CN112961261A - 阳春砂根茎多糖及其制备方法和抗氧化的应用 - Google Patents
阳春砂根茎多糖及其制备方法和抗氧化的应用 Download PDFInfo
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Abstract
本发明公开了阳春砂根茎多糖及其制备方法和抗氧化的应用。发明人通过提取阳春砂根茎粗多糖,然后以葡聚糖凝胶柱色谱法纯化得到了阳春砂根茎多糖AVRP‑1。AVRP‑1具有抗氧化活性,对DPPH和羟自由基的半数有效浓度(EC50)分别为4.50 mg/mL和2.47 mg/mL,有望用于开发成为功能性食品和保健药品,也可以更为充分地利用阳春砂,利于变废为宝,提高阳春砂整体的经济价值,利于增收。
Description
技术领域
本发明涉及天然产物及其制备方法和应用,特别涉及具有抗氧化功效的阳春砂根茎多糖及其制备方法和应用。
背景技术
砂仁为姜科植物阳春砂Amomum villosum Lour.、绿壳砂Amomum villosum Lour.var xanthioides T.L. Wu et Senjen或海南砂Amomum longiligular T.L. Wu的干燥成熟果实。具有化湿开胃,温脾止泻,理气安胎的功效。用于湿浊中阻,脘痞不饥,脾胃虚寒,呕吐泄泻,妊娠恶阻,胎动不安。其中阳春砂仁是砂仁的主要来源,为药食同源药材,需求量大。砂仁的药用部位多为种子团,目前国内外关于砂仁的研究也多集中于种子团。砂仁为多年生草本植物,需每周期更新种植,以提升砂仁产量和品质,因此产生大量的砂仁植株,这些植株大多被随意丢弃或焚烧,对环境造成一定污染。
多糖是由10个以上单糖通过糖苷键连接而成的聚合物,具有广泛的生物活性,如:抗氧化、免疫调节、抗肿瘤和降血糖活性等。天然多糖因具有良好的生物活性和无毒性、良好的生物降解性和生物相容性等特点,成为目前国内外研究的热点。关于砂仁多糖的研究较少,且多集中于提取方法和粗多糖的生物活性。由于阳春砂仁是主要的药用部分,一般认为阳春砂果实最具有价值。因此,关于阳春砂除果实外,其他部位多糖的研究未见报道。
发明内容
本发明的目的在于克服现有技术的至少一种不足,提供一种阳春砂根茎多糖及其制备方法和应用。
本发明所采取的技术方案是:
本发明的第一个方面,提供:
一种阳春砂根茎多糖AVRP-1,其制备方法包括:
S1)将阳春砂根茎粉末采用水提醇沉法提取多糖粗提液,分离出沉淀后复溶,采用Sevage试剂除蛋白,干燥后得阳春砂根茎粗多糖;
S2)将所述粗多糖以阴离子交换层析法纯化,经透析和干燥后得AVRPD-1;
S3)使用Sephadex G-100凝胶柱对AVRPD-1进行分离,蒸馏水洗脱,洗脱流速为3mL/10 min,10 min/管,收集流出物经冷冻干燥后得到目标阳春砂根茎多糖AVRP-1。
在一些实例中,所述Sevage试剂为氯仿:正丁醇体积比=4:1的混合液。
在一些实例中,所述阴离子交换层析法使用DEAE-52纤维离子交换柱进行分离,依次使用浓度为0、0.1、0.3、0.5、1.0 M的NaCl溶液洗脱,洗脱流速为1 mL/min,10 min/管,收集0.1 M NaCl溶液洗脱下来的组分,浓缩后使用截留分子量为3500 Da的透析袋透析,透析留存物干燥得到阳春砂根茎多糖AVRPD-1。
在一些实例中,所述干燥为冷冻干燥。避免高温对结构的破坏。
在一些实例中,所述阳春砂根茎多糖AVRP-1由摩尔比为4.14:4.98:5.10:23.77:17.30:28.51:16.19的甘露糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖和阿拉伯糖这7种单糖组成,糖醛酸含量为43.59%、相对分子质量为3372.01 kDa。
本发明的第二个方面,提供:
一种具有抗氧化功效的组合物,其添加有本发明第一个方面所述的阳春砂根茎多糖AVRP-1。
在一些实例中,所述组合物为口服制剂。
在一些实例中,所述组合物为功能性食品。
在一些实例中,还包括可接受的辅料。
本发明的第三个方面,提供:
本发明第一个方面所述的阳春砂根茎多糖AVRP-1在制备抗氧化添加剂中的应用。
在一些实例中,所述抗氧化添加剂应用于食品。
本发明的有益效果是:
本发明的一些实例,首次分离得到了阳春砂根茎多糖AVRP-1,其中糖醛酸含量为43.59%,经高效凝胶渗透色谱法(HPGPC)测定,其相对分子质量为3372.01 kDa,经高效液相色谱法(HPLC)分析,AVRP-1主要由甘露糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖和阿拉伯糖这7种单糖组成,摩尔比为4.14:4.98:5.10:23.77:17.30:28.51:16.19。
本发明一些实例的阳春砂根茎多糖AVRP-1具有抗氧化活性,对DPPH和羟自由基的半数有效浓度 (EC50) 分别为4.50 mg/mL 和2.47 mg/mL,有望用于开发成为功能性食品和保健药品,为充分开发利用阳春砂提供了理论依据。
本发明的一些实例,可以更为充分地利用阳春砂,利于变废为宝,提高阳春砂整体的经济价值,利于增收。
附图说明
图1是阳春砂根茎多糖AVRP-1的DEAE纤维柱层析洗脱图;
图2是阳春砂根茎多糖AVRP-1的Sephadex G-100分子筛洗脱图;
图3是阳春砂根茎多糖AVRP-1的HPGPC洗脱曲线(A)和葡聚糖标准品标准曲线图(B);
图4是阳春砂根茎多糖AVRP-1的单糖组成图(A)和9种标准品的液相色谱图(B);
图5是阳春砂根茎多糖AVRP-1的红外光谱图;
图6是阳春砂根茎多糖AVRP-1的抗氧化能力测定图,其中,A是AVRP-1对DPPH清除率的图;B是AVRP-1对羟自由基清除率的图。
具体实施方式
下面结合实施例对本发明作进一步详细的描述,但本发明的实施方式不限于此。在未作特别说明的情况下,本发明所采用的试剂、设备和方法均为本技术领域常规市购的试剂、设备和常规使用的方法。
阳春砂根茎多糖AVRP-1的制备
S1) 称取200 g粉碎后的阳春砂根茎粉末,加入4 L 95%乙醇,80 ℃回流提取3 h,重复操作3次,最后将粉末烘干,得阳春砂根茎脱脂粉末。200 g阳春砂根茎脱脂粉末加4 L水,95 ℃提取3 h,重复3次,再将水提液于60 ℃下减压浓缩,得浓缩液。向浓缩液中加入4倍体积的无水乙醇,于4 ℃静置12 h,离心收集沉淀。将沉淀复溶于蒸馏水中,得多糖溶液,向溶液中加入4倍体积的Sevage试剂(氯仿-正丁醇 4:1/v/v),剧烈震荡后离心,取上清液,重复操作,直至溶液中无变形蛋白质出现,冻干后得AVRP;
S2) 称取AVRP 100 mg,配制成浓度为20 mg/mL 的溶液,经DEAE-52纤维素离子交换柱分离,依次用浓度为0、0.1、0.3、0.5、1.0 M NaCl溶液进行洗脱,洗脱流速为1 mL/min,10 min/管,洗脱曲线如图1所示;使用全自动馏分收集器收集,采用苯酚-硫酸法进行监测,得到0和0.1 M NaCl洗脱下的2个组分,收集含量更多的0.1 M NaCl溶液洗脱下来的组分,60 ℃减压浓缩,用截留分子量为3500 Da的透析袋透析,冷冻干燥得AVRPD-1;
S3) 取AVRPD-1 20 mg,配制成5 mg/mL的溶液,经葡聚糖凝胶Sephadex G-100柱分离,蒸馏水洗脱,洗脱流速为3 mL/10 min,洗脱时间为10 min/管,洗脱曲线如图2所示;使用全自动馏分收集器收集,采用硫酸-苯酚法测多糖含量,合并同一组份,经浓缩、冷冻干燥后得到阳春砂根茎多糖AVRP-1。
阳春砂根茎多糖AVRP-1的组分测定
采用硫酸苯酚法测定AVRP-1的总糖含量,Bradford法测定AVRP-1的蛋白质含量,m-间羟基联苯法测定AVRP-1的糖醛酸含量。测定结果显示,AVRP-1的总糖含量为90.34%,蛋白质含量为0.64%,糖醛酸含量为43.59%,说明经过Sevage试剂除蛋白和DEAE-52纤维柱、Sephadex G-100柱层析进行分离纯化后,多糖较纯,AVRP-1中糖醛酸含量较高。
阳春砂根茎多糖AVRP-1的分子量测定
利用高效凝胶渗透色谱法(HPGPC)测定样品纯度及相对分子质量。
取已知相对分子量(M w)的葡聚糖标准品(5000、12000、25000、50000、150000、410000 Da)配制成2.0 mg/mL的溶液,经0.22 μm滤膜过滤后进样,进样量20 μL,每个样持续时间30 min。色谱条件为:色谱柱型号为PolySep-GFC-P4000,流动相为蒸馏水,流速为1.0 mL/min,柱温35 ℃,检测器为Shimadzu 的蒸发光检测器(ELSD-LT Ⅱ),检测器温度为60 ℃,Gain值为10。以标准葡聚糖相对分子质量的对数log M w为纵坐标,保留时间为横坐标,绘制标准曲线并得到回归方程。取AVRP配制成2.0 mg/mL的溶液,在相同条件下进样,记录保留时间。根据样品峰形鉴定AVRP的纯度,从标准曲线中求得具体M w。
结果如图3所示,由结果可知,AVRP-1只有单一的洗脱峰,且峰型对称,可以证明AVRP-1为纯多糖。线性回归方程为y=-0.7494x+10.332,R2=0.9971,式中y为葡聚糖相对分子质量的对数,x为保留时间,经计算,阳春砂根茎多糖AVRP-1的相对分子质量为3372.01kDa。
阳春砂根茎多糖AVRP-1的单糖组成分析
采用PMP柱前衍生化-高效液相色谱法(HPLC)测定AVRP的单糖组成。
混合标准单糖的PMP衍生化:分别取0.05 mL 2 mM的单糖标准品(甘露糖、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、岩藻糖)溶液于耐压管中混合,加入配制好的0.5 M PMP甲醇溶液和0.3 M NaOH溶液各0.45 mL,混匀后于70 ℃反应30min,冷却后加入0.46 mL 0.3 M的HCl溶液中和,然后加入1 mL三氯甲烷溶液,振摇,离心,弃三氯甲烷层,重复3次以去除过量的PMP,水层过0.45 μm滤膜后于HPLC检测。HPLC条件:Shimadzu高效液相色谱仪,Agilent ZORBAX XDB-C18柱,波长为250 nm,进样量为20 μL,流速为1 mL/min,流动相为0.05 M磷酸盐缓冲液(pH 6.7,A)-乙腈(B)。洗脱梯度为:0-40 min16% B等度洗脱,40-41 min 16% B降至14% B,41-54 min 14% B等度洗脱,54-55 min 14%B升至16% B,55-70 min 16% B等度洗脱。
AVRP-1样品的衍生化:称取AVRP-1样品5.0 mg于耐压管中,加入配制好的3 M TFA溶液 2mL,油浴锅内120 ℃水解6 h,冷却后加入甲醇减压浓缩至干,重复3次以除去多余的TFA,加入0.8 mL蒸馏水溶解。取0.1 mL完全水解的AVRP-1样品于反应管中,加入0.5 M PMP甲醇溶液和0.3 M NaOH溶液各0.1 mL,混匀后70 ℃水浴反应30 min,冷却后加入0.105 mL0.3M HCl溶液中和,加入0.2 mL蒸馏水稀释,然后加入0.6 mL三氯甲烷溶液,振摇,离心,弃三氯甲烷层,重复3次以除去过量的PMP,水层过0.45 μm滤膜后于HPLC检测。结果如图4所示,AVRP-1是由甘露糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖和阿拉伯糖这7种单糖组成,摩尔比为4.14:4.98:5.10:23.77:17.30:28.51:16.19。
阳春砂根茎多糖AVRP-1的红外光谱分析
取AVRP-1干燥粉末,使用傅里叶红外光谱仪(UATR Two, PerkinElmer, theNetherlands)在4000-450 cm-1范围内进行测定。
结果如图5所示,AVRP-1在3303.17 cm-1处的强吸收峰来自于O-H的伸缩振动。2932.27 cm-1处的吸收峰来自于C-H伸缩振动。1729.02 cm-1和1601.93 cm-1处的吸收峰分别来自于甲酯化COO-和游离COO-中的C=O伸缩振动,表明AVRP-1中存在糖醛酸,这一结论与单糖组成分析和糖醛酸含量测定结果相符。1200-1000 cm-1区被称为多糖的“指纹图谱”区。该区域中,1144.15 cm-1处的吸收峰来自于非对称的C-O-C伸缩振动,1073.90 cm-1处的吸收峰来自于糖环上的C-C-O伸缩振动,1017.01 cm-1处的吸收峰来自于侧链C-C-O的伸缩振动。这一区域的三处吸收峰证明AVRP-1为多糖。低于1000 cm-1的吸收峰会提供更多关于复杂结构的信息。955.90 cm-1和893.75 cm-1处的吸收峰证明AVRP-1中存在β构型,834.09 cm-1处的吸收峰证明AVRP-1中存在α构型,这一结果表明AVRP-1中α和β构型的糖苷键连接方式是同时存在的。759.51 cm-1处的吸收峰表明AVRP-1中存在吡喃糖。在1651 cm-1和1555 cm-1附近未发现吸收峰,表明AVRP-1中极少存在甚至不存在蛋白质,这一结论与蛋白质含量测定结果相符。
阳春砂根茎多糖AVRP-1对DPPH自由基清除能力测定
吸取50 μL 0.1 mM DPPH乙醇溶液于96孔板中,加入50 μL不同浓度(1.0-5.0 mg/mL)的多糖样品溶液,该组设置为样品测定组;以无水乙醇代替DPPH乙醇溶液作样品对照组;以去离子水代替多糖样品溶液作空白对照组;以Vc代替多糖样品溶液作阳性对照。以上各组混合均匀后,避光反应30min,于517 nm处测定吸光值。清除率通过下式计算:
式中,A0为空白对照组,A1为样品测定组,A2为样品对照组。
结果如图6 A所示,当多糖浓度从1.0增加到5.0 mg/mL时,AVRP-1对DPPH自由基的清除能力逐渐增强,但随着多糖浓度增加,增长趋势逐渐减小。AVRP-1对DPPH的半数有效浓度(EC50)为4.50 mg/mL。
阳春砂根茎多糖AVRP-1对羟自由基清除能力测定
于96孔板的检测孔中依次加入8 μL 18 mM FeSO4溶液,8 μL 18 mM水杨酸溶液,52 μL不同浓度(1.0-5.0 mg/mL)的多糖样品溶液,该组设置为样品测定组;检测孔中加入8μL 18 mM水杨酸溶液,60 μL去离子水,该组设置为空白组;检测孔中加入8 μL18 mM FeSO4溶液,8 μL 18 mM水杨酸溶液,52 μL去离子水,该组设置为对照组;检测孔中加入8 μL去离子水,8 μL 18 mM水杨酸溶液,52 μL多糖样品溶液,该组设置为样品对照组;以Vc代替多糖溶液作为阳性对照组。以上组别均加入32 μL 0.1% H2O2启动反应,静置30 min,于510 nm处测定吸光值。清除率通过下式计算:
式中,A0为空白组吸光度,A1为对照组吸光度,A2为样品对照组吸光度,A3为样品组吸光度。
结果如图6 B所示,当多糖浓度从1.0增加到5.0 mg/mL时,AVRP-1对羟自由基的清除能力逐渐增强,但随着多糖浓度增加,增长趋势逐渐减小,之后趋于平稳。AVRP-1对羟自由基的EC50为2.47 mg/mL。
实验结果表明阳春砂根茎多糖AVRP-1具有良好的抗氧化能力和自由基清除能力。
以上是对本发明所作的进一步详细说明,不可视为对本发明的具体实施的局限。对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的简单推演或替换,都在本发明的保护范围之内。
Claims (10)
1.一种阳春砂根茎多糖AVRP-1,其特征在于:其制备方法包括:
S1)将阳春砂根茎粉末采用水提醇沉法提取多糖粗提液,分离出沉淀后复溶,采用Sevage试剂除蛋白,干燥后得阳春砂根茎粗多糖;
S2)将所述粗多糖以阴离子交换层析法纯化,经透析和干燥后得AVRPD-1;
S3)使用Sephadex G-100凝胶柱对AVRPD-1进行分离,蒸馏水洗脱,洗脱流速为3 mL/10min,10 min/管,收集流出物经冷冻干燥后得到目标阳春砂根茎多糖AVRP-1。
2.根据权利要求1所述的阳春砂根茎多糖AVRP-1,其特征在于:所述Sevage试剂为氯仿:正丁醇体积比=4:1的混合液。
3.根据权利要求1所述的阳春砂根茎多糖AVRP-1,其特征在于:所述阴离子交换层析法使用DEAE-52纤维离子交换柱进行分离,依次使用浓度为0、0.1、0.3、0.5、1.0 M的NaCl溶液洗脱,洗脱流速为1 mL/min,10 min/管,收集0.1 M NaCl溶液洗脱下来的组分,浓缩后使用截留分子量为3500 Da的透析袋透析,透析留存物干燥得到阳春砂根茎多糖AVRPD-1。
4.根据权利要求1所述的阳春砂根茎多糖AVRP-1,其特征在于:所述阳春砂根茎多糖AVRP-1由摩尔比为4.14:4.98:5.10:23.77:17.30:28.51:16.19的甘露糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖和阿拉伯糖这7种单糖组成,糖醛酸含量为43.59%、相对分子质量为3372.01 kDa。
5.一种具有抗氧化功效的组合物,其特征在于:其添加有权利要求1~4任一项所述的阳春砂根茎多糖AVRP-1。
6.根据权利要求5所述的组合物,其特征在于:所述组合物为口服制剂。
7.根据权利要求5所述的组合物,其特征在于:所述组合物为功能性食品。
8.根据权利要求5~7任一项所述的组合物,其特征在于:还包括可接受的辅料。
9.阳春砂根茎多糖AVRP-1在制备抗氧化添加剂中的应用,其特征在于:所述阳春砂根茎多糖AVRP-1如权利要求1~4任一项所述。
10.根据权利要求9所述的应用,其特征在于:所述抗氧化添加剂应用于食品。
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CN113667032A (zh) * | 2021-09-13 | 2021-11-19 | 中山大学 | 硒化阳春砂仁多糖及其制备方法和应用 |
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