Abstract

In this work, an efficient method for the rapid extraction and separation of antioxidant phenols was developed and optimized. The method was then applied to extract and separate nine phenols from 37 varieties of raspberry, in which their antioxidant activities were further investigated. First, the extraction was conducted using ultra-sonication, which was then further separated using reversed-phase high-performance liquid chromatography/ultraviolet (RP-HPLC/UV) analysis. In this step, several key parameters (volume of the extraction reagent, time of extraction, and the temperature of extraction) affecting its efficiency were investigated and optimized using the response surface methodology (RSM) combined with the Box–Behnken design (BBD) so that the optimal conditions were obtained. According to the overall results of the optimization study, the optimal conditions were chosen as follows: volume of extraction reagent = 2.0 mL, time of extraction = 50.0 min, and temperature of extraction = 50 °C. The optimal conditions were then applied to extract nine phenols, including gallic acid, catechin, chlorogenic acid, vanillic acid, syringic acid, cumaric acid, ferulic acid, rosemary acid, and quercetin from 37 raspberry varieties. The extracted phenols were characterized and their antioxidant activities, including DPPH− and ABTS− free radical scavenging and intracellular reactive oxygen species (ROS) activity, using HepG2 cells as the model, were subsequently studied. The findings suggested that although their contents varied among most raspberry varieties, these phenols significantly contributed toward their antioxidant capacity and scavenging intracellular ROS activities. This study provides a scientific and theoretical basis for the selection of raspberry varieties and product development in Qinghai province.

Highlights

  • Raspberry (Rubus corchorifolius L. f ) belongs to the family Rosaceae, the subfamily Rosoideae, and the genera Rubus

  • Studies describing the methodology for determining phenolic compounds in raspberry plants grown in the Qinghai-Tibetan Plateau region using reversed-phase high-performance liquid chromatography/ultraviolet (RP-HPLC/UV) have not been found

  • The “lack of fit F-value” of 7.79 implied that it chlorogenic acid, vanillic acid, syringic acid, cumaric acid, ferulic acid, rosemary acid, and quercetin was significant, with a 3.80% probability that this occurred due to noise

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Summary

Introduction

Raspberry (Rubus corchorifolius L. f ) belongs to the family Rosaceae, the subfamily Rosoideae, and the genera Rubus. MS [20] can be used to determine the total phenol content, which provides information on the molecular mass and structural features of components, and they are considered to be more useful than other methods of separation, identification, and quantification of the characteristic compounds Since these methods are quite costly when achieving short analysis times, or long analysis times with common equipment, many studies [21,22,23] have used high-performance liquid chromatography/ultraviolet (HPLC/UV) detection, which is less costly, decreases the runtime, is comparably convenient to operate, minimizes wear on various HPLC system components, and is suitable for routine analysis for the determination of phenolic compounds. Studies describing the methodology for determining phenolic compounds in raspberry plants grown in the Qinghai-Tibetan Plateau region using reversed-phase high-performance liquid chromatography/ultraviolet (RP-HPLC/UV) have not been found. To the best of our knowledge, this is the first reported extraction, separation, and analysis of phenols and their antioxidative activities from 37 varieties of raspberry grown in the Qinghai-Tibetan Plateau region, and this study provides a scientific. Molecules 2019, 24, x FOR PEER REVIEW basis for the selection of raspberry varieties and product development

Optimization of the Extraction
Optimization of HPLC Separation
Validation of the Method
Analysis of 9 Phenols from 37 Raspberry Varieties
Scavenging Abilities Against Intracellular Free Radicals
Instruments
Materials and Reagents
Preparation of Standard Solutions
Sample Preparation
Experimental Design and Data Analysis
Biochemical Assays
Assays of the Intracellular Activities of Phenols
Conclusions
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