CN114766665A - 茯苓多糖原花青素组合物在抑制胃肠道AGEs形成中的应用 - Google Patents
茯苓多糖原花青素组合物在抑制胃肠道AGEs形成中的应用 Download PDFInfo
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Abstract
本发明公开了茯苓多糖原花青素组合物在抑制胃肠道AGEs形成中的应用，属于保健食品或药品领域。本发明发现茯苓多糖与原花青素组合使用后能显著提升胃肠道中抑制AGEs形成的效果，且两者组合的抗氧化能力显著强于单独的茯苓多糖或原花青素。茯苓多糖与原花青素或含茯苓多糖与原花青素的组合物可用于制备抑制AGEs形成或抗氧化或治疗、延缓或改善AGEs相关疾病的保健食品或药品，在抗氧化、抗衰老、抗糖尿病等领域具有广泛的应用前景。
Description
技术领域
本发明属于保健食品或药品领域，涉及茯苓多糖原花青素组合物在抑制胃肠道AGEs形成中的应用。
背景技术
食品在热加工过程中会发生美拉德反应，生成大量的美拉德反应产物，例如晚期糖化终末产物(advanced glycation end products，AGEs)。AGEs在体内有两个来源，一是通过进食将食物中存在的AGEs摄入体内，二是过量的糖和蛋白质在体内合成AGEs。AGEs与各种慢性疾病密切相关，在体内积聚会引发糖尿病的各种并发症。
茯苓多糖是茯苓主要活性成分之一，来源广泛。其具有抗肿瘤、抗炎、保肝、调节机体免疫力等药理作用，广泛应用于食品、医药及保健品等领域。茯苓多糖可以通过抑制自由基过氧化反应，降低脂质过氧化作用，减少脂褐质形成发挥延缓衰老的作用。
原花青素(oligomeric proantho cyanidins，OPC)是一种有着特殊分子结构的生物类黄酮，是目前国际上公认的清除人体内自由基有效的天然抗氧化剂。一般为红棕色粉末，气微、味涩，溶于水和大多有机溶剂。实验证明，OPC的抗自由基氧化能力是维生素E的50倍，维生素C的20倍，并吸收迅速完全，口服20分钟即可达到最高血液浓度，代谢半衰期达7小时之久。
发明内容
本发明的目的在于提供茯苓多糖原花青素组合物在抑制胃肠道AGEs形成中的应用。本发明的目的还在于提供茯苓多糖在抑制胃肠道AGEs形成中的应用。
本发明的目的通过下述技术方案实现：
本发明发现茯苓多糖在胃肠道中能抑制AGEs形成，茯苓多糖与原花青素组合使用后能显著提升胃肠道中抑制AGEs形成的效果，且两者组合的抗氧化能力显著强于单独的茯苓多糖或原花青素。基于该发现，茯苓多糖或含茯苓多糖的组合物具有抑制胃肠道AGEs形成的应用。茯苓多糖与原花青素或含茯苓多糖与原花青素的组合物具有抑制胃肠道AGEs形成的应用。茯苓多糖与原花青素或含茯苓多糖与原花青素的组合物具有制备抗氧化的保健食品或药品的应用。
进一步的，茯苓多糖或含茯苓多糖的组合物具有制备抑制AGEs形成或制备治疗、延缓或改善AGEs相关疾病的保健食品或药品的应用。茯苓多糖与原花青素或含茯苓多糖与原花青素的组合物具有制备抑制AGEs形成或制备治疗、延缓或改善AGEs相关疾病的保健食品或药品的应用。所述的AGEs相关疾病包括糖尿病、阿尔茨海默病、动脉粥样硬化等疾病。
一种抑制AGEs形成或治疗、延缓或改善AGEs相关疾病的保健食品或药品，包含茯苓多糖。
一种抑制AGEs形成或抗氧化或治疗、延缓或改善AGEs相关疾病的保健食品或药品，包含茯苓多糖和原花青素。
在一些实施方案中，所述的茯苓多糖为碱溶性茯苓多糖。
本发明的有益效果在于茯苓多糖原花青素组合物比单独的茯苓多糖或者原花青素具有更强的抗氧化能力，并且在胃肠道抑制AGEs生成的效果更显著。
本发明的茯苓多糖原花青素组合物在胃肠道表现出显著的抗AGEs生成作用和极强的自由基清除能力，在抗氧化、抗衰老、抗糖尿病等领域具有广泛的应用前景。
附图说明
图1是胃肠消化阶段的总荧光AGEs含量。
图2是胃肠消化阶段的戊糖素含量。
图3是胃肠消化阶段的精氨嘧啶含量。
图4是胃肠消化阶段的吡啶类衍生物含量。
图5是胃肠消化阶段的交联素含量。
图6是样品的DPPH自由基清除率。
图7是样品的ABTS自由基清除率。
图8是样品的HRSA自由基清除率。
图1-5中，a-d表示组内差异，A-D表示组间差异，不同字母表示差异有统计学意义(p<0.05)；
图6-7中，a-d不同字母表示差异有统计学意义(p<0.05)；
图8中，a-c不同字母表示差异有统计学意义(p<0.05)。
具体实施方式
以下实施例用于进一步说明本发明，但不应理解为对本发明的限制。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
下述实施例中所使用的茯苓多糖为碱溶性茯苓多糖，其通过下述方法制备得到：将茯苓粉加入到石油醚(固液比1:5)中于60℃水浴中加热回流脱脂12h，收集残渣，并将溶剂挥干。再加入80％乙醇(固液比1:5)于50℃下，磁力搅拌30min，离心(4000rpm，10min)，收集残渣，此过程重复几次，直至上清液无色，旋蒸将无水乙醇蒸出，得脱脂茯苓粉末。将其置于烧杯中，加入少量蒸馏水，搅拌使其充分混匀，以1:50固液比于80℃水浴中浸提1h，过程中多次搅拌，并对残渣重复水提一次，离心以去除水溶性物质，收集残渣。以1:50固液比例加入0.6mol/L氢氧化钠溶液，于4℃下静置提取8h。离心得上清液，并用10％的醋酸溶液中和至pH＝6，以大量蒸馏水、无水乙醇、丙酮洗涤胶状沉淀反复脱盐后冷冻干燥得到碱溶性茯苓多糖。
下述实施例中所使用的原花青素通过下述方法制备得到：通过已经建立的原花青素提取工艺(中国专利ZL 02115423.6)以莲房为原料进行提取，得到原花青素粗提物，将原花青素粗提取物用乙酸乙酯萃取3次，收集乙酸乙酯相，在40℃减压旋转蒸发浓缩，冷冻干燥，得到精提原花青素，用于下述实验。
下述实施例中所配制的相关溶液，如未说明均为用水配制。
实施例1：AGEs生成抑制剂筛选
(1)在4mL人工唾液(simulated salivary fluid，SSF，配制方法见表1)储备液中分别加入原花青素、茯苓多糖、原花青素茯苓多糖组合物(原花青素、茯苓多糖质量比1∶2)，终浓度保持在0.3g/L。再加入25μL 0.3mol/L的CaCl2、加超纯水补至5mL。加入200mg牛血清白蛋白(bovine serum albumin，BSA)、11.36μL 8.8mol/L的乙二醛(glyoxal，GO)，建立牛血清白蛋白-乙二醛模型。混匀2分钟，作为口腔组。对照组不含原花青素、茯苓多糖、原花青素茯苓多糖组合物。
表1模拟消化液原液的制备
| 成分 | SSF(mmol/L) | SGF(mmol/L) | SIF(mmol/L) |
| KCl | 15.1 | 6.9 | 6.8 |
| KH<sub>2</sub>PO<sub>4</sub> | 3.7 | 0.9 | 0.8 |
| NaHCO<sub>3</sub> | 13.6 | 25 | 85 |
| NaCl | — | 47.2 | 38.4 |
| MgCl<sub>2</sub>(H<sub>2</sub>O)<sub>6</sub> | 0.15 | 0.1 | 0.33 |
| (NH<sub>4</sub>)<sub>2</sub>CO<sub>3</sub> | 0.06 | 0.5 | — |
(2)在步骤(1)中加入3.75mL人工胃液(simulated gastric fluid，SGF，配制方法见表1)储备液，用0.1mol/L HCl溶液调pH值至2.0。然后加入0.25mL胃蛋白酶(80000U/mL)、2.5μL 0.3mol/L的CaCl2，最后加超纯水补至10mL，在37℃恒温振荡箱内进行消化2h，作为胃消化组。
(3)在步骤(2)中加入5.5mL人工肠液(simulated intestinal fluid，SIF，配制方法见表1)储备液，然后加入2.5mL胰酶(800U/mL)、1.25mL新鲜猪胆盐(160mmol/L)、20μL0.3M CaCl2，用NaOH(1mol/L)调pH值至中性灭酶。用超纯水补至20mL，置于37℃恒温振荡箱内进行消化2h，消化结束后煮沸灭酶，作为肠消化组。
(4)采用BSA-GO模型，用日立F-4700荧光分光光度计在激发波长(λex)＝370nm和发射波长(λem)＝440nm处的荧光强度测定样品不同阶段AGEs的含量，也在不同的激发/发射波长下对四种特定的AGE产物(戊糖素335nm/385nm，精氨嘧啶335nm/400nm，吡啶类衍生物366nm/442nm和交联素379nm/463nm)进行了测定。
AGEs抑制率(％)＝(1-A样/A对照)×100％
结果见图1-5和表2。
图1为胃肠消化阶段的总荧光AGEs含量图，横坐标代表消化时间，纵坐标代表荧光值，口腔阶段0h，胃阶段1h、2h，肠阶段3h、4h。0h～1h产生荧光AGEs较少，从第2h开始，荧光AGEs大量生成。茯苓多糖和原花青素对荧光AGEs均有抑制能力，但茯苓多糖原花青素组合物的抑制能力比单独的茯苓多糖和原花青素的抑制能力更强。
四种特定的AGEs产物在胃肠消化阶段的荧光值如图2-5所示，茯苓多糖和原花青素对四种特定的AGEs产物均有抑制能力，但茯苓多糖原花青素组合物的抑制能力均比单独的茯苓多糖和原花青素的抑制能力更强。
表2胃肠消化阶段的荧光AGEs抑制率
注：上标字母不同的同类别组间数据差异有统计学意义(p<0.05)。
由表2可以看出，在胃阶段和肠阶段，茯苓多糖和原花青素都可以抑制荧光AGEs生成，但茯苓多糖和原花青素的荧光AGEs抑制率均弱于茯苓多糖原花青素组合物，说明在胃肠消化阶段茯苓多糖原花青素组合物较于单一茯苓多糖原花青素显著提高了抗AGEs能力。
实施例2：1,1-diphenyl-2-picrylhydrazyl(DPPH)自由基清除率
方法如下：
(1)将上述用水稀释2倍后的肠消化组样品溶液0.2mL与3.8mL 0.1mmol/L DPPH乙醇溶液混合，作为样品组。
(2)将上述用水稀释2倍后的肠消化组样品溶液0.2mL与3.8mL乙醇溶液混合，作为对照组。
(3)将0.2mL H2O与3.8mL 0.1mmol/L DPPH乙醇溶液混合，作为空白组。
(4)将0.2mL H2O与3.8mL乙醇溶液混合，用来调零。
(5)将步骤(1)、(2)、(3)、(4)各组溶液放入黑暗条件下反应30min。
(6)用酶标仪测定各组反应液在波长517nm处的吸光值，样品组、对照组、空白组数据均需减去调零。
DPPH自由基清除能力(％)＝[1-(A样-A对照)/A空白]×100％。
结果如图6所示，对照组未加抑制剂，无DPPH自由基清除能力，茯苓多糖DPPH自由基清除率为13.73％，原花青素DPPH自由基清除率为28.23％，茯苓多糖原花青素组合物DPPH自由基清除率为47.57％。该组合物的DPPH自由基清除能力强于原花青素和茯苓多糖DPPH自由基清除能力，说明原花青素和茯苓多糖在清除DPPH自由基方面具有协同作用。
实施例3：2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt(ABTS)清除率
方法如下：
(1)配制7.4mmol/L ABTS。
(2)配制2.6mmol/L K2S2O8。
(3)取步骤(1)、(2)配制的溶液各0.2mL混合，在黑暗环境下，室温反应12h。
(4)用甲醇稀释30倍，酶标仪测定734nm处的值为0.7±0.02。
(5)取步骤(4)溶液1mL分别与0.1mL甲醇和用水稀释2倍后的上述肠消化组样品溶液0.1mL混合10s，摇匀，静置6min。分别作为对照组和样品组。
(6)用酶标仪测定各组反应液在波长734nm处的吸光值，用甲醇调零。
ABTS清除率(％)＝(A对照-A样)/A对照×100％。
结果如图7所示，对照组未加抑制剂，无ABTS清除能力，茯苓多糖ABTS清除率为22.6％，原花青素ABTS清除率为58.7％，茯苓多糖原花青素组合物ABTS清除率为80.95％。相较于单独的原花青素和茯苓多糖，该组合物的ABTS清除能力更显著。
实施例4：羟自由基(Hydroxyl radical-scavenging activity，HRSA)清除率
方法如下：
(1)1.5mL上述肠消化组样品溶液中加入1.5mL 9mmol/L FeSO4溶液、1.5mL9mmol/L水杨酸-乙醇溶液、1.5mL 8.8mmol/L H2O2(Ax组)。
(2)1.5mL H2O中加入1.5mL 9mmol/L FeSO4溶液、1.5mL 9mmol/L水杨酸-乙醇溶液、1.5mL 8.8mmol/L H2O2(A0组)。
(3)1.5mL样品中加入1.5mL 9mmol/L FeSO4溶液、1.5mL 9mmol/L水杨酸-乙醇溶液、1.5mL H2O(Ax0组)。
(4)步骤(1)、(2)、(3)各组溶液混匀后分别在37℃下反应10min。
(5)用酶标仪测定各组反应液在波长510nm处的吸光值，用H2O调零。
HRSA清除率(％)＝[A0-(Ax-Ax0)]/A0×100％。
结果如图8所示，茯苓多糖几乎没有HRSA自由基清除能力，原花青素HRSA自由基清除率为52.02％，茯苓多糖原花青素组合物HRSA自由基清除率为86.68％。该组合物的HRSA自由基清除能力强于原花青素和茯苓多糖HRSA自由基清除能力，说明原花青素和茯苓多糖在清除HRSA自由基方面具有协同作用。
以上所述仅表达了本申请的具体实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本申请技术方案构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。
Claims (10)
1.茯苓多糖或含茯苓多糖的组合物在抑制胃肠道AGEs形成中的应用。
2.茯苓多糖与原花青素或含茯苓多糖与原花青素的组合物在抑制胃肠道AGEs形成中的应用。
3.茯苓多糖与原花青素或含茯苓多糖与原花青素的组合物在制备抗氧化的保健食品或药品中的应用。
4.茯苓多糖或含茯苓多糖的组合物在制备抑制AGEs形成的保健食品或药品中的应用。
5.茯苓多糖或含茯苓多糖的组合物在制备治疗、延缓或改善AGEs相关疾病的保健食品或药品中的应用。
6.茯苓多糖与原花青素或含茯苓多糖与原花青素的组合物在制备抑制AGEs形成的保健食品或药品中的应用。
7.茯苓多糖与原花青素或含茯苓多糖与原花青素的组合物在制备治疗、延缓或改善AGEs相关疾病的保健食品或药品中的应用。
8.一种抑制AGEs形成或治疗、延缓或改善AGEs相关疾病的保健食品或药品，其特征在于：包含茯苓多糖。
9.一种抑制AGEs形成或抗氧化或治疗、延缓或改善AGEs相关疾病的保健食品或药品，其特征在于：包含茯苓多糖和原花青素。
10.根据权利要求1-7任一项所述的应用或权利要求8或9所述的保健食品或药品，其特征在于：所述的茯苓多糖为碱溶性茯苓多糖。
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