Study On Extraction And Function Of Active Ingredient From Bee Pollen

Pollen is male reproductive cells of plants. It is rich in various nutrients and hailed as natural complete nutrients internationally. Maize pollen polysaccharides are capable of antitumor, antioxidant, enhancing the immune function. But stiff pollen wall hindered dissolution of polysaccharides and lowered extraction efficiency. Dynamic high pressure micro-fluidization (DHPM) is a rising high pressure homogenization technique. Plant cells could be disrupted by violent treatment conditions, and the active ingredient was little damaged.Dynamic high pressure micro-fluidization technology was applied on maize pollen wall-breaking in this study. Maize pollen wall-breaking effect and yield of polysaccharides were compared with dry grinding and enzymatic hydrolysis wall-breaking. Then anti-oxidative function of maize pollen polysaccharides was evaluated in order to prepare for future research and development of maize pollen polysaccharides from now on. At the same time, the pollen function factor cyclic hydroxamic acid which contributed to treatment of prostate diseases was done qualitative and quantitative analysis. The main contents and conclusions were as follows:1. The content of maize pollen polysaccharides was determined by selected anthrone-sulfuric acid method with glucose as standard. The results were as follows: The absorption spectrum of maize pollen polysaccharides was consistent with glucose, and the linear relationship of standard curve was good (R2=0.9996). The recovery, precision and stability of this method were good, so anthrone-sulfuric acid method was ideal method which could be used to determine pollen polysaccharides content.2. Polysaccharides were extracted from broken maize pollen by dry comminution, and optimum extract conditions were obtained by orthogonal experiment. Results showed that pH value of extract had most significant impact on yield of polysaccharides. The optimum conditions were as follows:pollen by dry comminution and got over 120 mesh, extract of pH 7.0, ratio of solid-liquid 1:15, 70℃of extraction temperature for 3h, and extraction two times. The yield of polysaccharides was 7.239% under these conditions.3. Maize pollen wall was broken by enzymolysis with composition of pectinase and cellulase enzyme, and optimum enzymolysis conditions were obtained by orthogonal experiment. Results showed that pectinase addition was principal factor that affected yield of polysaccharides. The optimum conditions were as follows: pectinase addition of 300U/g pollen, cellulase addition of 150U/g pollen,20% of pollen concentration, pH4.5,50℃of enzymolysis temperature for 5h. The yield of polysaccharides was 7.194% under these conditions.4. Maize pollen wall was disrupted with wet method using DHPM technology, and the parameters were optimized by Box-Benhnken response surface method. The results showed that the linear effect and surface effect of homogenization pressure and extraction temperature on polysaccharides yield were significant. The optimum conditions were as follows:homogenization pressure 127.35 MPa, homogenizing one time, ratio of solid-liquid 1:20,72.41℃of extraction temperature for 1.74 h and extraction two times. Under these conditions the yield of maize pollen polysaccharides was theoretical maximum of 8.086% which was consistent with validation experiment results. Compared with dry comminution and enzymolysis wall-breaking extraction, extraction time was shortened 1.26 h and the yield of polysaccharides increased 0.847%.5. IR spectra scan results showed that infrared spectrum of maize pollen polysaccharides extracted by three wall-breaking methods was basically identical, and major functional groups had no differences. This indicated that the structure of pollen polysaccharides was not destroyed by DHPM. Pollen wall-breaking effect of three wall-breaking methods was observed by SEM. The results showed that:a few of incompletely broken pollen grains were still observed after dry comminution and enzymolysis treatment, and particles size was not uniform, the powder characteristics of raw materials were observed. However, pollen cells had been completely broken into lots of pieces after treatment by DHPM, and particles size was significantly smaller, and all of the contents overflowed. Therefore, wall-breaking effect of DHPM was best.6. The mechanism of maize pollen broken after DHPM treatment was preliminarily discussed as follows:in the high pressure homogenization process, the organizational structure of pollen cells was completely broken as a result of violent treatment conditions, and the original coarse grain pollen emulsion was processed into particles fine emulsion. The surface area of pollen particles was increased so that particles could fully contact with extraction solvent, thereby the extraction efficiency was enhanced.7. Cernilton extract was done qualitative analysis by UV spectral scan and HPLC-MS, and it was ultimately determined as cyclic hydroxamic acid whose purity was about 94%. Maize pollen DIBOA extract was purified by Sephadex G-25 and G-10 combined column. The content of maize pollen DIBOA was about 0.029% (290μg/g) after HPLC quantitative analysis and calculation.8. Antioxidant abilities of crude and refined maize pollen polysaccharides PPMC were measured as DPPH scavenging activity, activity against hydroxyl radical and superoxide anion scavenging activity. The investigation demonstrated that they possessed free radical scavenging activity, and scavenging effect enhanced with increasing of polysaccharides concentration. Scavenging activity of crude and refined polysaccharides on DPPH was better than the scavenging activity of hydroxyl radical and superoxide anion. In the same concentration, scavenging activity of refined polysaccharides PPMC was slightly better than crude polysaccharides.