魔芋葡甘露聚糖的超声提取及高效液相色谱分析
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摘要
魔芋学名为Amorphophallus rerieri，为单子叶植物纲，天南星科耐荫性多年生草本植物的块茎。魔芋精粉是由鲜魔芋块茎经过干燥、机械粉碎、风选等工序得到的一种精细产品。它的主要有效成分为魔芋葡甘露聚糖(Konjac Glucomannan，KGM，简称葡甘聚糖)。KGM是由D-葡萄糖和D-甘露糖残基通过β-1，4糖苷键聚合而成的高分子多糖，它具有独特的流变性、增稠性、胶凝性和成膜性，在食品、医药、化工等方面具有很好的应用价值。
KGM含量是评价魔芋精粉质量的一个关键指标，国内外研究证实，KGM是一种优良的低热量、低脂肪、高纤维素的水溶性膳食纤维，对营养不平衡有重要调节作用。由于其具有降血糖、降血脂、降血压、逆转脂肪肝、通便减肥、抗癌、增强免疫功能及抗衰老等医药保健功效，因而引起全世界的高度重视。
目前，KGM多采用35℃水浴水浸提取法，提取时间长、提取率低，制约了KGM的规模生产和广泛应用。因此，有必要建立一种提取率高，简便易行的方法。近年来，超声波在天然产物提取中已经显示出越来越多的优势，利用其机械性能、热学性能和强大的空化效应，可加速细胞内物质的释放、扩
第四军医大学硕士研究生学位论文
一
散及溶解，提高提取效率。因此，本文用探头式超声仪和水浴式超声仪（超
声波清洗器）对KGM的提取进行了实验研究，并分别与常规水提法进行了比
较。结果表明，前者固定超声频率20kHz，功率80W，作用时间7min时为最
佳操作条件，超声法比常规法提取液中KGM含量明显提高”（HO．01），提高
率为10．sl％；后者固定超声频率40kHZ，功率100W，超声水提lh，KGM含量明
显高于常规水提4h（HO．001），提高率为6．5％。说明超声水提法可提高魔
芋精粉中KGM的提取效率。同时进行了方法学考察，结果表明精密度、回收
率良好；线性关系和重现性亦较好。
KGM的测定方法有重量法、分光光度法、旋转豌度法、薄层色谱法、气
相色谱法（GC）。这些方法或多或少地存在着不同的缺陷，重量法虽不需要大
型仪器，但也较繁琐，需要多次烘烤，且由于甘露糖腺沉淀不完全会引起测
定结果偏低；薄层色谱法虽测定结果较准确，但制板用时长，且也需要较昂
贵的薄层扫描仪；分光光度法测定结果也较准确，但魔芋精粉中同时含有KGM
和淀粉，用酸处理均可水解为葡萄糖和甘露糖，因此也会给测定结果带来误
差；气相色谱法的重现性和准确性也较好，并能分别测出多糖的各种单糖含
量，主要困难是糖类本身没有足够的挥发性，必须将其转化为挥发性衍生物
才能进行测定且要求条件高，其操作麻烦、费时，且需要昂贵仪器。
高效液相色谱法（HPLC）测定植物多糖及单糖组成的报道较多，它具有
快速、方便、分辨率高、分离效果好、重现性好和不破坏样品等优点，特别
适用于某些糖类的测定。HPLC测定糖类化合物时，糖本身在紫外区无吸收，
多采用示差折光检测器门I）检测，但它的检测灵敏度低且不能用于梯度洗
脱。通过衍生使糖类化合物变成具有紫外吸收或荧光物质，是提高糖类HPLC
检测灵敏度的主要手段。HPLC测定样品中的单糖可用糖分析柱和氨基键合相
柱，但其价格昂贵，分析成本高，尤其是糖分析专用柱。
为此，本文用硫酸将KGM水解为葡萄糖和甘露糖，使其与卜苯基－3－甲
3
第四军医大学硕士研究生学位论文
一
基－卜毗哇咐酮（PMP）衍生化后，选用价格较低的C；。硅胶柱，进行高效液
相色谱法测定 KGM含量及其组成分析。色谱条件：ZORBAX Extend－C；s分析柱，
250 urn紫外检测，不同浓度的乙氰磷酸盐缓冲液梯度洗脱，流速 1．0 mL·min‘，
时间 30 min。测定时取不同体积的葡萄糖甘露糖标准混合液（加内标物鼠李
糖），室温真空干燥，用 PMP衍生化等一系列处理后离心，取上清液 10 p L进
行HPLC分析。以标准单糖衍生物峰面积与内标物峰面积之比为Y，标准单糖
的量（nmol）为X作图作标准曲线，其相关系数r均大于0．999，相同体积
的葡萄糖、甘露糖衍生化产物进样5次，葡萄糖、甘露糖、鼠李糖的进样精
密度（RSD，n＝5）分别为0．25％，0．09％，0．52％。不同体积的葡萄糖、甘露糖
和鼠李糖衍生物进样精密度分别为1．29％，0．65％，1．19％。结果表明，KGM
是由葡萄糖和甘露糖组成，H者物质的量比为1：1．67，魔芋精粉中KGM的
含量为79．54％。可见，高效液相色谱可以同时测定KGM含量并判断其组成成
分。
本实验结果表明，超声波能够缩短KGM的提取时间，提高提出率，且精
密度，回收率，重现性和线性关系均较好。经过摸索我们选用价格较低的C；s
硅胶柱，分段梯度洗脱，250 urn紫外检测，通过对操作条件的优化，30 min
分离测定了3种单糖，用于KGM中单糖的测定获得了满意结果，可用于魔芋
精粉的质量评价。
Amorphophallus rerieri is one of shade tolerance Perennial herb stem tubers, which belongs to Monocotyledoneae, Araceae. Konjac refined flour is a fine product made of fresh Amorphophallus rerieri stem tuber processed by drying, mechanical pulverizing and wind picking. Its main ingredient is Konjac Glucomannan (KGM), which is a macromolecule polysaccharide conjugated by D-glucose and D-Mannose residue with P-1,4 glucosidic bond. It has lots of unique characteristics such as rheological, thickening, gel formation and phragmoid character, so it is practical valuable in foodstuff, medicine and chemical engineering area.
Content of KGM is a key index to judge the quality of Konjac refined flour. Studies at home and aboard demonstrate that KGM is a kind of excellent low calorie, low fat and high cellulose hydrosoluble food fiber, which has a very
important modulating effect on nutritious imbalance. It is helpful to the health of the people worldwide for its blood sugar lowering, blood lipid lowering, blood pressure lowering, fatty liver reversing, purging, body weight reducing, anti-cancer effects, immunological function enhancing and anti-consenescence effects.
Nowadays, KGM is mainly extracted by 35 C water bathing and soaking Extraction, which needs longer time and gets lower extract rate, hence restricts the production and widespread application of KGM. Therefore, it is necessary to establish an efficient and easy method evaluating its quality. Ultrasonic wave shows its superiority in extraction of natural products in resent years. It can improve the extract effect by its mechanical function, thermotics function and powerful cavitation effect to accelerate intracellular material' s release, diffusion and dissolving. We investigate the extraction method for KGM by means of head ultrasonic instrument and water bathing ultrasonic instrument (ultrasonic wave washer), and compare them with conventional water extract method, respectively. The result shows the former can increase content of KGM in extract liquid by 10.81%, more than conventional water extract method (p<0.01), the optimal performance condition was fixed as follows: ultrasonic frequency 20kHz, power
80W, 7min. The latter can increase content of KGM by 6.5% using fixed
ultrasonic frequency 40kHz, power 100W, Ih compared with conventional water extract method for 4h (p<0.001), These results demonstrated that ultrasonic wave water extract method could improve extract effect of KGM in Konjac refined flour. In the mean time, the methodology studies showed that the elegant precision, recycle rate, better linear relation and repeatability have been obtained. Nowadays, there are many methods to determine the content of KGM such as weighing method, spectrophotography, circumrotating viscosimetry, thin-layer chromatography and gas chromatography (GC). However, all these methods have their defects. For example, weighing method needs to be roasted many times even though it doesn't need expensive equipment, which is relatively tedious and may result in lower content because of incomplete deposition of mannohydrazone; thin-layer chromatography can get more nicety results, but it needs longer time to make board, in addition it also needs expensive thin-layer scaning densitometer; spectrophotography can also get precise results, but KGM and starch coexist in Konjac refined flour, when they are processed by acid, both of them can be dehydrated into glucose and mannose,leading to error of the experimental results; Gas chromatography has better repeatability and precision, and it can determine various monosaccharide's content of polysaccharide. The main problem is that saccharide itself doesn't have enough volatility, it must be transformed into
volatility derivative to be determined, precise condition, complex manipulation, longer time and expensive equipment must be needed.
There are many reports concerned with determining plant's monosaccharide and polysaccharide by high performance liquid chromatography (HPLC), which has the characteri
KGM含量是评价魔芋精粉质量的一个关键指标，国内外研究证实，KGM是一种优良的低热量、低脂肪、高纤维素的水溶性膳食纤维，对营养不平衡有重要调节作用。由于其具有降血糖、降血脂、降血压、逆转脂肪肝、通便减肥、抗癌、增强免疫功能及抗衰老等医药保健功效，因而引起全世界的高度重视。
目前，KGM多采用35℃水浴水浸提取法，提取时间长、提取率低，制约了KGM的规模生产和广泛应用。因此，有必要建立一种提取率高，简便易行的方法。近年来，超声波在天然产物提取中已经显示出越来越多的优势，利用其机械性能、热学性能和强大的空化效应，可加速细胞内物质的释放、扩
第四军医大学硕士研究生学位论文
一
散及溶解，提高提取效率。因此，本文用探头式超声仪和水浴式超声仪（超
声波清洗器）对KGM的提取进行了实验研究，并分别与常规水提法进行了比
较。结果表明，前者固定超声频率20kHz，功率80W，作用时间7min时为最
佳操作条件，超声法比常规法提取液中KGM含量明显提高”（HO．01），提高
率为10．sl％；后者固定超声频率40kHZ，功率100W，超声水提lh，KGM含量明
显高于常规水提4h（HO．001），提高率为6．5％。说明超声水提法可提高魔
芋精粉中KGM的提取效率。同时进行了方法学考察，结果表明精密度、回收
率良好；线性关系和重现性亦较好。
KGM的测定方法有重量法、分光光度法、旋转豌度法、薄层色谱法、气
相色谱法（GC）。这些方法或多或少地存在着不同的缺陷，重量法虽不需要大
型仪器，但也较繁琐，需要多次烘烤，且由于甘露糖腺沉淀不完全会引起测
定结果偏低；薄层色谱法虽测定结果较准确，但制板用时长，且也需要较昂
贵的薄层扫描仪；分光光度法测定结果也较准确，但魔芋精粉中同时含有KGM
和淀粉，用酸处理均可水解为葡萄糖和甘露糖，因此也会给测定结果带来误
差；气相色谱法的重现性和准确性也较好，并能分别测出多糖的各种单糖含
量，主要困难是糖类本身没有足够的挥发性，必须将其转化为挥发性衍生物
才能进行测定且要求条件高，其操作麻烦、费时，且需要昂贵仪器。
高效液相色谱法（HPLC）测定植物多糖及单糖组成的报道较多，它具有
快速、方便、分辨率高、分离效果好、重现性好和不破坏样品等优点，特别
适用于某些糖类的测定。HPLC测定糖类化合物时，糖本身在紫外区无吸收，
多采用示差折光检测器门I）检测，但它的检测灵敏度低且不能用于梯度洗
脱。通过衍生使糖类化合物变成具有紫外吸收或荧光物质，是提高糖类HPLC
检测灵敏度的主要手段。HPLC测定样品中的单糖可用糖分析柱和氨基键合相
柱，但其价格昂贵，分析成本高，尤其是糖分析专用柱。
为此，本文用硫酸将KGM水解为葡萄糖和甘露糖，使其与卜苯基－3－甲
3
第四军医大学硕士研究生学位论文
一
基－卜毗哇咐酮（PMP）衍生化后，选用价格较低的C；。硅胶柱，进行高效液
相色谱法测定 KGM含量及其组成分析。色谱条件：ZORBAX Extend－C；s分析柱，
250 urn紫外检测，不同浓度的乙氰磷酸盐缓冲液梯度洗脱，流速 1．0 mL·min‘，
时间 30 min。测定时取不同体积的葡萄糖甘露糖标准混合液（加内标物鼠李
糖），室温真空干燥，用 PMP衍生化等一系列处理后离心，取上清液 10 p L进
行HPLC分析。以标准单糖衍生物峰面积与内标物峰面积之比为Y，标准单糖
的量（nmol）为X作图作标准曲线，其相关系数r均大于0．999，相同体积
的葡萄糖、甘露糖衍生化产物进样5次，葡萄糖、甘露糖、鼠李糖的进样精
密度（RSD，n＝5）分别为0．25％，0．09％，0．52％。不同体积的葡萄糖、甘露糖
和鼠李糖衍生物进样精密度分别为1．29％，0．65％，1．19％。结果表明，KGM
是由葡萄糖和甘露糖组成，H者物质的量比为1：1．67，魔芋精粉中KGM的
含量为79．54％。可见，高效液相色谱可以同时测定KGM含量并判断其组成成
分。
本实验结果表明，超声波能够缩短KGM的提取时间，提高提出率，且精
密度，回收率，重现性和线性关系均较好。经过摸索我们选用价格较低的C；s
硅胶柱，分段梯度洗脱，250 urn紫外检测，通过对操作条件的优化，30 min
分离测定了3种单糖，用于KGM中单糖的测定获得了满意结果，可用于魔芋
精粉的质量评价。
Amorphophallus rerieri is one of shade tolerance Perennial herb stem tubers, which belongs to Monocotyledoneae, Araceae. Konjac refined flour is a fine product made of fresh Amorphophallus rerieri stem tuber processed by drying, mechanical pulverizing and wind picking. Its main ingredient is Konjac Glucomannan (KGM), which is a macromolecule polysaccharide conjugated by D-glucose and D-Mannose residue with P-1,4 glucosidic bond. It has lots of unique characteristics such as rheological, thickening, gel formation and phragmoid character, so it is practical valuable in foodstuff, medicine and chemical engineering area.
Content of KGM is a key index to judge the quality of Konjac refined flour. Studies at home and aboard demonstrate that KGM is a kind of excellent low calorie, low fat and high cellulose hydrosoluble food fiber, which has a very
important modulating effect on nutritious imbalance. It is helpful to the health of the people worldwide for its blood sugar lowering, blood lipid lowering, blood pressure lowering, fatty liver reversing, purging, body weight reducing, anti-cancer effects, immunological function enhancing and anti-consenescence effects.
Nowadays, KGM is mainly extracted by 35 C water bathing and soaking Extraction, which needs longer time and gets lower extract rate, hence restricts the production and widespread application of KGM. Therefore, it is necessary to establish an efficient and easy method evaluating its quality. Ultrasonic wave shows its superiority in extraction of natural products in resent years. It can improve the extract effect by its mechanical function, thermotics function and powerful cavitation effect to accelerate intracellular material' s release, diffusion and dissolving. We investigate the extraction method for KGM by means of head ultrasonic instrument and water bathing ultrasonic instrument (ultrasonic wave washer), and compare them with conventional water extract method, respectively. The result shows the former can increase content of KGM in extract liquid by 10.81%, more than conventional water extract method (p<0.01), the optimal performance condition was fixed as follows: ultrasonic frequency 20kHz, power
80W, 7min. The latter can increase content of KGM by 6.5% using fixed
ultrasonic frequency 40kHz, power 100W, Ih compared with conventional water extract method for 4h (p<0.001), These results demonstrated that ultrasonic wave water extract method could improve extract effect of KGM in Konjac refined flour. In the mean time, the methodology studies showed that the elegant precision, recycle rate, better linear relation and repeatability have been obtained. Nowadays, there are many methods to determine the content of KGM such as weighing method, spectrophotography, circumrotating viscosimetry, thin-layer chromatography and gas chromatography (GC). However, all these methods have their defects. For example, weighing method needs to be roasted many times even though it doesn't need expensive equipment, which is relatively tedious and may result in lower content because of incomplete deposition of mannohydrazone; thin-layer chromatography can get more nicety results, but it needs longer time to make board, in addition it also needs expensive thin-layer scaning densitometer; spectrophotography can also get precise results, but KGM and starch coexist in Konjac refined flour, when they are processed by acid, both of them can be dehydrated into glucose and mannose,leading to error of the experimental results; Gas chromatography has better repeatability and precision, and it can determine various monosaccharide's content of polysaccharide. The main problem is that saccharide itself doesn't have enough volatility, it must be transformed into
volatility derivative to be determined, precise condition, complex manipulation, longer time and expensive equipment must be needed.
There are many reports concerned with determining plant's monosaccharide and polysaccharide by high performance liquid chromatography (HPLC), which has the characteri
引文
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[2]李恒，中国植物志(第十三卷第二分册)[M] 北京：科学出版社，1997，84-89
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