【作者】 姚秋萍；

【导师】 黄一帆；

【作者基本信息】 福建农林大学 ， 农产品加工及贮藏工程， 2009， 博士

【摘要】 油菜花粉是一种丰富的花粉资源,具有提高免疫活性、降血脂、抗肿瘤等生物学功能。目前对油菜花粉的研究侧重于基础和应用研究,而对于深入系统的研究较少。本研究以油菜花粉为实验材料,采用超微粉碎技术使花粉破壁,以普通粉碎方式作为对照,对比了两种粉碎方式对所得的超微粉和细粉中主要营养成分、有效成分的影响,及组织形态学上的差异;比较油菜花粉超微粉、细粉中槲皮素和山奈酚在实验动物体内的药代动力学参数;对油菜花粉超微粉多糖进行分级,筛选抗氧化和免疫活性部位,对活性部位多糖进行结构表征;建立油菜花粉超微粉黄酮类成分的HPLC指纹图谱,使这一丰富资源的开发和应用提高到新的水平,主要结果如下:1.分析比较了超微粉碎和普通粉碎方式对油菜花粉主要营养成分含量的影响,并比较了扫描电镜下超微粉和细粉形态学上的差异。结果表明:除粗纤维含量降低外,粗蛋白、粗脂肪、灰分、还原糖和总黄酮含量都有不同程度的提高。在电镜下观察,油菜花粉细粉基本可以看到完整的细胞形态,而超微粉的颗粒变小、大小均匀,绝大多数细胞壁破裂,基本无完整的细胞存在,破壁率为98.86%2.分别采用高效液相色谱法、比色法分析比较了油菜花粉超微粉和细粉中黄酮类成分（槲皮素、山奈酚）、不同浸提时间下多糖的溶出差异。结果表明:在相同的提取条件下,油菜花粉超微粉中槲皮素和山奈酚的溶出率比细粉分别提高了45.16%和27.86%。油菜花粉超微粉多糖的溶出量和溶出速度都大于细粉,浸提时间为10min时多糖的溶出率较细粉提高了252.54%;红外光谱结果显示油菜花粉超微粉和细粉多糖的主要基团无明显差异。超微粉碎可显著提高油菜花粉有效成分的溶出率。3.采用高效液相色谱法测定家兔灌胃给予油菜花粉超微粉和细粉后血浆中槲皮素和山奈酚的浓度,血药浓度-时间数据经药代动力学分析软件（PKS软件）处理,比较油菜花粉超微粉和细粉中槲皮素和山奈酚在家兔体内的药代动力学参数。结果表明:油菜花粉超微粉和细粉中槲皮素和山奈酚的药代动力学最佳模型均为二室开放模型。油菜花粉超微粉槲皮素的主要药动学参数:Ka=0.5793h^-1, t1/2α=2.43h,t1/2β=18.33h,AUC0→∞=0.3613μg·L^-1·h,Vd =804.8 L·kg^-1,Tpeak =2.5714h,Cmax=0.01908μg·mL^-1;油菜花粉细粉槲皮素的主要药动学参数:Ka=1.2790h^-1,t1/2α=2.63 h,t1/2β= 28.00h,AUC0→∞= 0.3411μg·L^-1·h,Vd = 997.3 L·kg^-1,Tpeak =3.048 h , Cmax =0.018769μg·mL^-1。油菜花粉超微粉山奈酚的主要药动学参数: Ka=0.4136h^-1, t1/2α=2.29h,t1/2β=2.97h,AUC0→∞=1.8897μg·L^-1·h,Vd =33.056L·kg^-1,Tpeak =3.6429h,Cmax=0.28079μg·mL^-1;油菜花粉细粉山奈酚的主要药动学参数:Ka=0.44362h^-1 , t1/2α=1.74 h,t1/2β= 1.99h,AUC0→∞=1.4599μg·L^-1·h,Vd = 31.418 L·kg^-1,Tpeak =3.7143 h,Cmax =0.24553μg·mL^-1。与细粉组比较,油菜花粉超微粉组血浆槲皮素和山奈酚的达峰时间缩短、达峰浓度提高,相对生物利用度较细粉分别提高了46.00%、29.44%。超微粉碎技术可提高油菜花粉有效成分槲皮素和山奈酚的生物利用度。4.采用不同截留分子量（100kD、50kD和10kD）的超滤膜对油菜花粉超微粉粗多糖进行分级纯化,分析了压力和时间对超滤过程的影响,确定最适操作条件,3种截留分子量的超滤膜运行时间分别控制在28、36和48min以内,操作压力分别为1.1、1.3和1.6MPa。油菜花粉超微粉粗多糖M按分子量分成M1>100kD、50kD<M2<100kD、10kD<M3<50kD及M4<10kD 4个级别,所得多糖质量分布比例约为2.3∶1∶1.1∶1.6,并获得各段分子量的多糖制品纯度均高于68%。通过体外抗氧化、体内抗氧化和免疫活性试验相结合对分级的产物跟踪筛选活性组分。体外清除（·OH）和（O2-·）的试验结果表明,油菜花粉多糖的抗氧化活性与其分子量分布密切相关,M3、M4两个级分的抗氧化作用较低,在后续试验中合并作为一个组分M3进行研究。由体内抗氧化、免疫活性试验筛选得到高活性组分M2和低活性组分M3,M2级分多糖较其它组分有较好的抗氧化和免疫活性,各组分的抗氧化和免疫活性大小顺序为M2>M>M1>M3。5. M2级分多糖先后经DEAE- SephadexA -25离子交换柱层析和Sephadex G^-100凝胶柱层析纯化得到RPP1-2。经高效液相凝胶色谱鉴定其纯度及计算分子量,RPP1-2为单一对称峰,重均分子量为65358Da。离子色谱测定RPP1-2中-SO42-含量为2.81%。紫外光谱、红外光谱和核磁共振分析结果表明,RPP1-2为糖蛋白缀合物,为α-糖苷键构型,分子结构中存在α-（1,3）、α-（1,6）糖苷键,RPP1-2由3种不同单糖残基组成的杂多糖,糖链中重复单位包含葡萄糖、甘露糖和半乳糖。6.采用高效液相色谱法对不同产地的油菜花粉超微粉的指纹图谱进行比较,应用中药色谱指纹图谱相似度评价系统软件对油菜花粉超微粉黄酮类成分HPLC指纹图谱进行分析评价。以芦丁为内标物,采用Diamonsil C18柱（250×4.6 mm,5μm）为分析色谱柱,乙腈-0.4%磷酸水溶液为流动相梯度洗脱,流速0.8 mL·min^-1,检测波长254 nm,柱温25℃。结果表明: 10批不同产地的油菜花粉超微粉黄酮类成分HPLC指纹图谱较相似,各成分得到较好的分离,并根据检测结果确定了10个共有指纹峰。该方法具有重现性好,特征性强,方法简便、快速等特点,能够客观反映油菜花粉超微粉的指纹特征,可以成为油菜花粉超微粉质量评价及控制的表征指标。更多还原

【Abstract】 Rape pollen, which had rich resources in China, was well-known for its bioactive functions such as enhancing immunity, hypoglycemic, antitumor, etc. Most of studies were performed on basis of the fundamental and applied researches, furtherly systematic researches on rape pollen were raraly reported. In this paper, ultra-fine powder technique was applied to break wall in rape pollen, in contrast of common powder technique. The effects of nutritional components, effective components, and the microscopic characteristics difference between ultra-fine powder and common powder were contrasted. Pharmacokinetics characteristics of both ultra-fine powdered and common powdered rape pollen were compared. The polysaccharide of ultra-fine powdered rape pollen were fractioned to get active part by anoxidation and immune tests, and the primary structure of active polysaccharide was studied . The fingerprint on flavonoids of ultra-fine powdered rape pollen was established by HPLC, which made rape pollen reach a new level in development and utilization. The main results were as follows:1. Rape pollen was treated respectively with ultra-fine powder technique and common powder technique, the effects of two methods on the main nutrient components were analyzed, and the characteristics of the two products were surveyed with the scanning electron microscope. The results indicated that the contents of proteins, fats, ashes, reducing sugar, flavonoids were found higher in ultra-fine powder than in common powder, contrary to fibre. The intact cell shape of the common powder could be basically observed, while the ultra-fine powder particles of rape pollen were more uniform in shape and became smaller in size, nearly no complete cells existed, and the cell wall-broken rate of the ultra-fine powder product reached 98.86%.2. High performance liquid chromatography（HPLC） method was employed to determine the concentration of Quercetin and Kaempferol in common powdered and ultra-fine powdered rape pollen. Phenol-sulfuric acid method was used to determine the content of polysaccharide, and the polysaccharide dissolubility in different time was compared. The results showed that the dissolution rates of Quercetin and Kaempferol of the ultra-fine powder were respectively increased by 45.16% and 27.86% compared with that of the common powder under the same condition. As compared to the common powder , the dissolution amount and the dissolution velocity of the ultra-fine powdered rape pollen polysaccharide were found to be increased, the dissolution rate of rape pollen polysaccharide was increased by 252.54% when extraction time was 10 min. The study of IR spectrum proved the primary groups of polysaccharide had no change. It was concluded that the ultra-fine powder technique could promote the dissolution rates of effective components of rape pollen.3. High performance liquid chromatography（HPLC） was applied to determine the concentrations of plamsa Quercetin and Kaempferol in two groups of rabbits after administering ultra-fine powdered and common powdered rape pollen （in water suspension） by gastrogavage respectively. The plasma concentration-time data of Quercetin and Kaempferol were dealt with Pharmaceutical Kinetics Software（PKS）. Pharmacokinetics characteristics of both ultra-fine powdered and common powdered rape pollen were compared. The results showed that the best pharmacokinetic model of Quercetin and Kaempferol in either group was two-compartment open model. The main pharmacokinetic parameters of Quercetin of ultra-fine powder were as follows: Ka= 0.5793 h^-1, t1/2α=2.43 h, t1/2β=18.33 h, AUC0→∞=0.3613μg·L^-1·h, Vd =804.8 L·kg^-1, Tpeak=2.5714 h, Cmax=0.01908μg·mL^-1; The main pharmacokinetic parameters of Quercetin of common powder were as follows: Ka=1.2790 h^-1, t1/2α=2.63 h, t1/2β= 28.00 h, AUC0→∞= 0.3411μg·L^-1·h, Vd = 997.3 L·kg^-1, Tpeak=3.048 h, Cmax =0.018769μg·mL^-1。The main pharmacokinetic parameters of Kaempferol of ultra-fine powder were as follows: Ka=0.4136 h^-1, t1/2α=2.29 h, t1/2β=2.97 h, AUC0→∞= 1.8897μg·L^-1·h, Vd= 33.056 L·kg^-1, Tpeak =3.6429 h, Cmax=0.28079μg·mL^-1; The main pharmacokinetic parameters of Kaempferol of common powder were as follows: Ka=0.44362 h^-1, t1/2α=1.74 h, t1/2β= 1.99 h, AUC0→∞=1.4599μg·L^-1·h, Vd = 31.418 L·kg^-1, Tpeak =3.7143h, Cmax =0.24553μg·mL^-1. Tpeak was shorter, Cmax was higher in ultra-fine powder group than in common powder group. The relative bioavailability of Quercetin and Kaempferol from ultra-fine powder compared with common powder were increased by 46.00%, 29.44%. It was indicated that the bioavailability of Quercetin and Kaempferol in rape pollen could be greatly improved by ultra-fine powder technique.4. Ultra-fine powdered rape pollen polysaccharide was separated and purified by the membrane（s100kD、50kD和10kD） with different cut-off molecular masses. The effects of parameters such as the pressure, the purification time on the ultrafiltration process were then investigated. The optimal conditions finally determined for ultrafiltration were the pressure of three membranes were 1.1、1.3和1.6MPa, the purification time were within 28、36和48min respectively. The polysaccharide was separated into four components(>100kD,50 kD^-100 kD,10kD-50 kD,and<10 kD) according to their molecular weight, whose mass distribution was about 2.3 to 1 to 1.1 to 1.6, and the purity of polysaccharide products were above 68%. The isolated component with high activity was sieved by combination of antioxidation in vitro and vivo, immune activity. The test results of eliminating （·OH） and （O2-·） showed that the antioxidant capability of rape pollen polysaccharides has close relationship with molecular weight distribution. M3 and M4 fraction almost had no antioxidation capability, so these two fractions united into one fraction were studied as M3 in following tests. Component with high activity M2 and component with low activity M3 were sieved by the antioxidation in vivo and immune activity test, M2 had better antioxidant and immune activity than other fractions, the order of antioxidant and immune activity among several fractions was M2>M>M1>M3.5. M2 was further purified to RPP1-2 with DEAE-SephadexA-25 chromatography and Sephadex G^-100 chromatography, the gle permeation chromatography proved that RPP1-2 was pure material, the weight average molecular weight of RPP1-2 was 65358Da. The content of SO42- group of measured by IC was 2.81%. UV, IR and NMR illustrated that RPP1-2 was a glycoprotein, containedα-glucosidic bonds, composed of glucose, mannose and galactose, there wereα-（1,3）、α-（1,6）linkage in RPP1-2.6. High performance liquid chromatography（HPLC） was applied to compare the fingerprints of ultra-fine powdered rape pollen from different places of production, constructing the HPLC fingerprinting, followed by analyzing and evaluating the HPLC fingerprint of ultra-fine powdered rape pollen using the computer-assisted system software specialized in evaluating resemblance of fingerprinting ultra-fine powdered rape pollen. Rutin was selected as the internal standard. Diamonsil? C18 column （250×4.6 mm,5μm） was used with a mixture of CH3CN-0.4% H3PO4 as mobile phase in a gradient mode, flowing rate was 0.8 mL·min^-1, wavelength was set at 254 nm, column temperature was 25℃. The chromatographic profiles of the samples from different regions were very similar, the chemical constituents of ultra-fine powdered rape pollen were all seperated perfectly under the chromatographic condition above, and the common HPLC fingerprints of ultra-fine powdered rape pollen contained ten components including rutin. The method was simple, rapid, stable and had a good reproducibility. HPLC fingerprint could objectively reflect the feature of ultra-fine powdered rape pollen, and be used to evaluate and control the quality of ultra-fine powdered rape pollen.更多还原