Isolation And Identification Of Polyphenols From Apple Pomace And Their Antioxidant And Anti-Inflammatory Characters In Vitro

Apple pomace is a byproduct of apple production, which has abundant polyphenols. Apple phenols has become the focus of investigation, which are shown to have a broad spectrum of pharmacological activities, such as anti-hypertension, antitumor, et al. At present, many reports on extraction of apple polypheols had been published. However, these methods have a lot of shortcomings, such as low efficiency, low purity of final product, making more serious pollution, et al. So, it is hard to apply to produce apple polyphenols industrially. Antioxidant and anti-inflammatory activities are the important physiological functions of apple polyphenols. However, study of relationship between polyphenols component and bioactivities is still lacking. This work focuses on isolation and identification of polyphenols from apple pomace and their antioxidant and anti-inflammatory characters in vitro. Firstly, different polar solvents were used to extract apple polyphenols from industrial pomace. On basis of yield and antioxidant activity, these afforded extracts were compared, and alcoholic microwave-assisted extraction process of polyphenols was optimized using response surface methodology (RSM). Then, different absorbent resins were selected and used to purify apple polyphenols by comparison of their absorption and desorption rates followed by optimization of purification processes. Six extracts were derived from different polar solvents consisted of water and ethanol, which phenol components were analyzed by high performance liquid chromatography (HPLC). Relationship between polyphenol constitutes and antioxidant activities in vitro were discovered through antioxidant assays. Finally, anti-inflammatory effect of purified extracts with abundant polyphenols was investigated using mouse macrophage cell line RAW264.7pretreated by lipid-polysaccharide (LPS).The main results were concluded as follows:1. Methanol was the best solvent to extract polyphenols from apple pomace, which yield reached upto2.85±0.11mg/g. Its DPPH-scavenging rate was (97.52±0.23)%, which was highest in all of solvents. Ethyl acetate was the inferior solvent, which yield was only0.38±0.04mg/g and its DPPH-scavenging rate was (73.89±0.81). It was shown that antioxidant activity of apple polyphenols was correlated not only with phenol quantity, but also with its polarities.2. On basis of pretreated experiment through single factor, alcoholic extraction of polyphenols from apple pomace was optimized using RSM. The optimial conditions of extraction process were as follows:microwave power650W, extraction time53s, ethanol content60%, ratio of pomace weight to volume of solvent1:20(g/mL). Validation tests indicated that the actual yield of polyphenols was62.68±0.35mg gallic acid equivalents (GAE) per100g dry apple pomace with RSD=0.86%(n=5) under the optimal conditions, which was in good agreement with the predicted yield and higher than those of reflux and ultrasonic-assisted extraction methods.3. NKA-Ⅱ was shown be the best macroporous absorbent resin through optimization experiments of absorption and desorption, which the optimal absorbent conditions were absorption rate1.06mL/min, pH value3.19, solution concentration1.42mg/mL and the optimal desorbent conditions were desorption rate0.91mL/min, ethanol content73.99%, pH value8.90. Validation tests suggested that the purity of apple polyphenols reached upto48.45%under these optimal conditions, which was applied to industrial production.4. Six phenol-enriched fractions, namely API-VI, were afforded from chromatography column using different elutions consisted of water and ethanol. The total phenolic content of APⅢ was the highest among these fractions, which was105.12±0.11mg/100g using Folin-Ciocalteu method. And its DPPH-scavenging capability was the strongest, which inhibitory rate reached (95.73±0.53)%. HPLC analysis showed that API-VI had notable difference in polyphenol components and their quantity. Procyanidin B2was the main component in APIII, which content was52.625mg/100g. antioxidant assay indicated that procyanidin B2had the strongest capability to scavenge DPPH (87.03±0.32)%followed by chlorogenic acid, syrigin,(-)-epicatechin, cinnamic acid, coumaric acid and phlorizin. It suggested that DPPH-scavenging capability of apple polyphenols was positive correlated with its content and procyanidin B2, chlorogenic acid, syrigin,(-)-epicatechin and other phenols were the key factors for antioxidant activity.5. Anti-inflammatory assay showed that fraction APIII had potential activity. HPLC analysis indicated that procyanidin B2, quercetin and syrigin were the main chemicals in APⅢ. The results of primary mechanism suggested that the quantity of procyanidin B2had positive correlation with anti-inflammatory effects. However, the inhibitory effect of procyanidin B2on COX-2expression did not depend on time. And the effect was synergic with quercetin but not with syrigin. Anti-inflammatory tests also exhibited that quercetin had antagonism with syrigin. Procyanidin B2did not repair the inflammation induced by LPS. It indicated that the inhibitory passage that procyanidin B2affected on COX-2maybe or should be PI3K-AKT but not HO-1.It is an important and scientific way to isolate and purify polyphenols from industrial apple pomace. The present work provided many significant findings for resonable development and utilization of polyphenols. It also estabilisded the theoretical foundation used to investigate on antoxidant and anti-inflammatory mechanism.