Studies On Procyanidins From Chestnut Shell And Their Anticancer Effects

Castanea Mollissima Blume (chestnut), a plant of the genus Fagaccac. In China, chestnut is planted widely, and the yield ranks first in the world, which account for three quarters of the world production. Chestnut shell is the castoff of processed chestnut, and normally it is deserted as garbage. In recent years, piocyandins extracted from chestnut shells (CSPC) have attracted much attention for their variety of pharmacological properties, including antioxidant activities, free radical scavenging and anticancer activities. In this study, the extraction, separation, purification of CSPC were investigatied, and some modern techniques were applied to identification of the compounds obtained. The stabilization and antioxidation of CSPC, and the effects of CSPC on cultured human hepatoma cell line HepG2 were researched, too. The main contents and conclusions were as follow:(1) The effects of ethanol concentration, ratio of solid to liquid, extraction temperature and extraction times on the extraction of chestnut shell polyphenol were studied in this study. Based on the single factor experiment, we optimized the extraction conditions of chestnut shell polyphenol with the response surface methodology. The conditions were determined as follow:concentration 59.7%, solid-liquid ratio 1:18 (g/mL), temperature 52.6℃and extraction 3 times. The yield of chestnut shell polyphenol was 76.35 mg/g. The best extraction conditions of CSPC were 51.1% ethanol, solid-liquid ratio 1:14.5 (g/mL), extraction temperature 67.3℃and extraction 3 times. The yield of CSPC was 40.23 mg/g.(2) Twelve kinds of macroporous resins were studied to compare their characteristics in absorbing and desorbing of CSPC. Based on static adsorption and desorption, AB-8 macroporous resin was selected to carry out dynamic adsorption experiments. The recovery of CSPC reached 94.2%, and the content of CSPC increased from 20.3% to 73.8%. Sephadex LH-20 gel chromatogram was used to separate and purify CSPCS, and different ratio of methanol to water was used as mobile phase with gradient of step elution, and 6 fracticns were gained.(3) UV, RI, ESI-MS, HPLC-MS and NMR were used in determining CSPC-2S, CSPC-3S and CSPC-4S fractions. The results show CSPC-2S, CSPC-3S and CSPC-4S fractions are the dimeric, trimeric, tetrameric procyanidins, respectively, which are the oligomeric procyanidins of monomeric procyanidins, catechin and (or) epicatechin linked together through C4-C8(cis) bonds.(4) CSPC exhibited strong ability of scavenging free radicals. The capacity of reduction and eliminating DPPH·,·OH, O2- gradually increased with the increase of CSPC concentration in the range of 25 mg/L to 200 mg/L. When the concentration of CSPC was 200 mg/L, the capacity of reduction was 0.841, and the DPPH·inhibition rate reached 89.7%, and the DPPH·eliminating rate of CSPC was better than BHT, but less than vitamine C. The clearance rate of O2 and·OH were 93.2% and 94.0%, respectively, when the concentration of CSPC was 200 mg/L, and the clearance rate was all higher than BHT and vitamine C. In addition, CSPC showed certain scavenging sodium nitrite and interrupting nitrosamine.(5) Stabilization of CSPC was studied in different conditions. CSPC reduces quickly when it is directly exposed to light, but it is stable in low pH valued and low temprature. Sodium benzoate is adverse to the stabilization of CSPC. NaHSO3 can protect CSPC from loss for light to some extent. The stabilization of CSPC will be destroyed when Ca2+, Fe2+ or Cu2+are added to its medium, whereas other metal ions, such as Na+、K+、Mg2+、Al3+, contribute to its stabilization.(6) CSPC could inhibit the growth of HepG2 cells effectively in a time and dose dependent manner, and the inhibitory rate of HepG2 cells was 81.72% after administrated 4 days, and observable changes in morphology were observed in the HepG2 cells treated with CSPC. Agarose gel electophoresis of fragmented DNA showed a ladder-like pattern. PI staining was conducted to evaluate cell cycle arrest. Flow cytometry analysis revealed HepG2 cells were blocked in GO/G1 period resulting in cells proliferation were inhibited. Which indicated that CSPC maybe induced the death of HepG2 cells via mitochondrial injury, and for the manner of HepG2 cells death, necrosis was primary, while apoptosis was supplementary.