归属如表1。
表1美网柄牛肝菌多糖(BRS-X)在13C NMR中的化学位移
1.3.5美网柄牛肝菌多糖BRS-X的连接位点分析
BRS-X的GC-MS结果显示,D-葡萄糖残基为4位取代,D-半乳糖为1,2,6位取代,如表2。其中,半乳糖与葡萄糖的比例为5:1,这与高效液相色谱的结果基本一致。因糖的特殊结构,2位和3位碳的空间位阻大,不易被甲基化,故实验中存在甲基化不完全。结合上述所有实验的结果并分析,解析BRS-X的结构如图6所示。
表2美网柄牛肝菌多糖(BRS-X)中单糖的连接位点
2.美网柄牛肝菌多糖BRS-X的体外免疫活性和抗肿瘤活性研究
2.1 BRS-X刺激下T、B和RAW264.7细胞的增殖活性
取对数生长期的T、B和RAW264.7细胞,用新鲜培养液稀释并转移至96孔板(100μL/孔),放入细胞培养箱(37℃,5%CO2,24小时)。空白对照组加入细胞培养液(100μL/孔),阳性对照组加入LPS(5μg/mL,100μL/孔),药物组(3个组)分别加入BRS-X(终浓度分别为5、10、20μg/mL,100μL/孔),放入细胞培养箱(37℃,5%CO2,24小时)。向每个孔中加入5μL CCK-8溶液并孵育3小时。测量450nm处的吸光度并记录结果。
2.2 BRS-X刺激下RAW264.7细胞的吞噬活性
取对数生长期的T、B和RAW264.7细胞,用新鲜培养液稀释并转移至96孔板(100μL/孔),放入细胞培养箱(37℃,5%CO2,24小时)。空白对照组加入细胞培养液(100μL/孔),阳性对照组加入LPS(5μg/mL,100μL/孔),药物组(3个组)分别加入BRS-X(终浓度分别为5、10、20μg/mL,100μL/孔),放入细胞培养箱(37℃,5%CO2,24小时)。每孔分别加100μL中性红溶液(0.075g/L),放入细胞培养箱(37℃,5%CO2,15分钟)。弃掉中性红溶液并用PBS清洗三次。每孔加入100μL细胞裂解液(无水乙醇:乙酸=1:1),裂解2小时。在酶标仪中检测吸光度,波长设置为540nm。
2.3 BRS-X体外抗肿瘤活性(S180、MFC、L929细胞)
操作步骤同2.1。药物组BRS-X的终浓度为2.5、5、10μg/mL。
2.4结果
2.4.1 BRS-X对T细胞增殖的影响
BRS-X对T细胞增殖活性影响的实验结果如图7所示。当BRS-X的浓度为20μg/mL时,增殖率达到最大为38.88%。当LPS的浓度为5μg/mL时,增殖率达到13.00%。
2.4.2 BRS-X对B细胞增殖的影响
BRS-X对B细胞增殖活性影响的实验结果如图8所示。当BRS-X的浓度为20μg/mL时,增殖率达到最大为30.97%,比LPS的增殖效果好。
2.4.3 BRS-X对RAW264.7细胞增殖的影响
BRS-X对RAW264.7细胞增殖活性影响的实验结果如图9所示。当BRS-X的浓度为10μg/mL时,增殖率达到最大为122.49%。当LPS的浓度为5μg/mL时,增殖率达到80.71%。
2.4.4 BRS-X对RAW264.7细胞吞噬的影响
BRS-X对RAW264.7细胞吞噬活性影响的实验结果如图10所示。当BRS-X的浓度为10μg/mL时,吞噬率达到最大为67.74%,比LPS的吞噬效果好。
2.4.5 BRS-X体外对S180细胞的影响
BRS-X对S180细胞生长影响的实验结果如图11所示。当BRS-X的浓度为10μg/mL、5μg/mL、2.5μg/mL时,抑制率分别为37.45%、32.70%、28.77%。
2.4.6 BRS-X体外对MFC细胞的影响
BRS-X对MFC细胞生长影响的实验结果如图12所示。当BRS-X的浓度为10μg/mL、5μg/mL、2.5μg/mL时,抑制率分别为23.95%、20.90%、20.50%。
2.4.7 BRS-X体外对L929细胞的影响
BRS-X对L929细胞生长影响的实验结果如图13所示。当BRS-X的浓度为10μg/mL、5μg/mL、2.5μg/mL时,抑制率分别为21.91%、40.77%、38.03%。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种美网柄牛肝菌多糖,其为由α-D-半乳糖和β-D-葡萄糖组成的杂多糖,其中α-D-半乳糖和β-D-葡萄糖的残基的摩尔比为5:1。
2.如权利要求1所述的多糖,其特征在于,所述多糖的化学结构中包含1,6-连接的α-D-半乳糖残基和4-连接的β-D-葡萄糖残基。
3.如权利要求1所述的多糖,其特征在于,所述多糖的化学结构中包含由1,6-连接的α-D-半乳糖残基组成的主链和由4-连接的β-D-葡萄糖残基组成的侧链。
5.如权利要求1-4任一项所述的多糖,其特征在于,所述多糖的重均分子量为100000-200000Da。
6.一种权利要求1-5任一项所述的美网柄牛肝菌多糖的制备方法,其包括如下步骤:
(1)取美网柄牛肝菌子实体粉末,热水浸提,所得水提物依次经浓缩、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)所得洗脱液用透析袋进行透析浓缩。
7.如权利要求6所述的制备方法,其特征在于,所述热水浸提步骤中,浸提温度为80-100℃;
浸提次数为1-5次
每次浸提时间为4-10小时;
水与美网柄牛肝菌子实体粉末的液料比为1-10:1;
所述醇沉步骤中,醇与水提物的浓缩液的体积比为1-10:1。
8.如权利要求6所述的制备方法,其特征在于,步骤(2)中,所述离子交换柱为纤维素柱,其填料为DEAE-52纤维素;洗脱所用洗脱剂为水。
9.如权利要求6-8任一项所述的制备方法,其特征在于,步骤(3)中,所述透析袋的截留分子量为5000-10000Da。
10.一种权利要求1-5任一项所述的美网柄牛肝菌多糖在制备增强免疫、预防和/或治疗肿瘤的药物中的应用。
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Effective date of registration: 20230118 Address after: 637260 Duofu Food Industrial Park, Xichong County, Nanchong City, Sichuan Province Patentee after: XICHONG HOPE LAND MUSHROOM TECHNOLOGY Co.,Ltd. Patentee after: SHAOGUAN STARWAY BIO-TECHNOLOGY Co.,Ltd. Address before: 637009 No.1 Shida Road, Shunqing District, Nanchong City, Sichuan Province Patentee before: CHINA WEST NORMAL University |