Research On Analysis And Preparation Of Active Ingredients From Pyracantha Fruit And Its Pharmaceutical Matrix

Pyracantha fortuneana is a wild evergreen shrub which belongs to the Pyracantha Roem. of Maloideae from the family Rosaceae. Recorded history and modern scientific research show that Pyracantha fruit is effective in invigorating the spleen, reducing blood lipids, being antioxidant and whitening. However, at present, systematic research on the efficacy and its material basis of Pyracantha fruit is still limited at home and abroad. In this paper, based on establishing the detection methods of main efficacy components from Pyracantha fruit, the synchronous extraction and preparation process of efficacy components from the flesh and seeds of Pyracantha fruit was optimized. At the same time, the preparation technology of high antioxidant activity extracts from the whole fruit based on DPPH total antioxidant capacity evaluation oriented was established. Furthermore, the preparation technology of separation and purification of total flavonoids and polysaccharides from Pyracantha fruit was further established. Moreover, on the basis of preliminary screening of antitumor activity, this research was mainly focused on the antitumor activity of total flavonoids from Pyracantha fruit in vitro and its possible mechanism. In addition, the interaction among main components of gel matrix which will be applied to development of Pyracantha fruit products was studied. The main research results were obtained as follows.(1) The analysis methods of contents of main efficacy components in Pyracantha fruit were established. Total procyanidins content from Pyracantha fruit was measured using Vanillin-HCl method. Total flavonoids was determined using Al Cl3 Colorimetric method, and various flavonoid content was detected using HPLC method.The content of polysaccharide and total phenol from Pyracantha fruit was detected, respectively, using the improved Phenol-H2SO4 method and Folin-Ciocalteu method which were both optimized with response surface method. Meanwhile, the antioxidant evaluation system in vitro based on DPPH method was established, and the stability of this method had been significantly improved.(2) The extraction technology of main efficacy substances from the fruit pulp and its peel, fruit seeds and the whole fruit of Pyracantha was established and optimized, respectively. With fruit peel and pulp as the raw material, the polysaccharides, total flavonoids and non-extractable polyphenols were extracted in sequence using conventional water extraction method, composite enzymatic pretreatment associated with ethanol refluxing extraction method, sulfuric acid-ethanol extraction method, respectively. After the extraction technology was optimized by response surface method, the yields of polysaccharides, total flavonoids and non-extractable polyphenols were 2.87%, 0.91% and 16.73%, respectively. With fruit seeds as raw material, the seed oil and non-extractable polyphenols were extracted sequentially using n-hexane and sulfuric acid-ethanol as solvents, and under the condition of optimization, the extraction yield of seed oil and non-extractable polyphenols was 10.24% and 7.08%, respectively. With the whole fruit as raw material, the extraction process was optimized and the extracts of high antioxidant activity was obtained based on DPPH total antioxidant capacity as the orientation index of extraction.(3) The total flavonoids from Pyracantha fruit was purified using HPD-100 C macroporous resin. After optimization of purification process, the content of purified total flavonoids was 2.3 times higher than that of the original, and the recovery rate was as high as 88.9%. The polysaccharides in Pyracantha fruit were carried out graded ethanol precipitation, and the antioxidant activity was compared. It was found that when graded ethanol precipitation occurred at ethanol concentration from low to high, the amount of polysaccharide obtained was the most at the ethanol concentration of 50%(V/V), while the antioxidant activity of polysaccharide was strongest at the ethanol concentration of 70%(V/V).(4) Using in vitro cell culture system, this research was mainly focused on in vitro antitumor activity of flavonoid extracts from Pyracantha fruit, based on preliminary screening of antitumor activity of above main separated and purified efficacy substances. The results showed that flavonoids extracts could effectively inhibit the proliferation of the mouse B16 melanoma cells, human hepatocellular carcinoma cells Hep G2 and human cervical cancer cells Hela, and that flavonoids extracts had time-dose dependence on the inhibition of cell proliferation. The apoptosis of Hep G2 cells induced by flavonoids extracts from Pyracantha fruit was further studied. It was found that when the concentration of flavonoids extracts from Pyracantha fruit is below 1600 ?g/m L, the early apoptosis rate and total apoptosis rate of induced Hep G2 cells increased with the increase in the dose, and when the concentration was higher than 1600 ?g/m L, the apoptosis rate decreased.(5) The correlation between material structure and gelling of tamarind xyloglucan(TSX)-gallic acid(GA) analogue system was studied adopting rheological method and differential scanning calorimetry(DSC) technique. It was found that GA ester bond is the main structural unit inducing TSX gelling. We also discussed the mechanism of gelling that was further explored by nuclear magnetic resonance(NMR) technology, which provided theoretical and experimental basis for the development of gel matrix of Pyracantha fruit products.Above research results have laid a solid foundation for the deep development and comprehensive utilization of the medicine food dual-purpose resource Pyracantha fruit, and also have provided more systematic experimental basis for the development of new Pyracantha fruit products.