Preparation Of Tea Polyphenols Nanoparticles And Its Antitumor Activities

Tea has a long history and a deep cultural heritage as a kind of characteristic economic crops in China. At the same time, it also is the most civilized and healthy beverages in current three welcome drinks by consumers in the world.Tea is rich in a lot of chemical components, including polyphenols, caffeine, tea pigment, tea polysaccharide, tea saponin, theanine and other natural products beneficial to human health, such as antioxidant, anti-mutation and anti-cancer biological activity, and its development prospects are also very bright.As the major effective ingredients green tea polyphenols possessed the antitumor activities. Tea polyphenols with high purity was prepared from green tea using the advanced extraction and separation technology of bioactive substances. Then, chtosan loaded tea polyphenols nanoparticles (CS-TP NPs) were prepared by ionic gelation method using two water-soluble chitosans as carriers of tea polyphenols. The preparation conditions were optimized and selected to obtain best size and encapsulation efficiency, and analyzed the size distribution of nanoparticles and in vitro characterization.The models of human hepatocarcinoma cell HepG2 and tumor animal were established by advanced biological technology and means to study antitumor activity of the CS-TP NPs. The methods of morphological observation by microscopy and transmission electron microscopy, microplate reader and flow cytometry were used to analysis the antitumor mechanism of CS-TP NPs.The main research results were as follows:1. Extraction in advance using superfine comminuted processing technology can significantly improve the effective dissolution of tea polyphenols. The results of this paper showed that comparison with raw materials, the dissolution volume of tea polyphenols in superfine comminuted green tea from the original of 156.34±4.22 g/kg increased to 272.67±9.56 g/kg. Separation and purification of tea polyphenols using organic solvent combined with macroporous resin to obtain a purity of 93.75% polyphenols, the product yield was 6.14%. HPLC analysis showed that compared to crude polyphenols, the purified polyphenols has a high content of catechins and low caffeine.2. Based on carboxymethyl chitosan and chitosan hydrochloride as a carrier, CS-TP NPs was preparated in solution by molecular self-assembly techniques, and optimized through the Box-Behnken experimental design and response surface methodology to obtain tea polyphenols nanoparticles with smaller particle size and high encapsulation efficiency. Detection with dynamic light scattering and observation by transmission electron microscopy (TEM) showed that the nanoparticles had an irregular shape, however, the distribution was more uniform, and the range of particle size was mostly in the 200 nm～400 nm. Zeta potential value was about 30 mV, and it present relatively stable colloidal state in solution. Nanoparticles prepared with chitosan was an excellent delivery carrier, and CS-TP NPs showed a strong sustained release in solution, the release time of polyphenols in nanoparticles was up to 48 h in PBS 7.4 buffer solution in vitro. CS-TP NPs were stable in room temperature when they were freeze-dried.3. The safety of CS-TP NPs was systematically evaluated by haemolyticus test, cytotoxicity test and acute toxicity experiment in vitro and in vivo. The results showed nanoparticles didn’t emerge haemolysis and conglomeration. The relative growth rate of L929 cells in CS-TP NPs was (100.03±0.06)%, and the toxicity grading results showed the non-toxic. Acute toxicity test showed that the maximum dose 3 g/kg BW of CS-TP NPs and 2.55 g/kg BW of the empty nanoparticles all have no effect on mice. It is suggested that CS-TP NPs and chitosan blank nanoparticles were safe and non-toxic to organism in this dose range.4. The results of anti-tumor effect in vitro showed that CS-TP NPs (0.5 mg/mL～1.0 mg/mL) significantly inhibited the growth of human hepatoma cell HepG2 with a dose and time dependent relationships. TEM and scanning electron microscope (SEM) results showed that HepG2 cell performed the typical apoptosis character of cell lessening, cytomembrane collapse, karyopyknosis, inregular changes of caryotype and nuclear fragmentation after addition of CS-TP NPs, which indicated CS-TP NPs could induce the apoptosis of human hepatocarcinoma cell lines HepG2. 5. The effect of CS-TP NPs on cell cycle and cell apoptosis of HepG2 was measured with flow cytometry. After the treatment for 24 h at 1.0 mg mL-1, the apoptotic cells at the early and late stage accounted for 23.83% and 3.30% of the total counted cells, significantly higher than the control of 2.44% and 2.89%, respectively. Under the same concentration, CS-TP NPs could keep the cell cycle of HepG2 cells to G0/G1 stage (control 0.62%, treatment 5.54%), block cell cycle to S phase, inhibit the proliferation and induce the apoptosis of the cancer cells.6. Mice transplanted with H22 liver tumor were established to research the antitumor effect of CS-TP NPs. The tumor volume, inhibition rate and morphological changes in the block in mice were detected and the antioxidant activity and pathological technology were utilized to detect the antitumor mechanism in vivo. The results showed that CS-TP NPs had significant antitumor effect on mice transplanted with H22 liver tumor in vivo. Mice were treated with 6.0 mg/kg and 12.0 mg/kg CS-TP NPs respectively, the tumor growth inhibition rate were 51.50% and 58.17%. Antioxidant results in vivo show that CS-TP NPs could significantly increase the activities of GSH-Px and T-AOC in mice serum, and decrese the content of H2O2 and SOD in liver. Results of tumor pathological section indicated that the connective tissues in the surrounding of tumor were increased after treatment with CS-TP NPs, and presented cell apoptosis state.