Synthesis and Functional Characterization of Caffeic Acid Glucoside

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Synthesis and Functional Characterization of Caffeic Acid Glucoside Using Leuconostoc mesenteroides Dextransucrase Seung-Hee Nam, Young-Min Kim, Marie K Walsh, Young-Jung Wee, Kwang-Yeol Yang, Jin-A Ko, Songhee Han, Thi Thanh Hanh Nguyen, Ji Young Kim, and Doman Kim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00344 • Publication Date (Web): 08 Mar 2017 Downloaded from http://pubs.acs.org on March 9, 2017

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Journal of Agricultural and Food Chemistry

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Synthesis and Functional Characterization of Caffeic Acid Glucoside Using

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Leuconostoc mesenteroides Dextransucrase

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Seung-Hee Nam,a,†,* Young-Min Kim,b, f, † Marie K. Walsh,c Young-Jung Wee,d Kwang-Yeol

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Yang,e Jin-A Ko,f Songhee Han,g Thi Thanh Hanh Nguyen,h Ji Young Kim,g and Doman

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Kimg, h, *

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a

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South Korea. b

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c

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Department of Food Science and Technology, Yeungnam University, Gyeongbuk 38541, South Korea

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Department of Nutrition, Dietetics, and Food Sciences, Utah State University, 8700 Old Main Hill, 750N 1200E, 84322-8700, Logan, UT, USA.

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Department of Food Science & Technology and BK21 Plus Program, Chonnam National University, Gwangju 61186, South Korea.

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Institute of Agricultural Science and Technology, Chonnam National University, Gwangju 61186,

Department of Plant Biotechnology, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, 61186, South Korea.

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Bio-energy Research Center, Chonnam National University, Gwangju 61186, South Korea

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ACS Paragon Plus Environment


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g

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Department of International Agricultural Technology, Seoul National University, Gangwon do 25354, South Korea

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Institute of Food Industrialization, Institutes of Green Bio Science & Technology, Seoul National University, Gangwon-do 25354, South Korea

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†

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*

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Seung-Hee Nam, Institute of Agricultural Science and Technology, Chonnam National University,

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Gwangju 61186, South Korea. TEL: +82-62-530-0620; Fax: +82-62-530-2149, E-mail: namsh1000@

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hanmail.net.

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Doman Kim, Graduate School of International Agricultural Technology, Seoul National University,

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Gangwon-do 25354, South Korea. TEL & Fax: +82-633-339-5736; E-mail: kimdm@ snu.ac.kr

These authors contributed equally to this work. Correspondence to:

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ABSTRACT: Caffeic acid was modified via transglucosylation, using sucrose and

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dextransucrase from Leuconostoc mesenteroides B‐512FMCM. Following enzymatic

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modification, a caffeic acid glucoside was isolated by butanol separation, silica gel

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chromatography, and preparative HPLC. The synthesized caffeic acid glucoside had a

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molecular mass-to-charge ratio of 365 m/z and its structure was identified as caffeic acid-3-O-

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α-D-glucopyranoside. The production of this caffeic acid-3-O-α-D-glucopyranoside at a

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concentration of 153 mM was optimized using 325 mM caffeic acid, 355 mM sucrose, and

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650mU mL-1 dextransucrase in the synthesis reaction. In comparison with the caffeic acid, the

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caffeic acid-3-O-α-D-glucopyranoside displayed 3-fold higher water solubility, 1.66-fold

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higher anti-lipid peroxidation effect, 15% stronger inhibition of colon cancer cell growth and

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11.5-fold higher browning resistance. These results indicate that this caffeic acid-3-O-α-D-

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glucopyranoside may be a suitable functional component of food and pharmaceutical

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products.

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KEYWORDS: caffeic acid, dextransucrase, Leuconostoc mesenteroides, transglucosylation

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■ INTRODUCTION

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Polyphenolic compounds are important bioactive substances that are prevalent in plants.

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Among them, caffeic acid (3,4-dihydroxycinnamic acid) is naturally found in various

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agricultural products such as fruit, vegetables, and coffee beans.1 It is widely present in

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higher plants in the free form, as well as in the form of glycosides, and esters.2 It has been

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well documented that caffeic acid has antioxidant,3,4 peroxy radical scavenging,5 and anti-

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mutagenic properties,6 as well as neuroprotective effects.7 In addition, caffeic acid also

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exerts an anti-proliferative activity against various types of cancer cells, such as those of

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cervical, lung, or colon cancer.8 Owing to various beneficial effects of caffeic acid, it has

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been used in functional foods or as pharmaceutical ingredients. However, such applications

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are limited by the low water solubility and instability of caffeic acid.

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To solve this problem, the enzymatic glycosylation of phenolic acids by enzymes such as

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glucansucrase has generated a considerable interest, because this modification improves the

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substance solubility, increases chemical stability, and modifies physiological effects of

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polyphenolic compounds.9–13 Usually, dextransucrase catalyzes synthesis of dextran from

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sucrose by transglucosylation,14 but it can also catalyze a transfer of a glucose unit to other

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carbohydrates, or phenolic compounds via glycosidic linkages in an acceptor reaction.15

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These enzymatic acceptor reactions were used to modify various bioactive compounds to

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improve their physical and functional properties.9–13

With sucrose and dextransucrase

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from L. mesenteroides B-512FMCM, acarbose and salicin were glycosylated to alleviate

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consequences of diabetes and reduce blood coagulation, respectively.9,12 Recently,

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epigallocatechin gallate (EGCG) and quercetin glucosides synthesized using the L.

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mesenteroides B-1299CB BF563 dextransucrase were shown to be stable to UV radiation

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and to have, respectively, 12- or 100-fold higher water solubility values than quercetin or

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EGCG themselves.12,13 Furthermore, the hydroquinone glucoside, synthesized using L.

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mesenteroides dextransucrase, had a higher antioxidant activity and higher inhibitory effect

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on lipid peroxidation than hydroquinone itself and, therefore, it could be suitably used as a

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potential skin-whitening agent in cosmetic.11

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In this study, caffeic acid glucoside was synthesized from sucrose and caffeic acid, using L.

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mesenteroides B-512FMCM dextransucrase. The synthetic caffeic acid glucoside was further

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purified by butanol partitioning and using silica gel chromatography, or by preparative high-

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performance liquid chromatography (HPLC). Its structure was confirmed by nuclear

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magnetic resonance (NMR) and matrix-assisted laser desorption ionization time-of-flight

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mass spectrometry (MALDI-TOF MS). In addition, the optimal production conditions for this

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caffeic acid glucoside were identified by the response surface methodology (RSM).

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Furthermore, the physico-chemical and functional properties of this caffeic acid glucoside

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were characterized with respect to its water solubility, resistance to browning, antioxidant

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activity, and inhibitory effect on lipid peroxidation. These experiments are important steps to 5



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further studies investigating chemical modifications of caffeic acid and functional

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applications of obtained derivatives, because until recently, there have been few report about

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the synthesis and activity of this class chemical compounds.16

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■ MATERIALS AND METHODS

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Materials. Caffeic acid, deuterium oxide, 1,1-diphenyl-2-picrylhydrazyl (DPPH), α-

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tocopherol, and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) were

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purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum, penicillin,

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streptomycin, and Dulbecco’s modified Eagle’s medium were purchased from GIBCO BRL

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(Grand Island, NY, USA). Silica gel beads (40–60 μm) were obtained from Acros Organics

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(Geel, Belgium). Other chemical substances were used chemically pure grade.

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Enzyme Preparation and Enzymatic Activity. Dextransucrase (EC 3.2.1.11) was

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obtained from L. mesenteroides B‐512FMCM grown on the LM medium containing 2%

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(w/v) glucose as previously described.13 Enzyme activity was measured at 28 °C with 100

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mM sucrose in a 20 mM sodium-acetate buffer (pH 5.2) for different reaction periods. 17 The

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reaction aliquots were placed on thin layer chromatography (TLC) silica gel 60 plates (Merck,

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Darmstadt, Germany) and developed twice, using the following solvent system: acetonitrile:

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water (85:15, v/v). The carbohydrates were visualized on the TLC plate by dipping into

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0.03% (w/v) N-(1-naphthyl) ethylenediamine and 5% (v/v) H2SO4 in methanol, followed by 6


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heating at 120 °C for 10 min.18 The released fructose content from sucrose was measured

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using the NIH densitometry Image Program (http://rsb.info.nih.gov/nih-image) with a

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standard compound. One unit (U) of activity was defined as the amount of enzyme that

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caused the release of 1 µmol of fructose per min at 28 °C in a 20 mM sodium-acetate buffer

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(pH 5.2).

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Synthesis, Purification, and Structural Elucidation of Caffeic Acid Glucoside. The

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reaction mixture (1 L) for the acceptor reaction comprising 325 mM caffeic acid, 355 mM

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sucrose, and B-512FMCM dextransucrase (650 U) was incubated in a 20 mM sodium-acetate

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buffer (pH 5.2) at 28 °C for 6 h and boiled for 10 min to stop the reaction. Reaction mixtures

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were placed on TLC plates and developed twice, using the following solvent systems:

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nitromethane: 1-propanol: water (4:10:3, v/v/v) or ethyl acetate: acetic acid: water (3:1:1,

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v/v/v) with caffeic acid, fructose, and sucrose as standard materials.

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The reaction mixtures were separated by 50% (v/v) butanol fractionation, as described for

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the case of hydroquinone glucoside previously.11 The sample was placed on top of silica gel

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column (4.0 × 75 cm) and caffeic acid glucoside was eluted by a 85% (v/v) solution of

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acetonitrile in water. The yields and purity of the obtained caffeic acid glucoside were

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determined by HPLC (LC-10AD; Shimadzu, Kyoto, Japan), using the following instruments

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and conditions: TSK-GEL, amide-80 column (Waters, Milford, MA, USA); 80% (v/v)

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acetonitrile solvent system; 1 mL min-1 flow rate; RID-10A RI detector (Shimadzu, Tokyo, 7



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Japan). Purified caffeic acid glucoside (5 mg mL-1) was mixed with 2,5-dihydroxybenzoic

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acid (1 mg mL-1), loaded on stainless steel plate, and dried at 25 °C. The molecular mass of

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the sample was determined by MALDI-TOF (Voyager DE-STR, Applied Biosystems, Poster,

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CA, USA) in the linear way with delayed extraction (75 laser shots) and acceleration voltage

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at 65 kV.

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Approximately 10 mg of purified caffeic acid glucoside was dissolved in DMSO-d6 (250

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µL) and placed into 3-mm NMR tubes. NMR spectra were acquired on an Avance-500

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instrument (Bruker, Pleasanton, CA, USA) at the National Center for Inter-University

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Research Facilities (Kanwak-Gu, Seoul, South Korea) running at 500 MHz and 125 MHz for

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determined by the homo nuclear correlation spectroscopy (COSY), hetero nuclear single

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quantum coherence (HSQC), and hetero nuclear multiple bond correlation (HMBC)

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analyses.12

H and

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C, respectively. The glucosidic linkage between caffeic acid and glucoside was

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Optimization of Caffeic Acid Glucoside Production by the Response Surface

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Methodology. The condition of the caffeic acid glucoside production was optimized using a

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Box-Behnken design with three independent variables, sucrose concentration, enzyme

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activity, and caffeic acid concentration. The synthesis of caffeic acid glucoside was optimized

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by the RSM and presented using a second-order polynomial equation as follows:19 y=β0+

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β1x1+β2x2+β3x3+β11x12+β22x22+β33x32+β12x1x2+β13x1x3+β23x2x3. Statistical analyses of the 8


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experimental design and data were performed using Design Expert 8.0.1 software (SAS

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Institute Inc.; Cary, NC, USA). The design included 20 cultivation conditions with six

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replicates at the central point. Here, the coefficient of determination (R2) or adjusted R2

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represented the fitness of the polynomial model equation. From a preliminary experiment, the

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following ranges for the three factors were selected to optimize the caffeic acid glucoside

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synthesis: L. mesenteroides dextransucrase, 30–1,238 mU; sucrose concentration, 10–700

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mM; caffeic acid concentration, 150–619 mM (Supplemental Table S1).

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Water Solubility and Browning Resistance of Caffeic Acid Glucoside. Excess

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amounts of caffeic acid or caffeic acid glucoside were mixed with 200 µL of water in an

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Eppendorf tube at room temperature. After a 1-h sonication at room temperature, each sample

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was diluted and then filtered through a 0.45 µm MFS membrane and the extent of water

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solubility was quantified by HPLC.20 Browning resistance was evaluated in 0.25% (w/v)

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water solutions of caffeic acid or caffeic acid glucoside. The sample solutions were exposed

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to 10-W irradiation at wavelength of 254 nm for 24 h at room temperature. Changes in

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absorbance at 460 nm were determined using a UV spectrophotometer.20

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Antioxidant Activity.

Antioxidant activity of caffeic acid and caffeic acid glucoside

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was detected using the stable radical DPPH.21 Samples containing 0.01–2.0 mM solutions of

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caffeic acid and caffeic acid glucoside in ethanol were reacted with a 100 µM DPPH solution

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and monitored at 517 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, 9



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USA). Ascorbic acid was used as a reference compound. Radical scavenging activity was

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expressed as the percentage of inhibition of DPPH radical concentration. IC50 designated the

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concentration of the compound at which the level of the free radical DPPH was reduced by

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50%.

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Anti-lipid Peroxidation Activity.

Inhibition of lipid peroxidation was determined

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using the High-Performance Chemiluminescent Analyzer (Tohoku Electronic Industrial,

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Tokyo, Japan) and an ARAL kit (ABCD GmbH, Berlin, Germany). According to total ion

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chromatogram (TIC) detection method, the antioxidant preparations of caffeic acid or caffeic

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acid glucoside were mixed with sample free radical-attached luminol. When antioxidant

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species in a sample were consumed, delayed photons were generated. The lag time was

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positively related to the antioxidant species content in the samples. Samples of caffeic acid or

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caffeic acid glucoside (50–500 µM), or vitamin E (10–100 µM) were mixed with the reaction

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buffer at 37 °C and levels of their antioxidant effect were then determined.22

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Growth Inhibition of Human Colon Cancer Cell (HT29).

HT29 human colon cancer

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cells, from a colonic adenocarcinoma of a female Caucasian, were purchased from the

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American Tissue Culture Collection (HTB-38™, Rockville, MD, USA). The cells were

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maintained in Dulbecco’s modified Eagle’s medium (Life Technologies, Inc., Grand Island,

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NY, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 U mL-1

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penicillin, and 0.1 mg mL-1streptomycin in a humidified atmosphere of 95% air and 5% CO2 10


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at 37 °C. Inhibition of cell growth was assessed by MTT assay according to Murad et al.23

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Briefly, cells were plated onto a 96-well plate at a density of 2 × 104/well in 100 µL of

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medium. After 24 h, they were treated with a series of caffeic acid and its glucoside

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concentrations (0.01, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, and 10 mM) for 48 h. After treatment,

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medium containing caffeic acid and its glucoside were carefully removed, and 100 µL MTT

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solution (0.25 mg/mL in phosphate-buffered saline) was added to each well. After 4 h of

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incubation at 37℃, MTT was discarded and 200 µL of extraction buffer (90% dimethyl

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sulfoxide, 10% 100 mM glycine-NaOH, pH 10.0) were added to each well, followed by

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shaking for 30 min. Absorbance (A) at 570 nm was measured using a microplate reader.

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Percent cell viability was calculated as follows: cell viability (%) = (absorbance of the sample

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tested/absorbance of the medium only) × 100. Each experiment was repeated three times. The

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concentration of caffeic acid or its glucoside compound at which 50% of the cells survived

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(IC50) was also determined.

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■ RESULTS AND DISCUSSION

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Synthesis and Purification of Caffeic Acid Glucoside. After caffeic acid was reacted

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with sucrose in an acceptor reaction in the presence of L. mesenteroides B-512FMCM

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dextransucrase, a single reaction product, caffeic acid glucoside, was detected by TLC

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analysis (Figure 1). Caffeic acid glucoside was produced from the solution containing 500 11



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mM caffeic acid, 300 mM sucrose, and 1 U mL-1 dextransucrase. The resulting concentration

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of caffeic acid glucoside was 131.4 mM, indicating conversion yield of 26.3% based on the

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input amount of caffeic acid. The reaction mixtures were separated by butanol partitioning,

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which removed unreacted or hydrolyzed carbohydrates or enzymes into the lower layer,

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whereas caffeic acid and caffeic acid glucoside were concentrated in the upper layer (data not

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shown). Furthermore, caffeic acid and caffeic acid glucoside were eluted using an 85% (v/v)

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solution of acetonitrile in water by silica gel column chromatography. Reaction schematic

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diagram and HPLC chromatogram of caffeic acid glucoside showed purification profile of

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caffeic acid and caffeic acid glucoside (Figure 1B). The total yield of caffeic acid glucoside,

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which appeared as a brownish-yellow powder, comprised 15.3 g.

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Structural Elucidation of Caffeic Acid Glucoside. The number of glucose units attached

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to the synthesized compound was verified by MALDI-TOF MS analysis. The molecular

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weight of caffeic acid was increased by one glucose moiety. The NMR data (1H, 13C, COSY,

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HSQC, and HMBC) of caffeic acid and caffeic acid glucoside are shown in Table 1 and

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Supplemental Figure S1. The molecular mass of caffeic acid glucoside was observed at m/z

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at 365 (M + Na)+. In Table 1, a doublet signal at 5.23 ppm (J=3.5 Hz) was assigned to the

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anomeric proton, indicating that one glucosyl residue was α-linked to caffeic acid. According

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to these results, the structure of caffeic acid glucoside was confirmed as caffeic acid-3-O-α-D-

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glucopyranoside (Figure 2). 12


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There were some shifts of carbon signals at 130.90 ppm to C-1, 117.04 ppm to C-5, 118.91

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ppm to C-6, 137.75 ppm to C-1', 125.41 ppm to C-2', and 170.54 ppm to C-3'. By the

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difference of solvent, 1H and 13C data were slightly shifted compared with those reported by

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Lim et al.25 According to the HMBC data, the anomeric carbon of glucopyranoside was

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attached to the C-3 hydroxyl group of caffeic acid. The C-1'' of the glucosyl residue was

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detected at 100.18 ppm according to this HSQC data.

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Optimum Synthesis of Caffeic Acid-3-O-α-D-Glucopyranoside. Experimental design

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and corresponding RSM responses are presented in Table 2, Supplemental Figure S2. The

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caffeic acid-3-O-α-D-glucopyranoside synthesis was investigated using sucrose concentration

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(x1), dextransucrase activity (x2), and caffeic acid concentration (x3) as variables. The central

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composite design for the caffeic acid-3-O-α-D-glucopyranoside synthesis was ascertained for

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the following values of the three factors: sucrose, 355 mM; dextransucrase activity, 650 mU

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mL-1; caffeic acid, 325 mM. Values of “Prob > F” 1 mM caffeic acid and significant loss of viability was evident after 48 h of

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treatment.8

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In summary, in this study, we synthesized caffeic acid-3-O-α-D-glucopyranoside, using

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sucrose and dextransucrase from L. mesenteroides B-512FMCM. We made several important

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observations with regard to this product. First, the use of dextransucrase allowed a relatively

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high yield (~50%) of the glycosylated derivative, compared to the yields (6~16%) obtained

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with other enzymes such as sucrose phosphorylase or glucosyltransferase.2,25 Second, by our

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method, we obtained a single product, caffeic acid-3-O-α-D-glucopyranoside, whereas the

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use of other enzymes led to production of two or three caffeic acid glucosides. Third, caffeic

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acid-3-O-α-D-glucopyranoside possessed beneficial physical properties such as higher water

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solubility and higher resistance to browning. Lastly, caffeic acid-3-O-α-D-glucopyranoside 18


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showed antioxidant activity similar to that of caffeic acid, whereas its inhibitory effects on

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lipid peroxidation and HT-29 colon cancer cells growth were stronger. Thus, caffeic acid

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glucoside could be used as a novel functional ingredient in food or cosmetics. Further

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possibilities of the synthesis of caffeic acid derivatives with two or three attached glucoside

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group, using dextransucrase, are currently being explored in our laboratories to confirm the

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relationship between the number of attached glucoside moieties to the caffeic acid and

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physical stability of corresponding derivatives.

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■ ASSOICATED CONTENT

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Supporting Information

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This supporting information is available free of charge via the Internet at http://pubs.acs.org.

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Additional experimental data (Table S1–S2 and Figure S1–S3)

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■ AUTHOR INFORMATIONS

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Corresponding Authors

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* (Seung-Hee Nam) E-mail: namsh1000@ hanmail.net/ [email protected]

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* (Doman Kim) E-mail: kimdm@ snu.ac.kr

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Author contributions

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†

These authors contributed equally to this work. 19



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■ ACKNOWLEDGMENTS

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This work was supported by Basic Science Research Program through the National Research

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Foundation of Korea funded by the Ministry of Education (NRF-2016R1D1A1B03936148),

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Republic of Korea. In addition, this study was supported in part by the Basic Science

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Research Program through the National Research Foundation of Korea (NRF) funded by the

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Ministry of Education (NRF-2015R1D1A1A01056929 to D. Kim and Project No. 2010–

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0020141).

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■ ABBREVIATIONS USED

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EGCG, epigallocatechin gallate; HPLC, high-performance liquid chromatography; NMR,

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nuclear magnetic resonance; MALDI-TOF MS, matrix-assisted laser desorption ionization

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time-of-flight mass spectrometry; RSM, response surface methodology; COSY, homo nuclear

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correlation spectroscopy; HSQC, hetero nuclear single quantum coherence; HMBC, hetero

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nuclear multiple bond correlation; DPPH, 1,1-diphenyl-2-picrylhydrazyl, MTT, 3-(4,5-

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dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; TIC, total ion chromatogram.

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dextransucrase. Enzyme Microb. Technol. 2009, 45, 355–360.

of

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glucoside

using

Leuconostoc

mesenteroides

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(12) Moon, Y. H.; Lee, J. H.; Ahn, J. S.; Nam, S. H.; Oh, D. K.; Park, D. H.; Chung, H. J.;

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Kang, S.; Day, D. F.; Kim D. Synthesis, structure analyses, and characterization of novel

408

epigallocatechin gallate (EGCG) glycosides using the glucansucrase from Leuconostoc 22


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mesenteroides B-1299CB. J. Agric. Food Chem. 2006, 54, 1230–1237.

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(13) Moon, Y. H.; Lee, J. H.; John, D. Y.; Jun, W. J.; Kang, S. S.; Sim, J.; Choi, H.; Moon, J.

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H.; Kim, D. Synthesis and characterization of novel quercetin-α-D-glucopyranosides

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using glucansucrase from Leuconostoc mesenteroides. Enzyme Microb. Technol. 2007,

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40, 1124–1129.

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(14) Robyt, J. F.; Yoon, H. S.; Mukerjea, R. Dextransucrase and the mechanism for dextran biosynthesis. Carbohydr. Res. 2008, 343, 3039–3048.

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(15) Iliev, I.; Vassileva, T.; Ignatova, C.; Ivanova, I.; Haertle, T.; Monsan, P.; Chobert, J. M.

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Gluco-oligosaccharides synthesized by glucosyltransferases from constitutive mutants of

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Leuconostoc mesenteroides strain Lm 28. J. Appl. Microbiol. 2008, 104, 243–250.

419

(16) Desmet, T.; Soetaert, W.; Bojarova, P.; Kren, V.; Dijkhuizen, L.; Eastwich-Field, V.;

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Schiller, A. Enzymatic glycosylation of small molecules: challenging substrates require

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tailored catalysts. Chem. Eur. J. 2012, 18, 10786–10801.

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(17) Cho, S. K.; Eom, H.J; Moon, J. S.; Lim, S. B.; Kim, Y. K.; Lee, K. W.; Han, N.S. An

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improved process of isomaltooligosaccharide production in kimchi involving the

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addition of a Leuconostoc starter and sugars. Int. J. Food Microbiol. 2014, 170, 61–64.

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(18) Kim, J.; Nguyen, T. T.; Kim, N. M.; Moon, Y. H.; Ha, J. M.; Park, N.; Lee, D. G.;

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Hwang, K. H.; Park, J. S.; Kim, D. Functional properties of novel epigallocatechin

427

gallate glucosides synthesized by using dextransucrase from Leuconostoc mesenteroides 23



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B-1299CB4. J. Agric. Food Chem. 2016, 64, 9203–9213.

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(19) Kim, G. E.; Lee, J. H.; Jung, S. H.; Seo, E. S.; Jin, S. D.; Kim, G. J.; Cha, J. C.; Kim, E.

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J.; Park, K. D. Enzymatic synthesis and characterization of hydroquinone galactoside

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using Kluyveromyces lactis lactase. J. Agric. Food Chem. 2010, 58, 9492–9497.

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Cha, J.; Moon, Y. H.; Nam, S. H.; Xia, Y. M.; Kimura, A.; Kim, D. Synthesis and

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characterization of ampelopsin glucosides using dextransucrase from Leuconostoc

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mesenteroides B-1299CB4: Glucosylation enhancing physicochemical properties.

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acid with Bacillus subtilis X-23 α-amylase and a description of the glucosides. J.

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caffeic acid by a UDP-glucose: glucosyltransferase is maintained in planta. Biochem. J.

451

2003, 373, 987–992.

452

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characterization

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levansucrase. Appl. Microbiol. Biotechnol. 2009, 83, 1009–1016.

455

456

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25



Page 26 of 35

Table 1. NMR analysis of caffeic acid and caffeic acid-3-O-α-D-glucopyranoside Carbon position

Caffeic acid (δ)

Caffeic acid glucoside (δ1)

δC

δ1C

δH

(δ1-δ)

δH

Caffeic acid 1

125.69

130.90

(5.21)

2

114.64

114.22

(-0.42)

3

145.55

145.95

(0.4)

4

148.13

147.63

(-0.5)

5

115.74

6.76 (1H, d, J=8 Hz)

117.04

(1.3)

7.15 (1H, d, J=8.5 Hz)

6

121.15

6.97 (1H, dd, J=2, 8 Hz)

118.91

(-2.24)

6.89 (1H, d, J=8.5 Hz)

1'

144.60

7.42 (1H, d, J=16 Hz)

137.75

(-6.85)

7.13 (1H, d, J=16 Hz)

2'

115.10

6.17 (1H, d, J=16 Hz)

125.41

(10.31)

6.26 (1H, d, J=16 Hz)

3'

167.91

170.54

(2.63)

7.03 (1H, d, J=2 Hz)

6.98 (1H, s)

Glucose 1'' 2''

100.18 72.11

5.23 (1H, d, J=3.5 Hz) 3.32 (1H, m)

3''

72.77

3.76 (1H, m)

4''

69.79

3.19 (1H, m)

5''

74.00

3.50 (1H, m)

6''

60.70

3.61 (1H, m)

26


Page 27 of 35


Table 2. Central composite design matrix for the experimental design and predicted responses for caffeic acid-3-O-α-D-glucopyranoside synthesis caffeic acid-3-O-α-Dglucopyranoside synthesis

Coded levels Run no.

(mM) X1

X2

X3

Actual

Predicted

1

150

300

150

38.9

9.72

2

560

300

150

36.5

26.6

3

150

1000

150

134.7

113.8

4

560

1000

150

109.4

101.8

5

150

300

500

79.0

58.7

6

560

300

500

66.2

59.2

7

150

1000

500

131.4

113.4

8

560

1000

500

83.8

85.1

9

10.2

650

325

66.6

105.7

10

699.8

650

325

95.8

96.1

11

355

61.4

325

4.4

30.5

12

355

1238.6

325

126.5

139.8

13

355

650

30.7

0.8

27.5

14

355

650

619.3

41.8

54.5

15

355

650

325

164.9

151.9

16

355

650

325

148.4

151.9

17

355

650

325

156.5

151.9

18

355

650

325

147.1

151.9

19

355

650

325

157.0

151.9

20

355

650

325

144.3

151.9

Y = −268.1 + 0.39X1 + 0.44X2 + 1.05X3 – 0.00043X12− 0.00019X22− 0.0013X32 − 0.0001X1X2 − 0.00011X1X3 − 0.0002X2X3.

27



Page 28 of 35

Table 3. Water solubility and browning resistance of caffeic acid and caffeic acid-3-O-α-Dglucopyranoside Sample

a

Solubility in water (µM)

Relative browning (%)

Caffeic acid

6.5 + 0.05a

100 + 0.012a

Caffeic acid-3-O-α-Dglucopyranoside

20.5 + 0.12a

8.72 + 0.019a

Mean ± standard deviation (n = 3).

457

28


Page 29 of 35

458


Figure Legends

459

460

Figure 1. Analysis of the transglucosylation product by thin layer chromatography (A) and

461

high pressure liquid chromatography (B).

462

(A) Lane 1: sucrose; lane 2: fructose; lane 3: glucose; lane 4: enzyme; lane 5: standard caffeic

463

acid; lane 6: dextransucrase reaction digest. (B) Reaction schematic diagram and HPLC

464

chromatogram of caffeic acid glucoside performed with the TSK-GEL amide-80 column

465

(acetonitrile/water = 80:20 (v/v); flow rate, 1.0 mL min-1; room temperature) after butanol

466

separation and silica gel column chromatography. R: reaction mixture of caffeic acid and

467

dextransucrase; P1: purified caffeic acid glucoside; P2: caffeic acid.

468

469

Figure 2. HMBC correlation of caffeic acid glucoside between caffeic acid structures.

470

471

Figure 3. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity (A), inhibition

472

of lipid peroxidation (B) and growth inhibition of HT-29 colon cancer cells (C) by caffeic

473

acid and caffeic acid-3-O-α-D-glucopyranoside.

474

(A) Caffeic acid or caffeic acid-3-O-α-D-glucopyranoside in concentrations 0, 0.01, 0.05, 0.1,

475

0.25, 0.5, 0.75, 1.0, or 2.0 mM was mixed with a 100 µM DPPH in darkness at room

476

temperature for 10 min, and then the absorbance was monitored at 517 nm. Data are reported 29



477

as the mean ± SD of three independent experiments.

478

(B) Inhibition of lipid peroxidation was determined using the High-Performance

479

Chemiluminescent Analyzer (Tohoku Electronic Industrial, Tokyo, Japan) and ARAL kit

480

(ABCD GmbH, Berlin, Germany). Inhibition of lipid peroxidation by α-tocopherol (10, 25,

481

50, and 100 µM) was performed to produce the control set of data for comparisons. The

482

inhibitory effects of 50, 100, and 500 µM solutions of caffeic acid and caffeic acid-3-O-α-D-

483

glucopyranoside on lipid peroxidation were then determined.

484

(C) Growth inhibition of HT29 colon cancer cells by MTT assay. HT29 cell (2 × 104 per

485

well) was treated with a series of caffeic acid and caffeic acid-3-O-α-D-glucopyranoside

486

concentration (0.01, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10 or 20 mM) for 48 h. Data are means

487

± SD of six independent experiments.

30


Page 30 of 35

Page 31 of 35


(A)

Fig. 1A. Nam et al.,

31



(B)

Fig. 1B. Nam et al.,

32


Page 32 of 35

Page 33 of 35


OH HO

A

B

HO

O

HO

HO

HO

O

HO

O

O

HO

HO

Fig. 2. Nam et al.,

33



(A)

(B)

(C)

Fig. 3. Nam et al., 34


Page 34 of 35

Page 35 of 35


Caffeic Acid Glycosylation Caffeic acid + Sucrose, Dextransucrase (L. mesenteroides)

Purification (HPLC), Identification (NMR), & Optimzation (RSM) Caffeic acid Caffeic acid glucoside

Caffeic acid-3-O-α-D-glucopyranoside

Physical & Functional characterization ACS Paragon Plus Environment

Synthesis and Functional Characterization of Caffeic Acid Glucoside

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