A New Compound Isolated from the Reduced RiboseâTryptophan

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Bioactive Constituents, Metabolites, and Functions

A new compound isolated from the reduced ribose-tryptophan Maillard reaction products exhibits distinct anti-inflammatory activity Dan Qin, Lin Li, Jing Li, JinLong Li, Di Zhao, Yuting Li, Bing Li, and Xia Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01561 • Publication Date (Web): 12 Jun 2018 Downloaded from http://pubs.acs.org on June 13, 2018

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Journal of Agricultural and Food Chemistry

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A new compound isolated from the reduced ribose-tryptophan Maillard reaction

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products exhibits distinct anti-inflammatory activity

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Dan Qin1, 2, Lin Li1, 3, 6, Jing Li4, Jinlong Li5, Di Zhao1, Yuting Li3, Bing Li1, 6, *, Xia

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Zhang1, 6*

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381 Wushan Road, Tianhe District, Guangzhou, 510640, China

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2

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Bengbu 233100, China

College of Food Science and Engineering, South China University of Technology,

College of Food Science and Engineering, Anhui Science and Technology University,

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3

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Technology, Dongguan, 523808, China

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4

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200 Xiaolingwei Street, Nanjing 210094, China

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5

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Guangzhou, 510515, China

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6

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Product Safety, 381 Wushan Road, Guangzhou, 510640, China

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Co-corresponding authors:

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*

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*

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(+86)20-87112214

School of Chemical Engineering and Energy Technology, Dongguan University of

Center for Molecular Metabolism, Nanjing University of Science and Technology,

School of Laboratory Medicine and Biotechnology, Southern Medical University,

Guangdong Province Key Laboratory for Green Processing of Natural Products and

Bing Li, Tel: (+86) 20-87113252; E-mail: [email protected]; Fax: (+86)20-87113252 Xia Zhang, Tel: (+86) 20-87112214; E-mail: [email protected]; Fax:

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ACS Paragon Plus Environment


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Abstract: In this study a compound of 532.24 Da named BF-4 was separated from

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the ribose-tryptophan Maillard reaction products by solvent extraction and purified

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through reverse phase high performance liquid chromatography. The purified

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compound BF-4 was identified as 3-((1H-indol-3-yl) methyl)-8-(5-((1H-indol-3-yl)

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methyl)-6- oxomorpholin-2-yl)-9-hydroxy-1,7,4-dioxazecan-2-one in accordance with

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1D- and 2D-NMR spectra and LC-ESI-MS/MS analysis. BF-4 significantly reduced

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the production of NO, IL-6 and TNF-α in lipopolysaccharide-induced RAW 264.7

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cells, and inhibited NF-κB activation and mitogen-activated protein kinase (MAPK)

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phosphorylation through suppressing phosphorylation of IκBα, P65, P38 and c-Jun

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N-terminal kinase (JNK). The anti-inflammatory activity of BF-4 was comparable to

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dexamethasone and, more importantly, BF-4 showed less cytotoxicity than

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dexamethasone on the normal human liver cell LO2. The results indicate that BF-4 is

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a promising anti-inflammatory agent with pharmaceutical potential.

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Key Words: ribose; tryptophan; Maillard reaction; structure; anti-inflammatory

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activity

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INTRODUCTION

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The Maillard reaction is a combination of a series of non-enzymatic reactions,

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and it is one of the most common and complex reactions that take place mainly in

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food during thermal process 1. During Maillard reaction Schiff bases or imines are

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formed at the early stage and stabilized by Amadori rearrangements

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Amadori products are further reacted via dehydration, oxidation, cyclization and other

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reactions to form melanoidin, advanced glycation end products (AGEs), and other

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chemicals 4. The complex reactions ensure the various structures and bioactivities of

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Maillard reaction products, such as antioxidation

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anti-inflammatory activity

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bioactivity11, 12.

5, 6

2,3

. Then the

, antiproliferation

7, 8

, and

9, 10

, even though some AGEs also show undesirable

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To date, antioxidant Maillard reaction products (MRPs) have attracted more

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attention owing to their free radical scavenging ability, chelating capacity and

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reducing power13-16. Consequently, MRPs can promisingly be used as natural

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antioxidants in food products15. Meanwhile, it is also an attractive and promising way

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to discover new anti-inflammatory compounds from MRPs, however such studies

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have been seldom reported17. It would facilitate the development of anti-inflammatory

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MRPs by elaborately choosing reactants (amino acid, saccharide, etc.) and skillfully

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designing reaction conditions.

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Studies revealed that ribose was superior to hexoses in Maillard reaction as the

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overall reactivity increased regardless of high temperature and long reaction time 18.

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Besides, the amount of toxic Amadori adducts was decreased in moderated

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conditions(below 70°C), and ribose could increase the safety of Maillard reaction 19.

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Indole and its derivatives display multiple biological and pharmacological activities20.

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As the indole ring constitutes the basic structure of many therapeutic agents with

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anti-inflammatory potential 21, food-derived indole derivatives such as tryptophan will

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be a competent precursor to produce new compounds that inhibit excessive expression

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of inflammation molecules. Schiff base is unstable because of the C=N bond.

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Reduction reagent potassium borohydride was used to reduce the Schiff base from

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C=N to C-N and inhibit the generation of Amadori adducts, aiming to increase the

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stability of the MRPs.

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Inflammation is an essential part of the complex biological reactions in tissue

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responding to physical, chemical or biological injuries. Both acute and chronic

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inflammations play important roles in restoration of homeostasis 22as they are related

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to generation of nitric oxide (NO) and various inflammatory factors such as

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interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α)

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mediator that activates the expression of some inflammatory cytokines to promote the

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inflammatory response

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response. However, excessive inflammatory factors or cytokines often cause

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inflammatory activities, tissue necrosis, or inflammatory diseases

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extrinsic inflammatory pathways are connected with initiation or development of

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cancer27. Therefore, the levels of NO and other inflammatory cytokines are important

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indicators of inflammation degree28. Maintaining TNF-α and IL-6 at moderate levels

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is regarded as an effective treatment for tissues damage or inflammatory diseases 29-31.

25

23, 24

. TNF-α is a key

while IL-6 has pro-inflammatory properties in the immune

4


26

. Besides, some

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As a result, compounds with anti-inflammatory activity interest researchers due to

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their pharmaceutical potential.

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Our objectives are to obtain a new indole derivative with distinct

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anti-inflammatory activity from reduced tryptophan-ribose Maillard reaction，and then

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study the possible blockage of signaling pathway. The chemical structure of this

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compound was determined by LC-MS/MS and 1D- and 2D-NMR. This study would

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provide information on a food-derived Maillard reaction compound that can be easily

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obtained and presents satisfactory anti-inflammatory activity.

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MATERIALS AND METHODS

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Materials and Chemicals. D-Ribose, L-tryptophan and potassium borohydride

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were purchased from Acros (New Jersey, USA). LPS was purchased from Sigma (St.

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Louis., MO, USA). Griess reagent was obtained from Beyotime Biotechnology

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(Shanghai, China). ELISA kits (Mouse TNF-α and IL-6) were purchased from

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Neobioscience Technology Company (Shenzhen, China). Fetal bovine serum (FBS)

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and Dulbecco’s modified eagle’s medium (DMEM) were purchased from Gibco Life

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Technologies (Grand Island, NY).

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Preparation of Reduced Ribose-Tryptophan Maillard Reaction Products

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(RRT-MRP). The Maillard reaction was performed following the procedure 32

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described by Wang et al.

with modification. At first, ten millimole of L-tryptophan

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was dissolved in 20 mL methanol containing 10 mmol of KOH. Then 10 mL methanol

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contained 10 mmol of D-ribose was added in drops. The solution was stirred at 65°C

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for 2 h under nitrogen protection and then reduced by 30 mmol of potassium

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borohydride at room temperature for 24 h. The reaction products were obtained by

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vacuum evaporation and re-dissolved in distilled water at pH 7.0. Undissolved residue

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was removed through filtration and the filtrate was dialyzed against water with a 200

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Da membrane to remove small molecules. The final products were lyophilized (Christ,

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Alpha 2-4, Bruce Co., Osterode, Germany) and stored at 4°C for later use.

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Cytotoxicity to Murine macrophage cell (RAW264.7) and Human Normal

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Liver Cells LO2. Human normal liver cells (LO2) were obtained from Beijing Beina

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Chuanglian Biotechnology Institute (Beijing, China). RAW264.7 cells were purchased

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from the cell bank of Chinese Academy of Sciences (CAS, Shanghai, China). LO2

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and RAW264.7 cells were cultured in 10% FBS of DMEM supplemented in a

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humidified atmosphere (5% CO2 at 37°C). Cells were grown in culture with the initial

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density 5×104 cells/mL. And then, 100 µL of different fractions of RRT-MRPs with

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appropriate concentrations were added to 96-well plates. The supernatant was

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carefully removed after incubation for 24 h and then 100 µL DMEM medium and

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MTS solution (10 µL) were added to wells. After subsequent 4 h incubation, the

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absorbance of 490 nm was measured by a microplate reader (Infinite M200Pro, Tecan

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Thermofisher Fluoroskan Ascent FL, Zürich, Switzerland). The toxicity of

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RRT-MRPs was evaluated by the percentage of cell viability33.

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Anti-inflammatory Activity Assay.

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Determination of NO and Cytokines Production. Anti-inflammatory was

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analyzed according to the methods of Zhang et al .34 and Shen et al .35. At first, cells

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(1×106 cells/mL) at the exponential growth phase were transferred to 96-well plates

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and incubation for 24 h. Then cells were treated with 100 µL of LPS (2 µg/mL) and

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Maillard reaction products for 24 h. Freshly prepared medium was used as the blank.

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Accumulation of NO and concentration of TNF-α and IL-6 in supernatant were determined by the Griess reagent and ELISA kit, respectively.36 Quantitative Real-Time PCR (qRT-PCR) Analysis. The qRT-PCR analysis

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was conducted according to Prathap et al.37 and Zhang et al.

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modification. RAW264.7 cells were grown into 6-well plates at an initial

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concentration of 1.5×106 cells/mL. Incubation for 12 h, cells were treated by different

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concentrations of BF-4 (10, 20, or 40 µg/mL) with LPS (1 µg/mL), and fresh medium

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was used as the blank. After 8 h, the cells were washed with cold PBS three times and

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the total RNA was splitting using RNAiso plus (Takala Bio, Japan). And the quantity

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of RNA was detected by Nanodrop Spectrophotometer (Thermo Scientific). Then the

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reverse transcriptase was used for cDNA synthesis from RNA. The cDNA encodings

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inducible nitric oxide synthase (iNOS), IL-6, TNF-α and COX-2 genes were

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quantified by QPCR assay. The internal reference was GAPDH, and the specific

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primers used are described as below: GAPDH (forward, 5-GGCATTGCTCTCAATG

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ACAA-3, reverse, 5-TGTGAGGGAGATGCTCAGTG-3), iNOS (forward, 5-CGGC

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AAACATGACTTCAGGC-3, reverse, 5-GCACATGAAAGCGGCCATAG-3), IL-6

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(forward, 5-CCCCAATTTCCAATGCTCTCC-3, reverse, 5-CGCACTAGGTTTGCC

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GAGTA-3), TNF-α (forward, 5-TGGAACTGGCAGAAGAGGCAC-3, reverse, 5-C

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GAGGCTCCAGTGAATTCGG-3), COX-2 (forward, 5-ATAGACGAAATCAACAA

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CCCCG-3, reverse, 5-GGATTGGAAGTTCTATTGGCAG-3) , COX-1 (forward,

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36

with slight


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5-ATCCACTCATGCCCAACTCCT-3, reverse, 5-GTTCCTACCTCCACCAATCCG

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-3), Nfkb1 (forward, 5-GATGACAGAGGCGTGTATTAGGG-3, reverse, 5-AGGCT

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CCAGTCTCCGAGTGAA-3). Gene amplification was operated by the ABI StepOne

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Plus sequence detection system (Applied Biosystems, Foster City, CA, USA). Reverse

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transcription was programmed at 42 °C for 60 min, then keep 70°C for 5 min to

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denature the RT enzyme. RT enzyme was inactivated at 95 °C for 10 min, and then

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followed by 40 cycles. Each cycle contained 15 s for denaturation at 95 °C and 60 s

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for annealing and extension at 60 °C. The melt curve confirmed to the specificity of

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the PCR products. PCR data was normalized to internal reference (GAPDH)cycle

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threshold value from the same gene, and the delta-delta CT method38 was used to

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calculate the fold changes in gene expression.

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Western Blot Analysis. RAW264.7 cells were grown in 6-well plates at an

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initial concentration of 1.5×106 cells/well. Twelve hours later, cells were treated with

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different concentrations of BF-4 (10, 20, or 40 µg/mL) with LPS (1 µg/mL), and fresh

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medium was used as the blank. After 12 h, the cells were washed with cold PBS three

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times and then added RIPA buffer (Servicebio, China) with Phenylmethylsulfonyl

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fluoride ((Servicebio, China)), sodium orthovanadate (Servicebio, China) and

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protease inhibitor cocktail (Servicebio, China). Cells were scraped into ice-cold PBS

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and mixed with lysis buffer at 4 °C for 15 min. Then the lysis was centrifuged at 12

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000 g at 4 °C for 5 min and the supernatants were collected. Then, the proteins of

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RAW264.7 cells were extracted using the Protein Extraction Kit (Servicebio, China)

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according to the manufacturer’s instructions. A BCA kit (Servicebio, China) were

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used to conform the concentrations of the obtained proteins. Equal amounts of protein

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were separated on 10% SDS-PAGE and transferred onto polyvinylidene fluoride

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(PVDF) membranes (Millipore, Bedford, MA, USA). Then the PVDF was sealed with

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5% non-fat dried milk and incubated with various primary antibodies in 1xPBS with

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0.05% Tween 20 (Solarbio, China) at 4 °C overnight. The PVDF was washed four

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times (each for 10 min) using washing buffer (1x PBS with 0.05% Tween 20) and

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incubated with horseradish peroxidase- conjugated antibody (Servicebio, China) for

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one hour at room temperature. The PVDF was washed three times again with washing

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buffer and protein binding was visualized using ECL reagents (Servicebio, China).

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The protein bands intensity was quantified by Alpha software (Alpha Innotech, USA)

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and normalized to GAPDH.

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Purification of the Anti-inflammatory Substance. RRT-MRPs were suspended

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in water and partitioned with n-Butanol. The collected fractions were concentrated by

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a rotary vacuum evaporator (RV10 control V, IKA, Germany) and then freeze-dried to

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obtain the n-Butanol fraction (Buta-RRT) and water fraction (Aqua-RRT). The

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Buta-RRT samples were further purified by semi-preparative RPHPLC system (Prep

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150 LC, Waters, USA) after filtrated through a 0.22 µm filter. Subsequently, the

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Buta-RRT samples were injected into the semi-preparative RP-HPLC system with a

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SunFire TM Prep C18 OBDTM column (250 × 19 mm, 5 µm, Waters, USA) and

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programmatically eluted at a flow rate of 10 mL/min with the eluent of 0.1% formic

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acid (A) and methanol (B). The detailed gradient elution program was listed as

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follows: 0-5 min, 85% A: 15% B; 5–25 min from 15% B to 85% B; 25- 30 min, 15%

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A: 85% B; 31-35 min, 0 % A: 100% B; 36-38 min, 85% A: 15% B. Absorbance was

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monitored at 220 nm. Finally, four fractions (BF-1, BF-2, BF-3 and BF-4) were

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collected and freeze-dried, and their anti-inflammatory activity and toxicity were

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evaluated.

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The active fraction BF-4 was further partitioned by semi-preparative RP-HPLC

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system, using 70% methanol elution. The structure of the active compound BF-4 was

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identified by analytical LC-MS/MS and nuclear magnetic resonance (NMR). HPLC

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analysis of BF-4 was performed by an HPLC-PDA system (Agilent 1100 / Esquire

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HCT PLUS, Bruker, Germany) equipped with a Symmetry C18 column (250 × 4.6

201

mm, 5 µm, Waters, USA) at a flow rate of 0.2 mL/min and detected at 200−400 nm

202

using a photodiode array detector (PDA).

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LC-MS/MS. The molecular weight of BF-4 was measured by the Agilent1290 /

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ma Xisimpact LC-MS (Bruker, Germany) equipped with an electrospray ionization (+)

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and a SB-C18 column (RRHD 50 × 2.1 mm, 1.8 µm, Agilent, USA) at 25 °C. Briefly,

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BF-4 (2 µL) was subjected into the ESI and programmatically eluted at a flow rate of

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0.2 mL/min containing 0.1% formic acid (A) and methanol (B). The gradient program

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was as follows: 0-1 min: 85% A, 15% B; 1-3 min from 15% B to 85% B; 4-8 min: 15%

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A, 85% B; 8-9 min from 85% B to 15% B; 9-10 min 85% A, 15% B. The mass scan

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range was from 200 m/z to 1000 m/z. MS conditions in positive mode were sprayed at

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the voltage of 2.0 kV and capillary voltage of 3.5 kV.

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Nuclear magnetic resonance (NMR) spectroscopy. Analysis by 1D and 2D

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NMR39 was performed. NMR spectra were recorded in CD3OD (99.8%, Innochem

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Company, Beijing, China) on a 600 MHz Bruker (Bruker Corp, Switzerland). The free

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induction decay (FID) was processed with Bruker software. Signals were reported in

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ppm (δ) using the CD3OD as a reference. Coupling constants (J) were given in hertz

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(Hz). Multiplet patterns were designated the following abbreviations, or combinations

218

thereof: multiplet (m), doublet (d), triplet (t), quartet (q). Signal assignments were

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made from unambiguous chemical shifts and Correlation spectroscopy (H-H COSY),

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Heteronuclear single quantum coherence (C-H HSQC), Heteronuclear multiple bond

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coherence (C-H HMBC) and Distortionless enhancement by polarization transfer

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(DEPT-135) spectroscopy experiments.

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Statistical analysis. All experiments were performed in triplicates and data were

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expressed as mean ± SD (n = 3). Analysis of variance (ANOVA) method was used to

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determine the difference between data sets at a significant level (p < 0.05) using SPSS

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20.0 (SPSS Inc., Chicago, IL).

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RESULTS AND DISCUSSION

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Anti-inflammatory effect of reduced ribose-tryptophan Maillard reaction

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products

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Inhibitory Effect of RRT-MRPs on Cytokine Secretion. Preliminary tests

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showed that moderate reaction temperature and short reaction time lowered the

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content of undesired toxic by-products in ribose-tryptophan Maillard reaction (data

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not shown), which was consistent with the former reports

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ribose-tryptophan reaction was performed at 65°C and the products were further

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subjected to butanol extraction and liquid chromatographic separation to obtain

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18, 40

. Therefore,


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possible anti-inflammatory compounds. The reduced ribose-tryptophan Maillard

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reaction products (RRT-MRPs) after reaction were fractionated by n-butanol into

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solvent (Buta-RRT) and water (Aqua-RRT) portions at first. The results of

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cytotoxicity on RAW264.7 cells demonstrated that RRT-MRPs and two portions were

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nontoxic (cell viability exceeded 90%) to cells after 24 h at the concentration up to

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1000 µg/mL (Supplementary Fig. S1).

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Lipopolysaccharide (LPS) induces production of NO and other inflammatory 20, 41

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factors including TNF-α and IL-6 in macrophages

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molecules take part in many physiological and pathological processes in tissue.

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However, excessive amounts of these molecules are detrimental to cells and result in

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chronic inflammation, carcinogenesis and sepsis 42. Table 1 indicates that production

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of NO, IL-6, as well as TNF-α in RAW 264.7 cells induced by LPS was obviously

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inhibited by addition of RRT-MRPs in a dose-dependent manner. Production of NO

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was inhibited by all products while Buta-RRT exhibited the highest activity compared

250

with the others. Meanwhile, 250 µg/mL of Buta-RRT gave 3.36 µM NO which was

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similar with the positive control dexamethasone (DXM) did (50 µg/ mL DXM, 6.70

252

µM NO, p < 0.05). Similar data was observed in IL-6 and TNF-α analysis, which

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indicates that Buta-RRT had the best performance. It seemed that the compound in

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Buta-RRT showed the best efficacy comparing with the one in aqueous extraction and

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in total RRT.

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Purification of bioactive compounds in Buta-RRT

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. These inflammatory

Reversed phase high performance liquid chromatography (RP-HPLC) was

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introduced to fractionate the components in Buta-RRT (Supplementary Fig. S2).

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Dozens of components were separated after RP-HPLC treatment and four fractions

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after further purification among them with promising anti-inflammatory activity were

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selected for further analysis, namely BF-1, BF-2, BF-3, and BF-4. Figure 1A

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illustrates that all fractions (BF-1~BF-4) was safe for RAW 264.7 cells under 250

263

µg/mL (cell viability exceeded 90%). Similar result was observed in LO2 cells

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(normal liver cells, Fig. 1B) when BF-1 ~BF-4 compounds were applied. The LO2

265

cells showed tolerance for more than 100 µg/mL of these compounds. However, when

266

a commonly used anti-inflammatory reagent, dexamethasone, was added, the viability

267

of LO2 cells remarkably decreased even in the low dose of 25 µg/mL. It is suggested

268

that these four compounds were safer than dexamethasone in the tested condition.

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Table 2 indicates that production of NO, IL-6, as well as TNF-α in RAW 264.7

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cells induced by LPS was inhibited by addition of BF-1~BF-4 and inhibition effect

271

was does-dependent. BF-4 showed the highest activity among others at the same

272

concentration. Besides, 125 µg/mL of BF-4 presented a better anti-inflammatory

273

ability than DXM (6.70 µM, p < 0.05 for NO production, 5.64 ng/mL, p < 0.05 for

274

IL-6, and 78.32 µg/mL, p < 0.05 for TNF-α).

275

Liver participates pivotal functions in animal body such as detoxification, protein 43

and it is vulnerable to toxic agents44 .

276

synthesis, and production of metabolites

277

Therefore, the cytotoxicity to liver cells must be concerned when chemical

278

compounds are investigated. Our results proved that BF-4 was safe for the human

279

normal hepatocyte cell lines in the tested

range.

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Given its promising


280

anti-inflammatory activity, BF-4 is a potential bioactive compound deserving further

281

analysis.

282

Inhibitory Effect of BF-4 on mRNA Expression. Figure 2 shows that LPS

283

induced transcription change of five key genes was alleviated by adding purified BF-4

284

compound. Transcription level of iNOS, IL-6, and TNF-α genes significantly

285

decreased when 20 µg/mL of BF-4 was added in the culture medium of RAW264.7

286

cells, and the effective dose for COX-2, and NF-κB genes were 10, and 40 µg/mL,

287

respectively. Besides, 40 µg/mL of BF-4 was more effective than 20 µg/mL of

288

dexamethasone in inhibiting the transcription of iNOS and NF-κB genes.

289

Accumulation of NO was attributed to the transcription increase of iNOS gene, which

290

was observed in this study (Fig. 2A and Table 2). Since excessive NO is related to the

291

pathophysiology of a variety of diseases and inflammation42, it is attracting that

292

application of BF-4 was effective in reducing accumulation of NO in LPS induced

293

cells by inhibiting the transcription of iNOS gene. Similar result was observed for

294

IL-6 and TNF-α (Fig. 2B, 2C, and Table 2). IL-6 is a multifunctional cytokine that

295

plays a central role in both innate and acquired immune responses45. Figure 2 also

296

shows that the dose of 20 µg/mL of BF-4 was more effective for IL-6 than it for

297

TNF-α. A possible reason was that some inflammatory factors, for example, IL-6,

298

could involve in regulating the production of TNF-α from macrophages33.

299

Cyclooxygenase (COX) contains two isozymes, COX-1 and COX-2, which involves

300

in the biosynthesis of the pro-inflammatory prostaglandins. Inhibition of COX-1 as

301

LPS does often causes the gastrointestinal damage46, while lowering the expression of

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COX-2 helps to reduce inflammation or pain47. The results suggested that BF-4

303

showed inhibitor activity for LPS induced elevation of COX-2(Fig. 2D) and slightly

304

promotes the production of COX-1 under 20 µg/mL (Fig. 2E).

305

Effect of BF-4 on NF-κB Activation and MAPKs Phosphorylation. Western

306

blot analysis showed that BF-4 treatment dramatically inhibited expression of COX-2.

307

The regulatory regions of COX-2 gene are responsive to transcriptional factor

308

NF-κB48. In addition, activation of the NF-κB pathway results in increasing the

309

phosphorylation of IκBα and p6526, 49. In our study, BF-4 significantly suppression the

310

phosphorylation of IκBα (Figure 3C) as well as the degradation of IκBα (Figure was

311

not shown). BF-4 could inhibit the phosphorylation of P65 at the concentration of 40

312

µg/mL. The phosphorylation of IκBα is a key process for the activation of NF-κB50.

313

After NF-κB was activated, it would promote the transcription of corresponding

314

pro-inflammatory genes 51. Based on the above analysis, it could be inferred that BF-4

315

inhibits the NF-κB activation through decreasing the p-IκBα and p-P65.

316

BF-4 significantly suppressed the phosphorylation of JNK (Figure 3E). The

317

expression level of phosphorylation of P38(p-P38) was down-regulated gradually

318

(Figure 3D). JNK and p38 are two important ways of MAPKs, and they involve in the

319

expression

320

inflammatory factors. It indicated that BF-4 could also blocked the signaling pathway

321

of MAPKs.

of

inflammation-related

genes,

causing

the

overproduction

of

322

The results showed that BF-4 obviously inhibited LPS-induced phosphorylation

323

of IκBα and JNK. In LPS-induced RAW 264.7 cells, BF-4 exerts the anti-

15



324

inflammatory effects by suppressing the pathways of NF-κB and MAPKs.

325

Structural elucidation of BF-4

326 327

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BF-4 was under twice RP-HPLC purification treatment to ensure its purity. Final purity of this compound was 96%.

328

High resolution ESI-MS gave the m/z of 533.2406 for BF-4 (Fig. 4). As the 13C

329

NMR spectrum clearly showed 29 carbon signals (Fig. 5), a possible formula was

330

speculated as C29H33N4O6. Moreover, the chemical structure of BF-4 was constructed

331

by means of 1H,

332

ESI-MS/MS fragments.

333

13

C, DEPT-135, COSY, HSQC, and HMBC spectra and verified by

Figure 5 illustrates the 1D-NMR spectra of BF-4 dissolved in CD3OD. Two 13

334

carbonyl carbons (δC 173.46, 172.36 ppm) at the low field were observed in

335

spectrum and six secondary carbons (CH2, δC 63.09, 52.18, 50.88, 50.38, 27.99, 25.46

336

ppm) and six quaternary carbons (C, δC 137.99, 137.70, 129.20, 128.48, 110.55,

337

110.31 ppm) were determined with the assistance of DEPT-135 spectrum. Peak

338

integration in δH 7.7-7.0 ppm in

339

corresponded to two indole rings.

1

C

H spectrum produced ten protons which

340

H-H and H-C correlations were easily obtained in COSY and HSQC spectra (Fig.

341

6). Long range (3JC-H) H-C correlations were carefully designated in HMBC spectrum

342

and 2JC-H and 1JC-H signals were omitted with assistance of COSY and HSQC spectra

343

(Fig. 7). The chemical structure of BF-4 (Fig. 8) was determined based on the above

344

results and the complete assignment of chemical shift was listed in Table 3.

345

Connections between carbons were determined by means of COSY (3JH-H) and

16


Page 17 of 45


346

HMBC (3JC-H) signals and the key signals used for constructing the carbon skeleton

347

were listed in Fig. 5.

348

BF-4 structure agreed well with the ESI-MS/MS results (Fig. 4 and Table 4).

349

Bond breaking generated several abundant fragment ions, including 3-(1H-indol-3-yl)

350

propanoic acid fragment (M+, m/z 188.07), 2-(3-((1H-indol-3-yl) methyl) -9-hydroxy

351

-2-oxo-1,7,4-dioxazecan-8-yl)-6-oxo-3,6-dihydro-2H-1,4-oxazin fragment ([M+H]+,

352

m/z 402.17), 2-(2-hydroxy-1-(2-(methyleneamino) ethoxy) ethyl) -6-oxo-3,6-dihydro

353

-2H-1,4-oxazin fragment ([M+H]+, m/z 215.10), and 3-ethyl-1H- indol fragment

354

([M+H]+, m/z 146.06). Previous studies concluded that low molecular weight (Mw

355

was less than 1000 Da) Maillard reaction products (MRPs) had a higher effectiveness

356

on inhibiting oxidation and inflammation

357

(p-hydroxyphenyl)-2-butenal53

358

furan-2-yl]-methanol54 showed a applaudable anti-inflammatory activity. Those

359

compounds need higher temperature (130°C and 121°C) to prepare while the reaction

360

temperature of BF-4 was only 65°C.

and

5, 52

. Some purified MRPs such as 2,4-Bis

[5-(5,6-dihydro-4H-pyridin-3-ylidene-methyl)

361

Maillard reaction consists of a series of relatively chemical reactions that

362

commonly occur in food production processes, such as material autoclaving,

363

fermentation, and high temperature mixing. Besides, Maillard reaction was observed

364

in human body55. Being different from the commonly used conditions, the alkali

365

solution was adopted in this study to speed up the reaction since tryptophan was more

366

active in alkali than that in water, Moreover, the temperature was well controlled

367

below 70°C (at 65°C) to minimize the side reaction in which harmful compounds

17



368

Page 18 of 45

were produced 15.

369

Our study enriched the conclusion that low molecular weight (BF-4, 532.24 Da)

370

ribose-tryptophan Maillard reaction product exhibited a considerable anti-

371

inflammatory activity and more importantly, low cytotoxicity to RAW 264.7 and LO2

372

cells. Considering the easy reaction and recovery procedures, BF-4 becomes a

373

potential anti-inflammatory agent with specific chemical structure.

374

ACKNOWLEDGEMENTS

375

This work is supported by the research grants from National Key R&D Program of

376

China (No. 2016YFD0400203), National Natural Science Foundation of China (No.

377

31671961),

378

2017A030311021) and the Fundamental Research Funds for the Central Universities,

379

(SCUT No. 30915011101). The authors would like to thank these organizations for

380

financial supports.

381

Supporting Information description

382

Fig. S1 Viability of RAW 264.7 cells exposed to the RRT-MRPs

383

Fig. S2 Preparative HPLC chromatogram of RRT from the n-Butanol fraction.

384

Fig. S3 Effects of fractions from RRT-MRPs on LPS-induced production of iNOS (A),

385

IL-6 (B) and TNF-α (C). The group without any treatment was used as the Blank, and

386

LPS (2 µg/mL) was used as the control, DXM (50 µg/mL) was used as the positive

387

control. Different lower letters indicate significant differences (p < 0.05) in

388

multiple-range analysis among the groups. The data are shown as means ± SD.

389

Fig. S4 Effects of main compounds from n-butanol fraction on LPS-induced

Natural

Science

Foundation

of

Guangdong

18


Province

(No.

Page 19 of 45


390

production of iNOS (A), IL-6 (B) and TNF-α (C). The group without any treatment

391

was used as the Blank, and LPS (2 µg/mL) was used as the control, DXM (50 µg/mL)

392

was used as the positive control. Different lower letters indicate significant

393

differences (p < 0.05) in multiple-range analysis among the groups. The data are

394

shown as means ± SD.

395

19



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Page 29 of 45


Figure captions Fig. 1. The viability of RAW 264.7 cells (A) and LO2 cells(B). All experiments were run in triplicate, and data showed mean ± SD values. Fig. 2. Effects of BF-4 on LPS-induced mRNA expression of iNOS (A), IL-6 (B), TNF-α (C) and COX-2 (D). The group without any treatment was used as the Blank, and LPS (1 µg/mL) was used as the control, DXM (20 µg/mL) was used as the positive control. Different lower case letters indicate significant differences (p < 0.05) in multiple-range analysis among the groups. The data are shown as means ± SD. Fig. 3. Effects of BF-4 on LPS-induced proteins expression of COX-2 (A), p-P65 (B), IκBα (C), p-P38 (D)and p-JNK(E). The group without any treatment was used as the Blank, and LPS (1 µg/mL) was used as the control, DXM (20 µg/mL) was used as the positive control. Different lower case letters indicate significant differences (p < 0.05) in multiple-range analysis among the groups. The data are shown as means ± SD. Fig. 4. The LC-MS (A) and LC-MS-MS (B) of BF-4 Fig. 5. The 1H NMR spectrum,

13

C NMR spectrum and

DEPT135(Distortionless

Enhancement by Polarization Transfer) of BF-4 Fig. 6. Expanded region of the 2D 1H–1H COSY and ring carbon region of the 2D 1

H–13C HSQC spectrum of the purified compound BF-4.

Fig. 7. The expansions of HMBC spectrum of BF-4 Fig. 8. The probable structure of BF-4

29



Page 30 of 45

Table 1 Anti-inflammatory activities of fractions from RRT-MRPs on RAW 264.7 cells Chemicals

Concentration

Inflammatory factors concentrations

(µg/mL)

NO(µM)

IL-6 (ng/mL)

TNF-α (ng/mL)

Blank control

/

2.63±0.65

0.60±0.06

0.48±0.02

LPS1

2

16.76±0.36

9.89±0.46

113.63±3.91

DXM2+LPS

50

6.70±0.23

5.64±0.07

78.32±2.05

RRT3+LPS

62.5

13.90±0.23Aa

8.24±0.68Ba

110.13±4.49Ba

125

9.43±0.27Bb

5.79±0.08Bb

90.32±2.07Ab

250

5.09±0.36Bc

4.47±0.03Bc

75.36±0.66Bc

500

3.43±0.36ABd

3.27±0.16Bd

64.85±1.77Bd

1000

2.29±0.06Be

2.70±0.05Be

47.81±0.83Be

62.5

10.79±0.33Ba

6.30±0.03Ca

99.83±6.08Ca

125

7.20±0.55Cb

5.59±0.19Cb

91.97±1.69Bb

250

3.36±0.17Cc

4.32±0.05Cc

68.57±1.89Cc

500

2.28±0.54Bd

2.94±0.12Cd

51.28±0.66Cd

1000

1.76±0.12Cd

1.47±0.05Ce

39.37±0.29Ce

62.5

14.91±1.18Aa

9.06±0.40Aa

112.71±2.87Aa

125

12.58±1.28Ab

6.42±0.13Ab

98.22±3.89Ab

250

9.38±0.86Ac

5.96±0.08Ac

87.30±1.51Ac

500

5.15±1.86Ad

5.29±0.26Ad

71.53±0.48Ad

1000

3.70±0.25Ad

5.05±0.08Ad

55.16±1.01Ae

Buta-RRT4+LPS

Aqua-RRT5+LPS

30


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Effects of DXM(Dexamethasone), RRT(Reduced Ribose-Tryptophan Maillard Reaction Products), Buta-RRT (n-Butanol RRT), and Aqua-RRT (water RRT) on RAW264.7 which were treated with LPS (lipopolysaccharide) to reduce NO, IL-6, and TNF-α. The group treated with 10% FBS in DMEM (Blank control), LPS (2 µg/mL), and DXM (50 µg/mL) was used as controls. Data are presented as the mean ± SD (n = 3). Different capital letters indicate significant differences (p < 0.05) in a multiple-range analysis among the different treatment group. Different lowercase letters indicate significant differences (p < 0.05) in multiple-range analysis among the same group in different concentrations.

31



Page 32 of 45

Table 2 Effects of main compounds from n-butanol fraction on on RAW 264.7 cells Chemicals

Concentration

Inflammatory factors concentrations

(µg/mL)

NO(µM)

IL-6 (ng/mL)

TNF-α (ng/mL)

Blank control

/

2.63±0.65

0.64±0.02

0.48±0.02

LPS

2

16.76±0.36

12.99±0.14

113.63±3.91

DXM+LPS

50

6.70±0.23

6.64±0.09

78.32±2.05

BF-1+LPS

62.5

16.13±0.05Aa

13.00±0.26Aa

124.56±4.86Aa

125

15.58±1.05Aa

10.52±0.05Ab

102.66±0.36Ab

250

13.30±0.26Ab

9.82±0.16Ac

97.92±1.39Ac

62.5

11.21±1.03Da

8.89±0.24Ba

95.83±2.04Ca

125

7.88±0.43Cb

7.51±0.23Cb

87.16±3.98CDb

250

5.40±0.09Cc

6.52±0.22Cc

70.96±1.10Cc

62.5

15.12±1.31Ca

9.17±0.11Ba

103.39±4.54Ba

125

10.51±0.63Bb

8.75±0.24Bb

97.96±3.68ABb

250

6.64±0.25Bc

7.58±0.10Bc

81.96±3.25Bc

62.5

7.50±0.03Ea

7.27±0.25Ca

91.28±1.78Da

125

4.91±0.48Db

6.10±0.15Db

73.63±6.91BCd

250

2.34±0.45Dc

5.51±0.16Dc

62.56±1.01Cc

62.5

10.79±0.33Ba

6.30±0.03Aa

99.83±6.08Da

125

7.20±0.55Bb

5.59±0.19Bb

91.97±1.69Db

250

3.36±0.17Cc

4.32±0.05Cc

68.57±1.89Dc

BF-2+LPS

BF-3+LPS

BF-4+LPS

Buta-RRT4+LPS

Effects of DXM, BF-1, BF-2, BF-3 and BF-4 (Four fractions of Buta-RRT) on RAW264.7 which

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Page 33 of 45


were treated with LPS to reduce NO, IL-6, and TNF-α. The group treated with 10% FBS in DMEM (Blank control), LPS (2 µg/mL), and DXM (50 µg/mL) was used as controls. Data are presented as the mean ± SD (n = 3). Different capital letters indicate significant differences (p < 0.05) in a multiple-range analysis among the different treatment group. Different lowercase letters indicate significant differences (p < 0.05) in multiple-range analysis among the same group in different concentrations.

33



Page 34 of 45

Table 3. 13C NMR and 1H NMR spectral data for compound BF-4 Position

δC, type

δH, (J in Hz) α

2

124.69, CH

7.20, m

3

110.31, C

-

4

119.18, CH

7.64, d (7.9)

5

119.97, CH

7.06, dd (7.4, 7.4)

6

122.56, CH

7.13, dd (7.5, 7.5)

7

112.41, CH

7.37, d (8.1)

8

137.99, C

-

9

128.48, C

-

10

25.46, CH2

3.41, m

11

71.10, CH

3.80, dd (6.7, 6.9)

13

52.18, CH2

3.61, m

14

57.18, CH

2.93, m

16

173.46, C

17

81.93, CH

19

50.38, CH2

3.17, br d (11.1)

2.55, dd (10.8, 10.0)

20

50.88, CH2

3.31, m

3.04, m

22

66.27, CH

23

172.36, C

25

63.09, CH2

3.51, dd (6.5, 5.1)

3.42, dd (6.4, 5.1)

26

72.32, CH

3.60, m

34


δH, (J in Hz) β

3.29, m

2.56, m

Page 35 of 45


27

27.99, CH2

3.31, m

28

110.55, C

-

29

125.03, CH

7.13, s

31

112.18, CH

7.35, d (8.2)

32

122.32, CH

7.10, dd (7.5, 7.7)

33

119.70, CH

7.02, dd (7.6, 7.5)

34

120.00, CH

7.60, d (8.0)

35

129.20, C

-

36

137.70，C

3.31, m

Sample was measured in CD3OD. 600 MHz (1H) and 150 MHz (13C), TMS was used as the internal standard; chemical shifts are shown in the δ scale with J values in parenthesis, brs Broad singlet, d doublet, dd doublet of doublet, m multiple

35



Table 4. Most abundant fragment ions of BF-4 in ESI-MS/MS Peak number

m/z

Relative intensity (%)

1

146.0603

12.2

2

188.0713

100.0

3

189.0743

11.1

4

214.0951

10.6

5

215.1031

32.6

6

271.0929

14.2

7

358.1763

10.0

8

384.1559

14.0

9

402.1669

45.0

10

403.1693

9.7

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Fig. 1

37



Figure 2

38


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Figure 3

39



Figure 4

40


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Figure 5

41



Figure 6

42


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Figure 7

43



Figure 8

44


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TOC grafic

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A New Compound Isolated from the Reduced RiboseâTryptophan

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