Identification, Quantification, and Anti-inflammatory Activity of 5-n

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Bioactive Constituents, Metabolites, and Functions

Identification, quantification and anti-inflammatory activity of 5-n-alkylresorcinols from 21 different wheat varieties Jie Liu, Yiming Hao, Ziyuan Wang, Fang Ni, Yu Wang, Lingxiao Gong, Baoguo Sun, and Jing Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02911 • Publication Date (Web): 14 Aug 2018 Downloaded from http://pubs.acs.org on August 17, 2018

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Journal of Agricultural and Food Chemistry

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Identification, quantification and anti-inflammatory activity of

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5-n-alkylresorcinols from 21 different wheat varieties

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Jie Liu a, Yiming Hao a, Ziyuan Wang a, Fang Ni a, Yu Wang a, Lingxiao Gonga,

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Baoguo Suna, Jing Wang a,b*

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a

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Technology & Business University, Beijing 100048, China

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b

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Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing

Beijing Engineering and Technology Research Center of Food Additives, Beijing

Technology & Business University (BTBU), Beijing 100048, China.

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*Corresponding authors E-mail addresses: [email protected] (Jing Wang)

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ACS Paragon Plus Environment


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ABSTRACT

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The characteristic constituent and anti-inflammatory activity of 5-n-alkylresorcinols

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(ARs) from 21 wheat bran samples in China were investigated in this study. The

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amount of ARs ranged from 697 µg/g to 1732 µg/g in the tested samples, which were

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composed of five different homologues. Among these homologues, C19:0 and C21:0

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were the most abundant, followed by C17:0, C23:0 and C25:0. Moreover, the mRNA

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expression of IL-1β, IL-6 and TNF-α in LPS-activated RAW264.7 macrophage cells

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were significantly inhibited by ARs supplementation. The molecular mechanisms

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behind its anti-inflammatory activity could result from the suppression of nuclear

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factor-κB (NF-κB) and JNK/MAPK activation. ARs treatment notably decreased

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NF-κB p65 nuclear translocation and inhibitor κB (IκBα) kinase and JNK

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phosphorylation. Additionally, ARs homologues C17:0 had been proven to be the

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main active constituent. The results from this study could be used to promote the

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comprehensive utilization of wheat and its by-products in improving human health.

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INTRODUCTION 5-n-alkylresorcinols (ARs) are mixture of AR homologues with odd numbered

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alk(en)yl chain which contains 17–25 carbon atoms, resulting in different proportions

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of C17:0-C25:0 from different cereals1,2. ARs are found in a large amount in cereals

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including wholegrain and the bran of wheat or rye, which locate in the outer layers of

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the grain (between pericarp and testa)3,4. Epidemiological and experimental studies

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indicate that whole grain products possess protective activity against obesity,

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cardiovascular diseases and cancer5. ARs can also be used as a biomarker for

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wholegrain consumption and evidenced with various bioactivities2,5-7. Previous study

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showed that ARs could inhibit body weight gain induced by high-fat diet and hepatic

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triacylglycerol accumulation, as well as insulin resistance8. Horikawa et al indicated

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that ARs could inhibit LDL oxidation, increase fecal cholesterol excretion and prevent

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cardiovascular diseases9. The modification of chain length and structure of ARs, for

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example unsaturation and keto groups play an important role in its bioactivity 10.

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Moreover, it has been widely proved that excessive inflammation could induce

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chronic or systemic inflammatory diseases, such as neurodegenerative diseases and

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metabolic disorders11. However, relevant studies on the anti-inflammatory capacity

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and possible molecular mechanisms of ARs are limited. Furthermore, little is known

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about the possible structure-activity relationship behind the anti-inflammatory

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capacity of ARs.

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Therefore, for a better understanding of the characteristics of ARs, chemical

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composition and anti-inflammatory property of 21 ARs specimens extracted from

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different wheat varieties were analyzed. The molecular mechanism of its

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anti-inflammatory activity and its possible structure-activity relationship were also

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investigated in the current work.



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MATERIALS AND METHODS

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Materials and reagents. Twenty-one wheat samples collected from different

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regions of China, which were provided and identified by Dr. Xiaoping Yuan (China

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Grain Reserves Corporation). The corresponding information for these wheat samples

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was listed in Table 1. AR standards (C17:0, C19:0, C21:0, C23:0, and C25:0), Fast

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Blue RR, and dimethyl sulfoxide (DMSO) were obtained from Sigma (St Louis, MO,

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USA). Rabbit monoclonal antibodies against IL-1β, TNF-α, IκBα, p-IκBα (Ser32),

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p65, p-p65 (Ser536), JNK, p-JNK (Thr183/Tyr185), p38, p-p38 (Thr180/Tyr182),

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ERK1/2, p-ERK1/2 (Thr202/Tyr204), β-actin and Histone H3 were purchased from

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Cell Signaling Technology (Beverly, MA, USA).

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Isolation of individual ARs from wheat brans. ARs from wheat brans (10 g)

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were isolated and extracted by ethyl acetate with a ratio of 1:40 (w/v) and stirring

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continuously at room temperature for 24h and then filtered4. The supernatant was

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collected and ethyl acetate was removed from the supernatant by evaporation. Then

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the collected extract was applied to a silica gel column, with chloroform/diethyl ether

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(9:1, v/v) to obtain ARs complex. The composition of ARs were determined by HPLC

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using LC-20A SPD-20A columns (GL Sciences Inc., Tokyo, Japan) under the

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following conditions: the mobile phase was methanol/water (96:4); the flow rate was

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set at 0.5 mL/min; the UV detection wavelength was 280 nm.

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LC-MS analysis. The quantitative analysis of ARs was conducted according to

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previous report12 and examined using Agilent Technology 6420 Triple Quad LC-MS

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(Milford, MA, USA). LC analysis was performed at 40 °C by using an HPLC Inertsil

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ODS-SP C18 column (4.6mm × 250 mm id, 5 µm). The elution gradient (eluent A,

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water; eluent B, methanol) used was set as 96% of eluent B. The injection volume

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was 10 µL with a flow rate of 0.5 mL/min. MS conditions were: mode, positive ESI;


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cell accelerator voltage, 4 kV; collision energy, 30 eV; fragmentor, 135 V; Gas

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temperature, 300 ℃; Gas flow, 11 L/min; scan time, 217.5 ms/cycle. Quantification

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was performed using multiple reaction monitoring (MRM) modes and plotted using

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the corresponding peak areas at a specific retention times of each external AR

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standard, which showed in Figure 1.

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Cell culture and cell viability assay. RAW 264.7 cells were obtained from

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American Type Culture Collection (ATCC, Rockville, MD, USA), and were

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maintained in DMEM supplemented with 10% of fetal bovine serum (Gibco, Carlsbad,

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USA) and 1% of streptomycin/penicillin in a humidified atmosphere of 5% CO2 at

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37℃. Cell viability was assessed by MTT assay. RAW 264.7 cells with the density of

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6×104 cells/mL were placed in a 96-well flat-bottom plate for 24 h, and then exposed

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to a variety of concentrations of ARs (0.5, 1, 2, 4 µg/mL) for another 24 h incubation

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period. Subsequently, MTT solution (5 mg/mL) was added to each well for another 4

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h and the supernatant was aspirated slightly. After that, 100µL DMSO solution was

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added to dissolve the formazan crystals. The optical absorbance was measured to

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calculate cell viability at wavelength of 490nm.

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Real-Time PCR. Total RNA was extracted by using TrizoL reagent (Invitrogen,

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Carlsbad, CA, USA). The first-stand cDNA was synthesized by using an equal aliquot

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of 0.4 µg total RNA and IScriptTM reverse cDNA transcriptase kit (Bio-Rad).

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Quantitative RT-PCR was performed on the ABI 7900 HT detection system (Applied

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Biosystems, Carlsbad, CA, USA) according to our previously reported13. The PCR

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quantification was carried out independently in triplicate. Relative mRNA level was

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used to indicate the changes of target gene expression, and normalized to β-actin

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which represented a housekeeping gene. The primers designed for gene amplification

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were listed as following: IL-1β, 5’-GTTGACGGACCCCAAAAGAT-3’(Forward)



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and 5’- CCTCATCCTGGAAGGTCCAC-3’ (Reverse); IL-6,

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5’-CACGGCCTTCCCTACTTCAC-3’(Forward) and

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5’-TGCAAGTGCATCATCGTTGT-3’(Reverse); TNF-α,

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5’-AGCCGATGGGTTGTACCTTGTCTA-3’(Forward) and

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5’-TGAGATAGCAAATCGGCTGACGGT-3’(Reverse); β-actin,

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5’-TGTCCACCTTCCAGCAGATGT-3’ (Forward) and 5’-

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AGCTCAGTAACAGTCCGCCTAGA-3’(Reverse).

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Western blotting analysis. Whole cells lysates were centrifuged at 13,400 g at

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4 °C for 20 min. Lysate protein was quantified by using a protein assay kit (Pierce,

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Rockford, IL, USA). Nuclear proteins were extracted by a nuclear extraction kit

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(Beyotime, Nantong, Jiangsu, China). Different volumes of loading buffer were used

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to adjust the concentration of the protein samples, which were then denatured.

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According to our lab previously reported methods13, equal quantities of protein (40 µg)

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were separated by 12% SDS-polyacrylamide gel electrophoresis and then

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electro-blotted onto PVDF membranes under the condition of 1.5 A and 20 V for 60

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min. The membranes were incubated with blocking solution for 2 h and washed by

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0.1% TBST for 15 mins followed by incubation with target protein primary antibodies

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overnight at 4 °C. After washed with 0.1% TBST and incubation with secondary

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antibodies for another 1.5 h, the immunoreactivity were visualized using a

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chemiluminescence kit (Bio-Rad, Hercules, CA, USA) and scanned by ChemiDoc

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XRS+ (Bio-Rad). β-actin and Histone H3 were used as the internal control.

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Statistical analysis. All the experiments performed in our study were in triplicate.

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All results denote the means ± SD. Comparisons between different groups were

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conducted by one-way ANOVA and analyzed by Duncan’s test using SPSS 17

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software. A level of P value less than 0.05 was considered to be statistically


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significant.

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RESULTS AND DISCUSSION

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Identification and quantification of ARs from 21 wheat brans. The ARs

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composition analysis of 21 cultivars were characterized by HPLC-QQQ-MS analysis

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(Table 2). It has been reported that several factors, including genetic factor, climate,

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soil condition and grain filling can influence ARs composition4. In our study, ARs

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were found in all 21 wheat bran samples and the content was in the range of 697 µg/g

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to 1732 µg/g dry brain. Compared with all the other cultivars, sample XN979

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collected from Hebei province possessed the highest ARs content (1732 µg/g dry

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wheat brain), while sample LM598 which was collected from Anhui province had the

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lowest content of ARs (Table 2). Based on the retention time of standards, ARs

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homologous (C17:0 to C25:0) were confirmed from the extracts of wheat bran, which

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was also in accordance with previous reports4. As shown in Table 2, the total amount

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of ARs was different among different wheat cultivars, but the amount of homologues

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ARs C19:0 and C21:0 were found to be the most abundant, followed by ARs C17:0

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and C23:0, which was also in consistent with other reported results 14. Moreover, the

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C17:0/C21:0 ratio was considered as an index to distinguish the variety of cereal

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product. For example, if the C17:0/C21:0 ratio was around 0.01, 0.1, and 1.0, it could

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be indicated the variety of durum wheat, common wheat and rye, respectively2. In our

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study, the ratio of C17:0 to C21:0 was in the range of 0.0035 to 0.0591 which

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suggested that all the tested samples were wheat rather than rye (Table 2).

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Anti-inflammatory activity of the ARs. As an important cell wall component of

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Gram-negative bacteria, lipopolysaccharide (LPS) could activate macrophage produce

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inflammatory mediators (eg. iNOS, TNF-α and COX-2) and mimick inflammatory

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reaction in vivo15. For the determination of anti-inflammatory activity of ARs



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extracted from the 21 wheat brans, the expression level of mRNA from IL-1β, IL-6

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and TNF-α in LPS-stimulated RAW 264.7 mouse macrophage cells were examined.

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Under the tested concentration of 1 µg/mL, there was no significant cytotoxic effects

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of ARs on RAW 264.7 cells (Figure S1). However, significant difference of the

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inhibitory activities to IL-1β, IL-6 and TNF-α mRNA expressions were found among

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these 21 samples. These samples significantly inhibited IL-1β mRNA expression

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except for sample XN979 and sample JM22, and sample XD33 had the strongest

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inhibition for IL-1β mRNA expression (Figure 2A). As Figure 2B indicated, all

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samples could inhibit IL-6 mRNA expression. As shown in Figure 2C, all samples

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could suppress TNF-α mRNA expression with the exception of sample CY25, LM598,

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SL021, LX99 and sample MM367, and sample XN979 had the strongest inhibitory

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effect. In addition, considering the anti-inflammatory activity, cultivated area and

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economic exploring potential, sample HM9, which is one of the main varieties of

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wheat cultivated in China, might have the potential of application in the development

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of anti-inflammatory biochemical and functional food. HM9 could significantly

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decrease the mRNA expressions of IL-1β, IL-6 and TNF-α by 57.7%, 62.9% and

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29.9%, respectively, and the effect of sample HM9 on pro-inflammatory protein

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expression was conducted by Western-blotting. ARs extracted from sample HM9

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could significantly inhibit protein expression level of IL-1β and TNF-α with

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dose-effect relationship (Figure 3). In addition, HM9 could decreased the protein

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expression level of IL-1β and TNF-α by 72.7% and 72.1% at the dose of 4µg/mL,

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which further confirmed its anti-inflammatory activity.

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ARs attenuates LPS-induced inflammation through NF-κB and JNK/MAPK

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pathway. NF-κB and MAPK signaling pathway can be triggered by LPS, which leads

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to a series of signal transduction events activation 16. The role of ARs played on


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NF-κB signaling pathway was evaluated by Western-blotting analysis. Under

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inactivated condition, NF-κB (a heterodimer of p65 and p50) could combine with

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IκBα and become an integrated complex which is located in the cytoplasm. When

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treated with LPS, the phosphorylation of IκBα increased, NF-κB became activated

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which could translocate into the nuclei and produce pro-inflammatory cytokines16-17.

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In our study, there is no significant cytotoxic effects of HM9 to RAW 264.7 cells with

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the tested concentration up to 4 µg/mL (Figure S2). LPS could increase IκBα

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phosphorylation subsequently improve nuclear translocation of p65 compared with

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the control group. In addition, pre-treatment with ARs could attenuate LPS-mediated

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phosphorylation activation of IκBα and p65 in a dose-dependent manner, thereby

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inhibited nuclear translocation of p65 (Figure 4A). Furthermore, MAPK plays a

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critical role in the activation of NF-κB and management of cellular responses to

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stimulation by cytokines 17. To investigate the role of ARs inhibits NF-κB activation

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on MAPK pathway, Western-blotting was used to examine the MAPK

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phosphorylation on LPS-stimulated RAW 264.7 cells treated with ARs. The

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expression level of total p38, JNK, and ERK did not change, whereas it significantly

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decreased the expression level of phosphorylated JNK in LPS-induced RAW 264.7

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macrophages cell (Figure 4B). The ﬁndings were in consistent with an earlier report

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from Yunjo Soh that JNK but not the p38 and ERK pathway involved in the inhibition

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of Herbacetin on LPS-stimulated RAW264.7 cells inflammation 18. These findings

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suggested that the inhibition of LPS-induced expression of inflammatory cytokines

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(IL-1β, IL-6, and TNF-α) by ARs could be dependent on the NF-κB and JNK/MAPK

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pathway. Moreover, these activities may be attributed to the lipophilicity of ARs,

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which allows a better penetration of it into the lipid micelles, and affects the

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efficiency of its anti-inflammatory bioactivity14. Considering the different



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homologous presented in ARs, it is of great importance to study the anti-inflammatory

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activity of ARs homologous with different chain length and chain modifications.

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Correlations of anti-inflammatory activity and chemical compositions. To

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determine the contribution of single homologue in ARs on anti-inflammatory capacity,

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the Pearson correlation coefficient was calculated. There was no significant cytotoxic

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effect of AR homologues to RAW 264.7 cells at the tested concentration of 15 µM

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(Figure S3). The results showed there was no significant correlation between the AR

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homologues content and anti-inflammatory activity except for index TNF-α. The

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strongest correlative value was obtained between TNF-α and C17:0 content (R=-0.347,

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P< 0.05). These data suggested that maybe only C17:0 contributed to the TNF-α

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inhibition activity of ARs. Previous study also showed that C17:0 had higher

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solubility compared with other long-chain homologues19. Due to the higher absorption

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of C17:0 to cells, it might play a more important role in anti-inflammatory activities.

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In order to testify this hypothesis, five AR homologues (C17:0, C19:0, C21:0, C23:0

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and C25:0) were compared under the same concentration (15 µM) to verify their

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anti-inflammatory activity by detecting the TNF-α protein expression using

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Western-blotting. Only C17:0 has significantly decreased the LPS-induced TNF-α

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expression compared with the other homologues, which suggested the

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anti-inflammatory bioactivity of ARs maybe partly dependent on the content of C17:0

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in some extent (Figure 5). Furthermore, epidemiological studies showed that habitual

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high intake of whole-grain rye could result in a higher ratio of AR homologues C17:0

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to C21:0 in plasma, which could improve insulin sensitivity and induce a beneficial

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blood lipid profile 20. The above results showed that C17:0 could contribute to the

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wellness of human health, and further study on the molecular mechanism and

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bioavailability of ARs will be conducted in future.


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In summary, this work analyzed the influence of grown area and cultivar to the total

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ARs content, investigated related composition and anti-inflammatory activity of ARs

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from 21 different wheat varieties. Moreover, the molecular mechanisms of its

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anti-inflammation and structure-activity relationship were also investigated. ARs

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homologues C17:0 had been proven to be the main active constituent. The finding of

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this work evidenced that ARs could be used as a good dietary source with the function

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of anti-inflammation to improve human health.

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ACKNOWLEDGMENT

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This research was financially supported by the Chinese National Natural Science

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Foundation (Grant 31701640), Beijing Excellent Talents Funding for Youth Scientist

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Innovation Team (2016000026833TD01), Support Project of Natural Science

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Foundation Training (BTBU-LKJJ2017-26) and Support Project of High-level

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Teachers in Beijing Municipal Universities (IDHT20180506).

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ABBREVIATIONS

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ARs, 5-n-alkylresorcinols; ERK1/2, extracellular signal-regulated kinase; FBS, fetal

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bovine serum; IL-6, interleukin-6; IL-1β, interleukin-1β; JNK, c-Jun N-terminal

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kinase LPS; lipopolysaccharide; MAPK, mitogen-activated protein kinase; MRM,

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multiple reaction monitor; MTT,

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3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide; NF-κB, nuclear

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factor-κB; TNF-α, tumor necrosis factor-α

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CONFLICT OF INTEREST

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The authors declare that there are no conflicts of interest.

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REFERENCES

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Figure captions:

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Figure 1. LC-MS of alkylresorcinols in ethyl acetate extracted from wheat brans.

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Figure 2. The effects of ARs extracted from 21 wheat brans on the expression of

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pro-inflammatory cytokines. RAW 264.7 cells (6.0×104 cells/mL) were pretreated

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with 1µg/mL ARs for 24 h, followed by treatment with 10ng/mL LPS for 4 h. The

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genes expression of pro-inflammatory cytokines on mRNA levels (A) IL-1β, (B) IL-6

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and (C) TNF-α were determined by qPCR. All results were expressed as the means ±

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SD (n = 3). Results marked with (*) are significantly different between LPS positive

325

control and test samples (P < 0.05).

326

Figure 3. The effects of ARs extracted from HM9 on the expression of

327

pro-inflammatory cytokines. RAW 264.7 cells (6.0×104 cells/mL) were pretreated

328

with ARs (0.5, 1, 2, and 4 µg/mL) for 24 h, followed by treatment with 10ng/mL LPS

329

for 4 h. The genes expression of pro-inflammatory cytokines on protein levels (A)

330

IL-1β, (B) TNF-α were determined by Western-blotting. All results denote the means

331

± SD (n = 3). Results marked with the same letters are not significantly different (P
111

2

5-nonadecylresorcinol

8.2

376

377 > 111

3

5-heneicosylresorcinol

9.3

404

405 > 111

4

5-n-tricosylresorcinol

10.7

432

433 > 111

5

5-pentacosylresorcinol

12.7

460

461 > 111

348 349

Figure 1.



350

351 352

Figure 2.

353


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354

355 356

Figure 3.

357 358 359 360



361 362

Figure 4.

363 364


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365 366

Figure 5.



367

Page 22 of 26

Table 1. Collection information of 21 wheat samples Sample

Species

Location

latitude and longitude

Elevation（m）

Collection time

AK58

Aikang58

Xi’an, Shanxi, China

34°16′N 108°54′E

400

Aug 2016

XD33

Xidong33

Wulumuqi, Xinjiang, China

43°46’N 87°36‘’E

682

Aug 2016

CY25

Chuanyu25

Chengdu, Sichuan, China

30°05’N 102°54′E

500

Aug 2016

MM367

Mianmai367


30°05’N 102°54′E

500

Aug 2016

HM9

Hanmai9

Baoding, Hebei, China

38°30’N 115°01’E

14

Aug 2016

SL021

Shiluan021

Zibo, Shandong, China

35°55’N 117°32’E

34

Aug 2016

LX99

Liangxing99

Wuan, Hebei, China

36°28′N 113°45′E

378

Sep 2016

HG35

Hengguan35

Baoding, Hebei, China

38°30’N 115°01’E

14

Aug 2016

XD22

Xindong22


43°46’N 87°36‘’E

682

Aug 2016

XN979

Xinong979

Nanyang, Henan, China

34°40′N 112°21′E

72.2

Aug 2016

JM22

Jimai22

Zibo, Shandong, China

35°55’N 117°32’E

34

Aug 2016

LM35

Longmai35

Heihe, Heilongjiang, China

50°22′N 127°53′E

90

Aug 2016

BN207

Bainong207

Xi’an, Shanxi, China

34°16′N 108°54′E

400

Aug 2016


Page 23 of 26


CN24

Chuannong24


30°05’N 102°54′E

500

Aug 2016

LM598

LemaiL598

Huaibei, Anhui, China

33°16′N 116°23′E

32

Aug 2016

XC6

Xinchun6


43°46’N 87°36‘’E

682

Aug 2016

YN836

Yannong836

Shijiazhuang, Hebei, China

38°04′N 114°51′E

78

Aug 2016

CN42

Chuannong42


30°05’N 102°54′E

500

Aug 2016

ZM9023

Zhengmai9023

Wuhan, Hubei, China

30°52′N 114°31′E

23

Aug 2016

ZM7698

Zhengmai7698

Ningling, Henan, China

34°44′N 115°31′E

50

Aug 2016

DM98

Danmai98

Shijiazhuang, Hebei, China

38°04′N 114°51′E

78

Aug 2016



368

Page 24 of 26

Table 2. Quantification of Alkylresorcinols from 21 wheat samples Alkylresorcinols(µg/g dry brain)

369

Samples

C17:0

C19:0

C21:0

C23:0

C25:0

Total ARs

C17:0/C21:0

AK58

12±1.7

182±1.7

513±4.0

62.2±7.5

128±10

898±20

0.024±0.0032

XD33

22±1.0

217±2.1

494±0.71 99.2±4.1

141±7.4

973±12

0.044±0.0020

CY25

5.1±0.60

172±4.8

538±1.2

68.9±4.7

120±15

904±23

0.0094±0.0011

MM367

1.8±0.56

141±1.4

513±0.50 68.2±2.6

114±9.2

838±7.9

0.0035±0.0011

HM9

16±2.1

223±2.4

624±5.0

96.7±6.5

114±11

1073±19

0.026±0.0033

SL021

27±1.2

273±1.1

677±2.8

122±15

117±6.4

1217±19

0.040±0.0019

LX99

15±1.7

241±7.3

603±3.5

68.1±13

91.4±5.2

1018±13

0.024±0.0029

HG35

12±1.9

224±2.1

520±4.6

91.0±3.1

119±6.8

966±11

0.024±0.0038

XD22

17±1.2

228±3.1

499±2.5

81.3±6.6

137±12

961±16

0.033±0.0026

XN979

39±0.61

332±11

924±15

212±35

225±15

1732±58

0.042±0.0013

JM22

36±3.1

291±2.9

614±8.1

112±9.5

136±6.0

1189±11

0.059±0.0058

LM35

15±2.1

270±1.5

671±3.2

82.4±14

138±12

1177±18

0.022±0.0031

BN207

11±1.2

163±1.9

485±3.0

38.2±5.1

101±10

798±13

0.023±0.0024

CN24

9.2±1.1

170±6.7

510±2.9

50.3±5.9 93.3±6.3

834±9.8

0.018±0.0022

LM598

13±1.2

118±0.2

417±1.4

48.5±3.9

101±11

697±13

0.032±0.0028

XC6

17±1.3

223±13

540±5.3

97.5±9.7

119±4.6

996±18

0.031±0.0024

YN836

15±2.0

244±6.2

669±2.6

130±6.6

126±10

1184±17

0.022±0.0031

CN42

14±0.59

164±4.8

459±2.3

58.1±2.7

105±11

799±15

0.031±0.0013

ZM9023

26±1.0

264±7.3

517±2.2

82.1±15

138±12

1027±35

0.051±0.0019

ZM7698

20±3.0

220±5.5

520±3.4

119±6.8

151±8.5

1031±3.5

0.038±0.0061

DM98

26±2.6

263±12

629±4.1

83.5±13

93.5±5.5

1096±10

0.042±0.0043

Data expressed as mean values ± SD (n = 3).


Page 25 of 26


370

Table 3. Pearson correlation coefficient(r) between the alkylresorcinols homologues

371

content and anti-inflammatory activity.

372

373

Correlation

R

Correlation

R

Correlation

R

C17:0 versus IL-1β

0.064

C17:0 versus IL-6

0.003

C17:0 versus TNF-α

-0.347*

C19:0 versus IL-1β

-0.112

C19:0 versus IL-6

-0.159

C19:0 versus TNF-α

-0.142

C21:0 versus IL-1β

0.006

C21:0 versus IL-6

0.131

C21:0 versus TNF-α

0.390

C23:0 versus IL-1β

-0.019

C23:0 versus IL-6

-0.023

C23:0 versus TNF-α

0.218

C25:0 versus IL-1β

-0.002

C25:0 versus IL-6

-0.063

C25:0 versus TNF-α

-0.026

*P

Identification, Quantification, and Anti-inflammatory Activity of 5-n

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