Optimized Extraction of Phenolics from Jujube Peel and Their Anti

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Bioactive Constituents, Metabolites, and Functions

Optimized Extraction of Phenolics from Jujube Peel and Their Anti-inflammatory Effects in LPS-stimulated Murine Macrophages Bini Wang, Yuanyuan Hui, Longgang Liu, aiqing zhao, Yishiou Chiou, Fuxin Zhang, and Min-Hsiung Pan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06309 • Publication Date (Web): 10 Jan 2019 Downloaded from http://pubs.acs.org on January 10, 2019

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Journal of Agricultural and Food Chemistry

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Optimized Extraction of Phenolics from Jujube Peel and Their Anti-inflammatory

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Effects in LPS-stimulated Murine Macrophages

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Bini Wang§, Yuanyuan Hui§, Longgang Liu§, Aiqing Zhao§, Yi-Shiou Chiou﹟, Fuxin Zhang§,*

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and Min-Hsiung Pan§,﹟, Φ, ζ,*

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§College

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China.

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﹟

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Taiwan.

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ΦDepartment

of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an,

Institute of Food Science and Technology, National Taiwan University, Taipei 10617,

of Medical Research, China Medical University Hospital, China Medical

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University, Taichung 40402, Taiwan

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ζDepartment

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Taiwan

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*Corresponding author:

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Fuxin-Zhang, Professor

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College of Food Engineering and Nutritional Science, Shaanxi Normal University,

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Xi’an 710119, China

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E-mail: [email protected]

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Or

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Min-Hsiung Pan, Distinguished Professor

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Institute of Food Science and Technology, National Taiwan University, Taipei 10617,

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Taiwan.

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E-mail: [email protected]

of Health and Nutrition Biotechnology, Asia University, Taichung, 41354,

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Abbreviations: LPS, lipopolysaccharide; RSM, response surface methodology;

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ANOVA, analysis of variance; iNOS, inducible nitric oxide synthase; COX-2,

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cycoloxygenase-2; NO, nitric oxide; DMSO, dimethyl sulfoxide; NF-κB, nuclear

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factor-κB; MAPK, mitogen-activated protein kinase.

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ACS Paragon Plus Environment


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ABSTRACT: The extraction of phenolics from jujube peel (PJP) was optimized using

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response surface methodology (RSM). A Box−Behnken design was utilized to analyze

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the effects of NaOH concentration, temperature, and extraction time on the total

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phenolic content (TPC). The results showed that RSM could be an adequate approach

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for modeling the extraction of PJP. The optimal extraction condition for the highest

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TPC was obtained with 3.4 M NaOH concentration for 67 minutes at 50oC. Not only

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PJP but also phenolics from the jujube seed (PJS) contain considerable amounts of

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phenolics, particularly flavonoids. Quercetin and galangin were found to be the

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predominant phenolics. PJP markedly down-regulated the levels iNOS and COX-2

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proteins in macrophages by inhibiting the activation of NF-κB through interfering with

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the MAPK signaling pathways. Compared to PJS, PJP presented higher anti-

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inflammatory activities, reflecting increased amounts of TPC and total flavonoid

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content (TFC). These findings suggest that PJP could be a potential source of anti-

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inflammatory agents.

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KEYWORDS:

jujube,

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macrophages, MAPK

phenolics,

anti-inflammatory,

2


RAW

264.7

murine

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INTRODUCTION

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Jujube is the fruit of Ziziphus jujuba Mill., a thorny rhamnaceous plant widely

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distributed in northern China. It is generally recognized as an outstanding source of

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biologically active compounds related to both high nutritional and pharmacological

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effects. Dried jujube has been commonly used as a food, food additive, flavor, and also

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a traditional Chinese medicine for thousands of years. Today’s medical practitioners

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are seeking scientific proof of its exceptional properties. It has been claimed that the

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jujube has a wide range of health benefits including antiobesity,1antiepileptic,2 anti-

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insomnia,3 neuroprotective,4 antioxidant,5 anti-inflammatory,6,7 and anticancer

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properties.8–11 Phenolic compounds have been reported to possess various biological

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effects responsible for their chemopreventive properties (e.g., antioxidant and anti-

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inflammatory effects).12–14 Recent studies have shown that the jujube contains various

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bioactive compounds including phenolic.15–18 However, most of these studies were

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focused on the soluble phenolics in the jujube.

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In general, after the soluble phenolics are extracted from jujubes, the residues are

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discarded as waste. We have found previously that the phenolic fractions constitute the

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main powerful antioxidant compounds, particularly in jujube peel and seeds.19 These

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latter compounds, the byproducts of jujube processing, are usually discarded as waste.

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Thus, their phenolic fractions with powerful antioxidant activities have been ignored,

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underestimating the content of phenolic compounds present in the jujube. However, the

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compositions of the phenolics from jujube peel and seed (PJP and PJS, respectively)

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should be further analyzed to provide the material base for the evaluation of their bio3



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activities.

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To further utilize the jujube residues after extraction of soluble phenolics, the first

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step is to extract their insoluble-methanol phenolics. The extraction conditions may not

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be the same for different fruit materials since they are affected by several parameters

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such as solvent concentration, temperature, and extraction time.20 It is essential to

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optimize the extraction parameters to reach an accurate analysis. RSM is a statistical

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experimental protocol used in mathematical modeling. It has emerged as an ideal

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strategy for optimizing the extraction process because it requires fewer experimental

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measurements and provides a statistical interpretation of the data and the interaction

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amongst variables.21 Thus, the objective of this research was to study the effects of

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sodium hydroxide concentration, temperature, and extraction time on the extraction of

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PJP and to evaluate the phenolic composition and the anti-inflammatory effects of PJP

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and PJS. The anti-inflammatory mechanism of PJP and PJS were also explored to

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provide some theoretical basis for the development of the targeted anti-inflammatory

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foods.

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MATERIALS AND METHODS

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Chemicals and reagents. Gallic acid, rutin, quercetin and galangin were purchased

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from Sigma-Aldrich (Steinheim, Germany). LPS (Escherichia coli 0127: E8), naphthyl

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ethylenediamine dihydrochloride, and sulphanilamide were purchased from Sigma

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Chemical Co. (St. Louis, MO, USA). Other chemicals purchased were in the purest

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form commercially available (Sigma and Fluka Chemical Co., St. Louis, MO, USA; 4


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Merck, Darmstadt, Germany). HPLC grade water was purified by the Milli-Q system

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(Millipore, Bedford, MA, USA).

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Sample preparation. Jujubes (Ziziphus jujuba Mill. var.Goutouzao) were

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acquired from Renrenle supermarket (Xi’an, China). The jujube peel and seed were

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directly obtained by manual separation, and then dried and milled. The detail procedure

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is shown in the Supporting Information. Samples used for the analysis of phenolic

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composition and the evaluation of anti-inflammatory effects of PJP and PJS can be

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obtained by dissolving 100 mg extracts in 1 mL methanol and DMSO, respectively.

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Experimental design. RSM is applied to evaluate the relationship between a

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group of controllable experimental factors and observed results. Based on the single

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factor investigations, a three-factor, three-level Box and Behnken design (BBD) was

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used to optimize the extraction condition. The 17 experiments were conducted

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randomly (Table 1) to analyze the response pattern and to establish a model for PJP

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extraction. The experimental data obtained were fitted into a second-order polynomial

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model and regression coefficients were calculated as previously described.22 The

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optimized extraction condition of the independent variables was further applied to

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validate the model using the same experimental procedure as made previously. 22

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Determination of total phenolic contents (TPC) and total flavonoid contents

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(TFC). TPC was determined according a modified version of the Folin–Ciocalteu

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method reported by Wang et al.19 TFC was determined by the aluminum chloride

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method.23

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LC-MS/MS analysis. The chromatographic separation was performed with an 5



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ACE C18–PFP (2.1×150 mm, 3.0 μm) at a column temperature of 30 °C. The mobile

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phase consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in

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acetonitrile with gradient elution as follows: 0–1 min, 30% solvent B; 1–1.5 min, 30–

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50% solvent B; 1.5–3 min, 50–85% solvent B; 3–3.5 min, 85–90% solvent B; 3.5–4

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min, 90% solvent B. The flow rate was 0.3 mL/min. Detection was performed with

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selected reaction monitoring (SRM). The conditions of the ion source were optimized

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as follows: Capillary, 3.00 kV; Cone, 30.00 V; Source Temperature, 150 °C;

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Desolvation Temperature, 350 °C; Cone Gas Flow, 1 L/h; Desolvation Gas Flow, 650

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L/h.

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Cell culture and MTT assay. Murine macrophage RAW 264.7 cells purchased

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from the American Type Culture Collection (Rockville, MD, USA). They were cultured

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in the same conditions as our previous study.

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viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

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bromide (MTT) as our previously described. 24

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The effect of the test extracts on cell

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Nitrite and luciferase assay. The nitrite concentration in the culture medium was

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measured as an indicator of NO production, according to the Griess reaction as

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described previously.25 The luciferase assay was performed as described previously.26

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Western blot analysis. Protein from whole-cell lysates was resolved by 10%

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SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes

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(Immobilon P, Millipore, Bedford, MA), and then detected with primary antibodies

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followed

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immunocomplexs were visualized using enhanced chemiluminescence (Amersham,

by

horseradish-peroxidase

conjugated

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sendary

antibodies.

The

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UK).24 Statistical analysis. Data are presented as the mean ± standard error for the indicated number of independently performed experiments as previously described 26.

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RESULTS AND DISCUSSION

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Optimized extraction of PJP. RSM was applied to analyze the effects of NaOH

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concentration, temperature, and extraction time on the TPC of PJP. The predicted and

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experimentally measured responses for the 17 runs according to the experimental

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design are presented in Table 1. A close agreement between experimental and predicted

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values was found, indicating a satisfactory model developed. The ANOVA results for

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the model response indicated that the contribution of the model was significant, as

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shown in Table 2. The second-order polynomial model predicted by RSM demonstrated

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a significant fitting (p

Optimized Extraction of Phenolics from Jujube Peel and Their Anti

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