CN106565811B - 山药地上部分降血糖有效成分的提取方法及应用 - Google Patents
山药地上部分降血糖有效成分的提取方法及应用 Download PDFInfo
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Abstract
本发明公开了一种山药地上部分降血糖有效成分的提取方法及应用,属于山药地上部分有效成分的分离及应用技术领域。本发明的技术方案要点为:山药地上部分粗粉用乙醇渗漉提取并浓缩,提取物用有机溶剂萃取,然后经大孔吸附树脂、硅胶、葡聚糖糖凝胶等柱层析分离得到降血糖有效成分。本发明的山药地上部分中两种有效成分均对a‑葡萄糖苷酶均有显著的抑制活性,在相同条件下均远优于降血糖药阿卡波糖的活性。
Description
技术领域
本发明属于山药地上部分有效成分的分离及应用技术领域,具体涉及一种山药地上部分降血糖有效成分的提取方法及应用。
背景技术
由于人民生活水平的提高、饮食结构的改变以及少动多坐的生活方式等诸多因素,全球糖尿病发病率增长迅速,糖尿病已经成为继肿瘤、心血管病变之后第三大严重威胁人类健康的慢性疾病。糖尿病的发病过程中最先出现的症状往往是餐后高血糖,即糖尿病的早期征兆,特别是Ⅱ型糖尿病病人,餐后高血糖不仅极易诱发各种并发症,还会极大地提高糖尿病的死亡率。所以,降低餐后血糖是预防糖尿病、减少并发症和降低死亡率的重要措施之一。α-葡萄糖苷酶是碳水化合物消化过程的一种关键酶,它可将多糖或二糖水解成为能够供人体吸收的单糖,因此抑制α-葡萄糖苷酶的活性就能延缓食物中碳水化合物的分解,从而减少葡萄糖的摄入,达到抑制餐后血糖升高的目的。目前临床上的此类药物有阿卡波糖、伏格列波糖和米格列醇。现有的α-葡萄糖苷酶抑制剂种类较少,并伴有肠道副作用,因此人们仍不断致力于开发新型α-葡萄糖苷酶抑制剂。
山药(Disocorea opposita Thunb.)为薯蓣科薯蓣的干燥根茎,具有补脾养肺,固肾益精之功效,临床上广泛用于治疗消渴症、慢性腹泻和虚劳咳嗽等症状,是我国传统的药食同源中药之一,自古以河南焦作(古怀庆府)所产最为地道,故称“怀山药”,为著名的“四大怀药”之一。2006年河南省武陟县获批山药GAP种植基地,解决了野生山药资源短缺,资源可持续利用发展等问题。山药以地下根茎入药,地上部分常作为废弃物被遗弃,导致资源的极大浪费。目前对于山药的研究主要集中于地下根茎(入药部位)的研究,药理研究显示其具有降血糖、降血脂、抗氧化和抗肿瘤等活性。然而对于山药地上部分的研究非常少,未见其在降血糖方面的研究报道。
发明内容
本发明解决的技术问题是提供了一种山药地上部分降血糖有效成分的提取方法及应用。
本发明为解决上述技术问题采用如下技术方案,山药地上部分降血糖有效成分的提取方法,其特征在于具体步骤为:
(1)将阴干的山药地上部分粉碎后,用体积分数为90%-95%的乙醇渗漉提取,合并提取液,减压浓缩得到山药地上部分乙醇提取物,再将该乙醇提取物悬浮于蒸馏水中,依次用石油醚、乙酸乙酯和正丁醇萃取,分别收集石油醚萃取液和正丁醇萃取液,并减压浓缩得到石油醚萃取物和正丁醇萃取物;
(2)将石油醚萃取物经大孔吸附树脂柱层析,依次用体积分数为80%的乙醇和体积分数为90%的乙醇洗脱,收集体积分数为90%的乙醇洗脱液减压浓缩,得到乙醇洗脱产物,再将乙醇洗脱产物经常压硅胶柱层析,用石油醚-丙酮溶剂系统在体积比为20:1-1:1的范围内梯度洗脱,经TLC鉴定合并相同组分得到目标组分,目标组分经重结晶得到降血糖有效成分化合物1,该化合物1的结构式为;
(3)将正丁醇萃取物经大孔吸附树脂柱层析,依次用体积分数为40%的乙醇和体积分数为60%的乙醇洗脱,收集体积分数为60%的乙醇洗脱液减压浓缩,得到乙醇洗脱产物,再将乙醇洗脱产物经常压硅胶柱层析,用二氯甲烷-甲醇溶剂系统在体积比为10:1-1:1的范围内梯度洗脱,经TLC鉴定合并相同组分得到初始目标组分,将初始目标组分经ODS柱层析,用甲醇-水溶剂系统在体积比为40:60-100:0的范围内梯度洗脱,经TLC鉴定合并相同组分得到中间目标组分,将中间目标组分用葡聚糖凝胶柱色谱纯化,经甲醇洗脱得到降血糖有效成分化合物2,该化合物2的结构式为。
进一步优选,所述的大孔吸附树脂为Diaion HP-20型大孔吸附树脂,葡聚糖凝胶为Sephadex LH-20型葡聚糖凝胶。
根据上述方法提取的降血糖有效成分在制备降血糖药物或保健品中的应用。
根据上述方法提取的降血糖有效成分在制备α-葡萄糖苷酶抑制剂类药物或保健品中的应用。
本发明通过体外α-葡萄糖苷酶抑制活性研究发现山药地上部分中的两种有效成分化合物1和化合物2对α-葡萄糖苷酶均有显著的抑制作用,其IC50分别为0.37±0.0062mM和0.27±0.0027mM,明显远强于阳性对照药阿卡波糖的活性(IC50为61.57±1.40mM),并且对α-葡萄糖苷酶的抑制率均呈剂量依赖性。
附图说明
图1是实施例1提取的化合物1对α-葡萄糖苷酶抑制率随药物浓度的变化曲线;
图2是实施例2提取的化合物2对α-葡萄糖苷酶抑制率随药物浓度的变化曲线。
具体实施方式
以下通过实施例对本发明的上述内容做进一步详细说明,但不应该将此理解为本发明上述主题的范围仅限于以下的实施例,凡基于本发明上述内容实现的技术均属于本发明的范围。
实施例1
阴干的山药地上部分8.5kg,粉碎后,用体积分数为95%的乙醇渗漉提取,合并提取液,减压浓缩得总浸膏871.5g,将浸膏悬浮于2倍量水中,用5倍量的石油醚萃取,收集石油醚萃取液并减压浓缩,得到石油醚萃取物107.5g。将石油醚萃取物经Diaion HP-20型大孔吸附树脂柱层析,依次用体积分数为80%的乙醇和体积分数为90%的乙醇洗脱,收集体积分数为90%的乙醇洗脱液减压浓缩,得乙醇洗脱产物67.1g。将乙醇洗脱产物经常压硅胶柱层析,以石油醚-丙酮(体积比分别为20:1、10:1、5:1、2:1和1:1)溶剂系统洗脱,经TLC鉴定合并相同组分得到9个组分 (Fr.1-Fr.9),组分Fr.4经重结晶得到有效成分化合物1(5.4mg)。
化合物1的波谱数据:9,19-cyclolart-25-en-3β,24R-diol,白色无定形粉末,ESI-MS m/z 465 [M+Na]+。1H-NMR (400 MHz, CDCl3+DMSO) δ: 4.82 (1H, d, J = 5.5Hz, H-26), 4.70 (1H, s, H-26), 4.63 (1H, m, H-24), 4.31 (1H, d, J = 5.1 Hz,H-3), 1.62 (3H, s, H3-27), 1.24 (3H, s, H3-29), 0.93 (3H, s, H3-21), 0.86 (3H,s, H3-18), 0.85 (3H, s, H3-30), 0.71 (3H, s, H3-28), 0.47, 0.30 (each 1H, d, J = 3.5 Hz, H2-19); 13C-NMR (100 MHz, CDCl3+DMSO) δ: 148.4 (C-25), 109.7 (C-26),76.6 (C-3), 74.1 (C-24), 51.7 (C-17), 48.4 (C-14), 47.4 (C-8), 46.7 (C-5),44.8 (C-13), 40.1 (C-4), 35.4 (C-20), 35.3 (C-15), 35.1 (C-12), 32.5 (C-1),31.5 (C-23), 31.5 (C-22), 30.2 (C-2), 29.3 (C-19), 27.7 (C-16), 25.8 (C-10,11), 25.7 (C-7), 25.6 (C-28), 20.7 (C-6), 19.3 (C-9), 19.1 (C-30), 18.2 (C-21), 17.9 (C-18), 17.6 (C-27), 14.3 (C-29)。
实施例2
阴干的山药地上部分8.5kg,粉碎后,用体积分数为95%的乙醇渗漉提取,合并提取液,减压浓缩得总浸膏871.5g,将浸膏悬浮于2倍量水中,依次用5倍量的石油醚、乙酸乙酯和正丁醇萃取,收集正丁醇萃取液并减压浓缩,得到正丁醇萃取物112.0g。将正丁醇萃取物经Diaion HP-20型大孔吸附树脂柱层析,依次用体积分数为40%的乙醇和体积分数为60%的乙醇洗脱,收集体积分数为60%的乙醇洗脱液减压浓缩,得乙醇洗脱产物16.3g。将乙醇洗脱产物经常压硅胶柱层析,以二氯甲烷-甲醇(体积比分别为10:1、7:1、5:1、2:1和1:1)溶剂系统洗脱,经TLC鉴定合并相同组分得到10个组分 (Fr.1-Fr.10)。组分Fr.4经ODS柱层析,用甲醇-水(体积比分别为40:60、60:40和100:0)溶剂系统洗脱,经TLC鉴定合并相同组分得到组分Fr.4-1-Fr.4-4,组分Fr.4-3用Sephadex LH-20型葡聚糖凝胶柱色谱纯化,甲醇洗脱得到有效成分化合物2(4.5mg)。
化合物2的波谱数据:6-trans-[2''-O-(a-rhamnopyranosyl)]-ethenyl-5,7,4'-trihydroxyflavone,黄色无定形粉末,ESI-MS m/z 481 [M+Na]+。1H-NMR (400 MHz,CD3OD) δ: 7.83 (2H, d, J = 8.7 Hz, H-2', 6'), 7.61 (1H, d, J = 12.5 Hz, H-2''), 6.92 (2H, d, J = 8.7 Hz, H-3', 5'), 6.58 (1H, s, H-8), 6.50(1H, s, H-3), 6.34 (1H, d, J = 12.5 Hz, H-1''), 5.06 (1H, s, H-1rha), 3.95 (1H, d, J =3.4 Hz, H-2rha), 3.76 (1H, dd, J = 9.6, 3.4 Hz, H-3rha), 3.67 (1H, m, H-5rha),3.43 (1H, t, J = 9.5 Hz, H-4rha), 1.29 (3H, d, J = 6.2 Hz, H-6rha); 13C-NMR (100MHz, CD3OD) δ: 184.2 (C-4), 166.2 (C-7), 162.8 (C-2), 162.7 (C-5), 159.3 (C-4'), 156.8 (C-9), 148.6 (C-2''), 129.5 (C-2', 6'), 123.6 (C-1'), 117.2 (C-3',5'), 108.7 (C-6), 105.3 (C-10), 104.0 (C-3), 102.0 (C-1rha), 101,8 (C-1''),94.6 (C-8), 74.1 (C-4rha), 72.5 (C-3rha), 71.8 (C-2rha), 70.7 (C-5rha), 18.2 (C-6rha)。
实施例3
本实施例为实施例1和2提取的有效成分化合物1和化合物2对α-葡萄糖苷酶的抑制活性测试。
方法:微孔板法。
仪器:酶标仪,恒温培养箱,分析天平,各种型号移液枪。
试剂:α-葡萄糖苷酶、4-硝基苯-α-D-吡喃葡萄糖苷、阿卡波糖、磷酸盐缓冲液和二甲基亚砜。
测试方法:取96孔板,每孔加入2.0mmol/L的PNPG溶液(PBS溶解)80μL,再分别加入10μL不同浓度样品溶液(DMSO溶解),以DMSO为空白对照组,每组设3个平行孔,在酶标仪400nm下测定背景组吸光度(A值)。然后再加入10μL 1.62U/mL α-葡萄糖苷酶溶液(PBS溶解),置于恒温培养箱中,在37℃下恒温孵育30min,测定试验组吸光度(A值)。抑制率(%)用以下公式计算:
抑制率(%)= (ΔA空白-ΔA样品)/ΔA空白×100%
ΔA空白为空白组酶与底物反应后A值-背景A值;
ΔA样品为样品组酶与底物反应后A值-背景A值。
化合物1和化合物2对α-葡萄糖苷酶的抑制活性结果如下(表1):
表1 化合物1和化合物2对α-葡萄糖苷酶的抑制活性
化合物 | IC50 (mM) |
1 | 0.37±0.0062 |
2 | 0.27±0.0027 |
阿卡波糖 | 61.57±1.40 |
从表1可以看出,实施例1和2提取的有效成分化合物1和化合物2对α-葡萄糖苷酶具有明显的抑制活性,均远优于阳性对照药阿卡波糖的活性。
以上实施例描述了本发明的基本原理、主要特征及优点,本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明原理的范围下,本发明还会有各种变化和改进,这些变化和改进均落入本发明保护的范围内。
Claims (2)
1.山药地上部分降血糖有效成分的提取方法,其特征在于具体步骤为:
(1)将阴干的山药地上部分粉碎后,用体积分数为90%-95%的乙醇渗漉提取,合并提取液,减压浓缩得到山药地上部分乙醇提取物,再将该乙醇提取物悬浮于蒸馏水中,依次用石油醚、乙酸乙酯和正丁醇萃取,分别收集石油醚萃取液和正丁醇萃取液,并减压浓缩得到石油醚萃取物和正丁醇萃取物;
(2)将石油醚萃取物经大孔吸附树脂柱层析,依次用体积分数为80%的乙醇和体积分数为90%的乙醇洗脱,收集体积分数为90%的乙醇洗脱液减压浓缩,得到乙醇洗脱产物,再将乙醇洗脱产物经常压硅胶柱层析,用石油醚-丙酮溶剂系统在体积比为20:1-1:1的范围内梯度洗脱,经TLC鉴定合并相同组分得到目标组分,目标组分经重结晶得到降血糖有效成分化合物1,该化合物1的结构式为;
(3)将正丁醇萃取物经大孔吸附树脂柱层析,依次用体积分数为40%的乙醇和体积分数为60%的乙醇洗脱,收集体积分数为60%的乙醇洗脱液减压浓缩,得到乙醇洗脱产物,再将乙醇洗脱产物经常压硅胶柱层析,用二氯甲烷-甲醇溶剂系统在体积比为10:1-1:1的范围内梯度洗脱,经TLC鉴定合并相同组分得到初始目标组分,将初始目标组分经ODS柱层析,用甲醇-水溶剂系统在体积比为40:60-100:0的范围内梯度洗脱,经TLC鉴定合并相同组分得到中间目标组分,将中间目标组分用葡聚糖凝胶柱色谱纯化,经甲醇洗脱得到降血糖有效成分化合物2,该化合物2的结构式为。
2.根据权利要求1所述的山药地上部分降血糖有效成分的提取方法,其特征在于:所述的大孔吸附树脂为Diaion HP-20型大孔吸附树脂,葡聚糖凝胶为Sephadex LH-20型葡聚糖凝胶。
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