摘要
荞麦(Fagopyrum esculemum Moench)是一种适于在冷凉气候下生长的短季蓼科(polygonacoae)作物。植物分类学主要有苦荞(Fagopyrum tataricum Gaert)和甜荞(Fagopyrum esculentum Moench)两个品种。荞麦富含高生物价的蛋白质、维生素、矿物质元素、黄酮类化合物等，具有多种生物功能，如降血糖、降血脂、抗癌、抗氧化及清除自由基等。研究发现，荞麦种子中含有少量D-手性肌醇单体及大量的D-手性肌醇衍生物-荞麦糖醇(Fagopyritol)，能有效地调节血糖，改善糖尿病人(特别是Ⅱ型糖尿病人)症状。
D-手性肌醇(D-chiro-inositol，DCI)是肌醇立体异构体，它作为新一代胰岛素受体促敏剂，能有效地促进胰岛素功能，降低血糖、血甘油三酯水平等。进一步研究发现：D-手性肌醇在唯一的降血糖激素-胰岛素的信号传导过程中发挥极为重要的作用，当体内缺乏足够的D-手性肌醇时，会导致胰岛素抵抗现象。因此，补充D-手性肌醇可提高机体组织对胰岛素的敏感性，消除胰岛素抵抗，从根本上调节机体的生理机能和代谢平衡，从而降低糖尿病的发生率。
本研究首先采用三种不同的提取方法对荞麦中D-手性肌醇粗提工艺进行研究，包括不同溶剂提取法、微波法、超声波法。通过对不同提取方法的工艺条件进行研究，分别确定出最佳的工艺条件。实验结果表明，采用不同溶剂提取荞麦中D-手性肌醇时，乙醇作为溶剂提取效果最好，乙醇提取的最佳工艺条件：温度30℃，乙醇浓度50％，料液比1：20，时间1.5h，进行二级提取荞麦中D-手性肌醇的含量达到4.95mg／g；采用微波法提取荞麦中D-手性肌醇，经单因素及正交试验确定最佳工艺条件：微波功率245W，微波加热时间120s，乙醇浓度80％，料液比1：30，此时D-手性肌醇的含量达到5.11mg／g；采用超声波法时，确定最佳工艺条件：乙醇浓度50％，料液比1：15，提取时间30min，浸提温度50℃，此时D-手性肌醇的含量为5.19mg／g。通过比较三种不同的提取方法对D-手性肌醇提取效果影响，确定采用超声波法提取时，荞麦中D-手性肌醇提取效果最好。
在荞麦中D-手性肌醇粗提工艺基础上，为进一步提高荞麦提取物中D-手性肌醇的含量，去除提取物中可溶性碳水化合物及其它杂质。本研究采用两种不同方法作用于荞麦提取物进行研究。方法一采用不同种类微生物发酵荞麦提取物，增加荞麦籽粒水溶液中D-手性肌醇及其衍生物的含量，同时减少提取物中总糖含量，本研究采用酿酒酵母、毛霉、大肠杆菌、蜜蜂生球拟酵母、米曲霉、枯草芽孢杆菌、解酯假丝酵母、黑曲霉接种于荞麦提取物中进行培养，并跟踪测定提取物中各种成分的含量变化。结果表明，采用米曲霉培养5天后，荞麦提取液中D-手性肌醇含量达到6.07mg／g，且总糖含量降低较大，减少约53％，综合考虑为最符合条件的提取组分。采用活性炭柱层析纯化样品，D-手性肌醇及糖醇类物质可吸附于活性炭柱，然后进行梯度洗脱，经高效液相色谱法测定D-手性肌醇的纯度达到31.8％。此方法既简化D-手性肌醇纯化工艺，又可减少纯化过程中有机溶剂的使用量，降低生产成本。
方法二利用萌芽及酸水解方法提高荞麦中D-手性肌醇含量，荞麦中D-手性肌醇大部分以衍生物的形式存在。荞麦籽粒经萌芽后，D-手性肌醇单体含量明显增加，提高约8倍。因此，本研究采用萌芽后荞麦籽粒作为后续试验材料。酸水解使荞麦中D-手性肌醇衍生物-荞麦糖醇转化为D-手性肌醇，明显提高荞麦中D-手性肌醇的含量。同时大部分碳水化合物转化为不溶性副产物，有利于简化D-手性肌醇的纯化过程。实验结果表明，荞麦中D-手性肌醇最佳提取工艺为：HCl浓度为9N，温度为95℃，时间为18h，料液比为1：10。在此条件下，提取物中D-手性肌醇含量达到62.5mg／g，达到未萌芽、水解前提取含量的15倍左右。D-手性肌醇提取物纯化过程，用等体积水溶解，加入2倍体积95％乙醇，70℃搅拌1h，充分反应后，取出冷却至15℃，得到D-手性肌醇沉淀固体。所得沉淀物，D-手性肌醇纯度较低，可用适量的水溶解，加入乙醇溶液，通过反复的加热和冷却处理，进行重结晶。经多次的重结晶处理，D-手性肌醇的含量达到432.92mg／g。
Buckwheat which belongs to the Polygonaceace family adapts to cool, moist climates and short growingseason. Plant taxonomy mainly has two varities of Fagopyrum tataricum Gaert and Fagopyrum esculentumMoench. Buckwheat contains high biological value protein, vitamin, mineral, flavanoid and so on.Buckwheat contains many biological functions, such as degrade blood sugar, degrade blood fat, anticancer,antioxygen and eliminate free radical. Research discovers that buckwheat seed contains little DCImonomer and lots of DCI derivate- Fagopyritol. It can effectually regulate blood sugar and amelioratediabetic (especiallyⅡtype diabetic) symptom.
DCI is the stereo-isomer of inositol, as a new generation insuline acceptor which can effectuallypromote insulin function, degrade blood sugar, triglyceride level and so on. Further reserch discovers: DCIhas an important effect on the unique single conduction process of degrading blood sugar hormone-insulin.When vivo defects enough DCI, it can result in insulin resistance phenomenon. Therefore supplying withDCI can improve organism soma to insulin sensitivity and eliminate insuline resistance. Fundamental DCIaccommodates organism physiological functions and metabolic balance, so it degrades diabetic incidence.
In this study, adopting three kinds of extraction methods for researching extraction technology of DCI,methods include different solvents extraction method, microwave method and supersonic wave method.Research technology conditions with different extraction methods and definite optimal technologycondition. Experimental result indicates, when adopting different solvents extract DCI from buckwheat,solvent as alcohol has the best effect. The optimal technology condition: temperature 30℃, alcoholconcentration 50%, solid-liquid ratiol:20, time 1.5h, progress second order extraction, the highest yield ofDCI is 4.95mg/g; When microwave method extract DCI from buckwheeat, mono-factor and orthogonalexperiment that determine the optimal technology condition: microwave power 245W, microwavewarm-up time 120s, alcohol concentration 80%, solid-liquid ratio 1:30, the DCI content achieves5.09mg/g; When adopting supersonic wave method extract, the optimal technology condition, alcoholconcention 50%, solid-liquid ratio 1:15, extraction time 30min, extraction temperature 50℃, the DCIcontent achieves 5.19mg/g. Three different extraction methods is compared with influence to DCIextraction. Then determine the best effect for extracting DCI with supersonic method.
At the base of extracting crudely DCI from buckwheat, in order to improve DCI content in buckwheatextraction and reduce soluble carbohydrate and other impurities. The research adopts two differentmethods for buckwheat extraction.The first method adopts different kind microbes to ferment buckwheatextraction. The method can increase DCI and its derivation content in buckwheat seed solution, while itreduces sugar content in extraction. The research adopts Saccharomyces cerevisia, Mucor, Bacterium coli, Torulopsis apicola, Aspergillus oryzae Bacillus subtilis, Candida mycoderma, Black mold that inoculate inbuckwheat extraction. Determine content change of various compotent with tracing. Culturing withAspergillus oryzae for five days, the DCI content in buckwheat extraction that increases to 6.07mg/g, whilesugar content decrease 53%. It is the fittest condition of extraction composition for synthetic consideration.Adopting activated carbon column chromatography to purify sample, DCI and some sugar alcoholmaterials can adsorb to activated carbon column, then using gradient elution. Sample determines withHPLC, DCI content achieve 31.8%. The method can simply DCI techology of purifying, reduce solventdosage in purified process and degrade production cost.
The second method adopts sprout and acid hydrolysis method to improve DCI content in buckwheat,mostly DCI exists with derivation pattern. With sprouting buckwheat seed, DCI monomer contentincreased obviously, as eight times as before. Therefore the research uses sprouting buckwheat seed ascontinual experimental material. Acid hydrolysis causes DCI derivation invertion to DCI, obviouslyelevates DCI content in buckwheat: Meanwhile mostly carbohydrate invert to insoluble by-product, andmake simply for DCI purity procession. Experimental result indicates, the optimal technology of DCI: HClconcention 9N, temperature 95℃, time 18h, solid-liquid ratio 1: 10. At this condition, the DCI content ofextraction arrive to 62.5mg/g, about fifteen times with nonsproutand acid hydrolysis. Purify procession ofextraction that dissolves with same volume, adds 95% alcohol of two times volume, stirs 1 hour forsufficient reaction at 70℃, cools to 15℃, and gets DCI precipitate. The DCI purity of precipitate is lower,so the process that dissolves the precipitate and adds alcohol solution. Repetat heat and cool treatment,progress of recrystallization. After several recrystal treatments, the DCI content achieves 432.92mg/g.
引文
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