| 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.
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