Transglycosylation Improved Caffeic Acid Phenethyl Ester Anti

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Bioactive Constituents, Metabolites, and Functions

Transglycosylation improved caffeic acid phenethyl ester anti-inflammatory activity and water solubility by Leuconostoc mesenteroides dextransucrase Yao Li, Lan-hua Liu, Xiao-qin Yu, Yu-xin Zhang, Jing-wen Yang, Xue-Qin Hu, and Hong-bin Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01143 • Publication Date (Web): 27 Mar 2019 Downloaded from http://pubs.acs.org on April 1, 2019

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Journal of Agricultural and Food Chemistry

Transglycosylation improved caffeic acid phenethyl ester anti-inflammatory activity and water solubility by Leuconostoc mesenteroides dextransucrase

Order of authors: Yao Li1, Lan-hua Liu2, **, Xiao-qinYu 1, Yu-xin Zhang1, Jing-wen Yang1, Xue-qin Hu1, Hong-bin Zhang1, *

1

Department of Pharmaceutical Engineering, School of Food and Biological

Engineering，Hefei University of Technology, 193# Tunxi Road, Hefei, 230009, Anhui Province, P. R. China 2

Instrumental Analysis Center，Hefei University of Technology, 193# Tunxi Road,

Hefei, 230009, Anhui Province, P. R. China.

Corresponding author: *

Dr. Hong-bin Zhang

Tel: 86-551-62901968 Fax: 86-551-62901968

1

ACS Paragon Plus Environment


E-mail: [email protected]

**

Lan-hua Liu

Tel: 86-18655145510 Fax: 86-62904719 E-mail: [email protected]

2


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Abstract:

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Bio-glycosylation is an efficient strategy to improve the biological activity and

3

physicochemical properties of natural compounds for therapeutic drug development.

4

In this study, two caffeic acid phenethyl ester (CAPE) glucosides (G-CAPE and

5

2G-CAPE) were synthesized by transglycosylation with dextransucrase from

6

Leuconostoc mesenteroides 0326 with CAPE as an acceptor and sucrose as a donor.

7

The products were purified and the structures were characterized. The

8

physicochemical properties, anti-inflammatory activity and cytotoxicity of the two

9

CAPE glucosides were measured. The water solubility of G-CAPE and 2G-CAPE is

10

35 and 90 times higher, respectively, than that of CAPE. Compared to CAPE, the

11

mono-glycoside product showed superior anti-inflammatory effects, and its inhibition

12

rate of NO, IF-6, and TNF-α is 93.4%, 76.81%, and 56.58% in RAW 264.7

13

macrophages, respectively, at 20 µM. Also the cytotoxicity of both products was

14

significantly improved. These glycosylation-modified CAPEs circumvent some of the

15

flaws in CAPE application in anti-inflammatory drugs.

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17

Keywords:

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propolis, caffeic acid phenethyl ester, dextransucrase, transglycosylation,

19

anti-inflammation

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Introduction

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Inflammation is a non-specific immune process that responds to pathogen invasion

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and tissue injury. It is a multifaceted and complicated process that attracts immune

4

cells and inflammatory cells, activated by these stimuli, to the site of injury and

5

stimulates these cells to release a wide range of inflammatory mediators, which attract

6

more cells to the site of inflammation.1 Inflammation has been elucidated as

7

purposeful, powerful, and self-limited and as a balance between pro- and

8

anti-inflammatory signals.2 Persistence of the inflammatory response may be involved

9

in many human diseases, tissue destruction, and organ function loss.3 It has been

10

stated that chronic inflammation is a serious medical issue, as it is implied to be

11

involved in the pathogenesis of arthritis, cancer, and cardiovascular, autoimmune, and

12

neurological diseases.4 A number of studies have demonstrated that the elimination of

13

chronic inflammation is a crucial path to prevent various chronic diseases.

14

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There are many natural compounds that have been shown to target and interfere with

16

chronic inflammatory responses by different mechanisms, giving protection in many

5



17

pathologies.5,6 Propolis, a natural product from honey bees, has been popular in folk

18

medicine for centuries due to its beneficial effects on abscesses, canker sores, and

19

wound healing. Caffeic acid phenethyl ester (CAPE) is one of the most promising

20

active components in propolis. It possesses a multitude of beneficial biological

21

properties, covering anti-bacterial, antioxidant, anti-viral, anti-inflammatory, and

22

anti-cancer effects.7,8 For example, CAPE attenuated the inflammatory symptoms in

23

lipopolysaccharide (LPS)-induced skin inflammation animal model and consistently

24

reduced the expression of various inflammatory cytokines and chemokines in

25

macrophages stimulated with LPS, thereby protecting cells from damage and potential

26

death.8 It is inhibited LPS-induced activation of Nuclear factor (NF)-κB and

27

interferon- regulatory factor-3 (IRF-3), key transcription factors in the inflammatory

28

response, which control the production of several cytokines, such as tumor necrosis

29

factor- α (TNF-α) or interleukin-6 (IL-6).9 Additionally, recent research has shown

30

that CAPE blocks the binding of LPS to the TLR4/MD2 complex, which is a complex

31

between glycoprotein MD2 and TLR4 when glycoprotein MD2 expressed on the cell

32

surface and can bind with LPS to stimulate inflammation pathway, leading to a

6


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remarkable down-regulation of downstream signal activation and inflammatory

34

mediator expression.10 CAPE is a promising drug candidate for anti-inflammation

35

therapy, however its low bioavailability after oral ingestion is a limit for its potential

36

use as a clinical treatment. Although CAPE is a well-known anti-inflammatory agent,

37

there are some restrictions to its use due to its low solubility and cytotoxicity.

38

39

In order to solve such problems, we were interested in the enzymatic glycosylation of

40

phenolic hydroxyl groups, e.g., by glucosyltransferases (GTFs). Relative to chemical

41

and physical methods, bio-enzymatic modification owes its unique advantages, high

42

specificity, simple preparation and environmental friendliness. GTFs form a class of

43

enzymes that cleave the glycosidic bond of sucrose, transfer the glucose moiety to an

44

acceptor substrate, synthesize dextran or glucan, and release the fructosyl moiety.11

45

Oligosaccharides are synthesized by transferring a sucrose-derived glucose to other

46

carbohydrates. Mono-, di-, or higher glucose units can be linked to the acceptor. Many

47

studies have focused on the GTF-mediated glycosylation of phenolic compounds with

48

an incredible variety of sugar moieties to improve the physicochemical and biological

7



49

properties of the molecules, enhance their bioavailability, ameliorate their biological

50

properties, or improve the stability of anti-autooxidation molecules. 12, 13, 14 De-Xing

51

Hou et al. used transglucosylation by L. mesenteroides B-512FMCM to improve the

52

water solubility, anti-lipid peroxidation effects, and browning resistance of caffeic

53

acid.15 Acarbose analogues, which show potent inhibition of α-glucosidase, an

54

enzyme related to diabetes, have been obtained by glycosylation by Leuconostoc

55

mesenteroides B-512FMC.16 The glycosylated epigallocatechin gallate has been

56

synthesized using L. mesenteroides B-512FMC, and these glucosides show improved

57

stability upon UV radiation and solubility in liquid water.17 Hydroquinone glucoside

58

has been synthesized using L. mesenteroides dextransucrase and has been suggested

59

to be a potential skin-whitening agent owing to its superior scavenging activity.18 In

60

addition, the glycosyl moiety may affect its pharmacokinetics, cause bio-specificity at

61

the molecular level, and even target precise mechanisms of action.19,20

62

63

In this study, we applied bio-glycosylation of CAPE to improve its biological activity

64

and physicochemical properties. The mono-glucoside G-CAPE and the novel 8


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di-glucoside 2G-CAPE were synthesized by glycosylation reactions mediated by

66

dexYG P473S/P856S21 with CAPE as an acceptor and sucrose as a donor. The

67

glucosides were purified and characterized by 1H-NMR, 13C -NMR, ESI MS spectra,

68

and 1H-13C HMBC. Furthermore, the anti-inflammatory activity, the solubility, and

69

the cytotoxicity were measured. We evaluated the inhibitory effect of CAPE and its

70

glycosides on LPS-induced inflammatory responses in RAW 264.7 macrophages. In

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the process, we show the mono-glucoside product is a promising anti-inflammatory

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pro-drug, superior to CAPE and 2G-CAPE; G-CAPE can therefore be used in the

73

development of anti-inflammatory drugs. Since there are few reports on the synthesis

74

and activity of such compounds, these experiments are of great significance for

75

further study of the structural modification of CAPE and the functional application of

76

the derivatives.

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Materials and methods

79

Materials

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CAPE was purchased from Energy Chemical (Shanghai, China). Fetal bovine serum

81

(FBS) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) were

82

obtained from Sigma-Aldrich (USA). High-performance liquid chromatography

83

(HPLC)-grade water and acetonitrile were purchased from Aladdin (Shanghai, China).

84

Toll-like receptor 4 (TLR4)-specific Escherichia coli LPS was purchased from Alexis

85

Biochemical (USA). All of the other chemicals were of analytical reagent grade and

86

purchased from Aladdin. All of the enzymatic reactions were performed in reaction

87

buffer containing 5 mM calcium acetate (pH 5.4), unless stated otherwise.

88

89

Enzyme preparation and activity assays

90

Double mutant, recombinant, C-terminally truncated dextransucrase (DSR, EC2.4.5.1)

91

of L. mesenteroides 0326 was obtained as described in our preceding work.22 The

92

purity of the enzymes was checked by SDS-PAGE, and enzyme concentrations were

93

measured by the Bradford method.23 To determine the activity of dextransucrase on

94

sucrose as both glucosyl donor and acceptor substrate, one unit of enzyme activity

10


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was defined as the amount of enzyme that catalyzed the formation of 1 µmoL of

96

fructose per minute.22 Enzyme activity was measured by incubation of the enzyme at

97

25 °C in 5 mM calcium acetate (pH 5.4) with 100 mM sucrose as a substrate for 1 h;

98

the reaction was stopped by adding 3,5-dinitrosalicylic acid (DNS) solution, and the

99

fructose concentration was measured. The mixtures were boiled for 5 min and cooled

100

down. Colorimetric detection was performed with a spectrophotometer at a

101

wavelength of 520 nm. The activity was calculated by DNS methods.21

102

103

Enzymatic glucosylation of CAPE using DSR

104

To synthesize CAPE glucosides by DSR, incubation reactions were carried out in 10%

105

DMSO reaction buffer with 15 mM CAPE (>99% pure), 1M sucrose, and 100 U/mL

106

purified mutant DSR enzyme for 15 h. The reaction mixture was heated in boiling

107

water for 10 min to stop the transfer reaction, followed by centrifugation at 8,000×g

108

for 10 min to separate the supernatant. The supernatant fraction was then filtered with

11



109

a 0.22-µm syringe filter (Satorius, Germany) and analyzed by TLC and UPLC-MS to

110

confirm the glucosylation of CAPE.

111

112

TLC analysis

113

The glucosides were identified by thin-layer chromatography (TLC) with an HSG F254

114

silica gel plate (Yanyou, China) and ethyl acetate–methanol (4:1 (v/v)) upper layer as

115

a developing solvent. After developing, the TLC plate was dried and visualized using

116

a UV lamp in combination with a UV viewing box (Camag, Switzerland) at 254 nm.

117

All of the TLC analyses were performed in triplicate.

118

119

Purification of CAPE acceptor reaction products

120

The reaction mixtures were separated by 50% (v/v) ethanol fractionation. The sample

121

was placed on top of a silica gel column (4.0 × 75 cm), and CAPE glucosides were

122

eluted by ethyl acetate–methanol (5:1 (v/v)) solution. The purified transfer products

123

were analyzed by TLC method as described above to ensure removal of impurities.

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The yield, purity, and molecular mass of the obtained CAPE glucosides were

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determined by LC-MS (ACQUITY UPLC LCT Premier XE, Waters, Milford, MA,

126

USA).

127

128

Liquid chromatography time-of-flight mass spectrometry analysis

129

LC-MS analysis of the supernatant was carried out to identify the glucosylation

130

products. All of the samples were analyzed using a Waters ACQUITY UPLC Premier

131

XE equipped with an electrospray ionization (ESI) interface. A sample volume of 20

132

µL was used. Solvent A was water, and solvent B was acetonitrile. Separation was

133

achieved under the following conditions: an isocratic condition of 20/80 A/B (v/v); a

134

flow rate of 1 mL/min; column TSK-GEL amide-80, 5 µm (Waters, Milford, MA,

135

USA); monitoring at 323 nm. The eluate was introduced into the mass spectrometer.

136

The components were identified by a mass scan.

137

138

Nuclear magnetic resonance (NMR) spectroscopy

13



139

Approximately 50 mg of purified G-CAPE or 2G-CAPE was dissolved in 0.5 mL of

140

pure deuterated methanol (CD3 OD) and placed in 5-mm NMR tubes. One- and

141

two-dimensional 1H and 13C NMR spectra were then obtained on a Bruker-400 MHz

142

NMR spectrometer (Bruker Inc. Switzerland) operated at 400 MHz for 1H and 101

143

MHz for

144

2G-CAPE and the glycosidic bond in 2G-CAPE were confirmed by examining the

145

two-dimensional heteronuclear multiple bond correlation (HMBC) spectrum (376

146

MHz for

147

SiMe4 signal. All of the spectra were processed using MestReNov.10.1 (Mestrelabs

148

Research SL, Santiago de Compostela, Spain).

13

19

C at 25 °C. The bond between CAPE and glucose in G-CAPE or

F (1H,

13

C decoupled)). NMR spectra were internally referenced to an

149

150

Cell culture

151

Murine macrophage RAW264.7 cells (ATCC, USA) were cultured in Dulbecco’s

152

modified Eagle’s medium (DMEM, Hyclone, Miami, FL, USA), supplemented with

153

10% (v/v) FBS (Beyotime Biotechnology, China), 100 unit/mL penicillin, and 100

154

mg/mL streptomycin, at 37 °C in a humidified atmosphere containing 5% CO2. 14


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Water solubility determination

157

Excess amounts of CAPE, G-CAPE, or 2G-CAPE were suspended in 200 µL of

158

distilled water in a microcentrifuge tube at 25 °C. Then, the solubility of each

159

compound was maximized by a Scinentz-IID ultrasonic cleaner (Xinzhi, China).

160

Following sonication at 25 °C for 1 h, each sample was centrifuged at 12,000 ×g for

161

10 min and the supernatant was then filtered through a 0.22-µm membrane filter

162

(Satorius, Germany). The concentration of the compound in the supernatant was

163

estimated by UPLC (ACQUITY UPLC LCT Premier XE, Waters, Milford, MA, USA)

164

and the absolute water solubility was calculated.

165

166

Cell viability assay

167

Cytotoxicity of CAPE, G-CAPE and 2G-CAPE were evaluated by MTT assay in

168

RAW264.7 cells. Prior to the assay, the medium was changed. MTT (dissolved in

169

phosphate-buffered saline (PBS)) was added to the culture medium to reach 0.5

170

mg/mL. Following incubation at 37 °C for 4 h, the culture media containing MTT 15



171

were removed, and then DMSO was added to each well and the absorbance at 570 nm

172

was measured using a microplate reader (MQX200, Bio-Tek, USA) to evaluate the

173

cell viability.

174

175

Assay for NO production

176

NO production was quantified by nitrite accumulation in the culture medium using

177

the Griess reaction. LPS (1 µg/mL) was used to stimulate RAW264.7 cells, which

178

were pre-treated with compounds for 1 h. The isolated supernatants of cells with or

179

without stimulus were mixed with an equal volume of Griess reagent (Beyotime

180

Biotechnology, China) and the optical density at 540 nm was measured using a

181

microplate reader (MQX200, Bio-Tek, USA) to measure the nitrite production.

182

NaNO2 was used to generate a standard curve.

183

184

Measurement of cytokine production

185

Cytokine production was measured by enzyme-linked immunosorbent assay (ELISA)

186

by a mouse ELISA kit (TNF-α: R ＆ D SYSTEMS, DY410-05; IL-6: R ＆ D

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SYSTEMS, DY406-05) as previously reported.19

188

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Statistical analysis

190

Statistical analysis of the data (n = 3 independent experiments) was performed using

191

analysis of variance (ANOVA) and the means was compared by Tukey’s post hoc test.

192

All of the values are expressed as the mean ± SEM. Values sharing the same

193

superscript are not significantly different at P < 0.05.

194

195

Results and discussion

196

Enzymatic glycosylation of CAPE and purification of CAPE glycoside products

197

Although CAPE exerts significant anti-inflammatory activity，its cytotoxicity and low

198

solubility in water restrict its therapeutic applications.15 Therefore, we examined

199

whether CAPE could be used as a sugar transferee by LM 0326 and whether its

200

biological activity and physicochemical properties could be improved by

201

glycosylation. After CAPE was fermented with mutant dexYG P473S/P856S from L.

17



202

mesenteroides 0326, the metabolites were subjected to TLC analysis. We found that

203

CAPE was successfully transglycosylated (Figure 1A) and two reaction products were

204

detected (Figure 1 B, two spots expect CAPE on TLC plat). Then LC/MS was used to

205

further identify the product (Figure 2B and C); three peaks (two new products and

206

CAPE) were observed in HPLC chromatogram. Of the two peaks at 2 min and 3 min,

207

the corresponding mass were m/z = 445 [M-H]- and m/z = 607 [M-H]-, respectively,

208

showing that two different reaction products, mono-glycosides (G-CAPE) and

209

di-glucosides (2G-CAPE), were synthesized. These results show that CAPE

210

glucosides were successfully manufactured by the enzyme and CAPE is a new

211

receptor substrate for L. mesenteroides 0326. The reaction mixtures were separated by

212

ethanol fractionation and CAPE glucosides were eluted by ethyl acetate–methanol

213

(5:1 (v/v)) solution by silica gel column. The purified transfer products were verified

214

by TLC and LC/MS (MS are shown in Figure 1 C, others are not shown).

215

216

Structural analysis of CAPE glycosides

217

The presence and purity of CAPE glucosides with a single and a double glucose 18


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moiety attached were confirmed using LC-MS. The G-CAPE and 2G-CAPE

219

structures were determined by 1H, 13C, 1H-HCOSY, and HMBC analyses. The NMR

220

spectra of the two products are shown in Figure 2 and Figure 3, respectively, and the

221

results of 1H analysis can be found in the SI.

222

223

G-CAPE: The 10 downfield olefinic methine proton signals were observed from the

224

1

225

indicated the double bond in trans-configuration and six other signals (δ 6.8~7.5, H-2,

226

5, 6, 13, 14, 15, 16, 17) that resulted from two benzene moieties. An oxygenated

227

methylene proton signal (δ 4.37, 1H, t, J = 6.8 Hz, H-10) and a methylene proton

228

signal (δ 2.98, 1H, t, J = 6.8 Hz, H-11) were also observed. Combined with

229

spectrum analysis, we conclude the main structure of CAPE was not changed. A

230

doublet signal at 5.42 ppm (J = 3.5 Hz) was assigned to the anomeric proton,

231

indicating that one glucosyl residue was α-linked to CAPE. Using 2D NMR

232

spectroscopy (Figure 2), all 1H (SI) and 13C chemical shifts were assigned (Figure 2A).

233

It was observed that the H-1’ (5.42 ppm, J = 3.6 Hz) of the glucosyl residue was

H-NMR spectrum, which included two signals (δ 7.52 and 6.30, J = 16.0 Hz) that

19


13

C


234

coupled to C (147.73 ppm) and that the C (147.73 ppm) was coupled with H-5 (6.96

235

ppm), H-6 (7.08 ppm), and H-2 (7.21 ppm), indicating that the C (147.73 ppm) is C-4

236

of CAPE. According to these results, the structure of G-CAPE was determined, which

237

could be most appropriately referred to as α-D-Glcp-(1’→4)-CAPE (Figure 2C).

238

239

2G-CAPE: In Figure 3B, two doublet signals at 5.24 ppm (J = 3.5 Hz) and 5.45 ppm

240

(J = 3.6 Hz) were assigned to the anomeric protons, indicating that two glucosyl

241

residues were α-linked to CAPE. The two anomer carbon signals were observed from

242

the

243

heteronuclear multiple bond connectivity (gHMBC) spectrum, the anomer proton

244

signal (δ 5.45, H-1’) correlated with the oxygenated olefinic quaternary carbon signal

245

(δ 144.82, C-4) (shown in SI), indicating that the first glucose was linked to HO-4 of

246

CAPE. Moreover, the anomer proton signal (δ 5.24, H-1”) of the other glucosyl

247

residues coupled with C-3’, C-2”, C-5”, and C-6”, indicating that two glucose

248

moieties were linked by a 1-3 glycosidic bond (Figure 3B). These results reveal that

13

C-NMR, showing C-1’ and C-1” (Figure 3A) of two glucosyl residues. In the

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the structure of 2G-CAPE is α-D-Glcp-(1”→3’)-α-D-Glcp-(1’→4)-CAPE (Figure 3C).

250

This structure was confirmed by the 1 H-COSY (SI).

251

252

Glycosylation enhances the water solubility of CAPE

253

A comparison also was made between the water solubility of CAPE and its glycosides.

254

As shown in table 1, the solubility of CAPE was 5.98 µM, whereas the solubility of

255

G-CAPE and 2G-CAPE was 206.13 µM and 540.86 µM. The solubility of G-CAPE

256

or 2G-CAPE is 35 and 90 times higher, respectively, than that of CAPE. These data

257

imply that the linkage of glucosyl residues to CAPE enhances its water solubility, and

258

the influence is related to the number of attached glycosyl groups. With the

259

introduction of more glycosyl groups, the water solubility is enhanced. These results

260

are consistent with a previous study which showed that glucosides of EGCG or

261

caffeic acid possessed higher solubility than the non-glycosylated compounds.18,19,24

262

263

Cell cytotoxicity of CAPE glycosides 21



264

CAPE and CAPE glycosides showed different levels of cytotoxicity (Figure 4A). To

265

determine the concentration of CAPE and the glucosides that can be used without any

266

cytotoxicity, MTT assays were carried out on the murine macrophage cell line RAW

267

264.7.25 The cytotoxicity of CAPE, G-CAPE, and 2G-CAPE at different

268

concentrations from 1.25 to 100 µM was tested in the RAW264.7. There was a

269

significant difference between the results (Figure 4A). CAPE-treated cells showed a

270

considerably diminished cellular respiration at concentrations above 3.125 µM,

271

whereas G-CAPE only showed cytotoxicity at concentrations above 20 µM, and

272

2G-CAPE did not even show a significant cytotoxicity at concentrations below 80 µM.

273

These results indicate that the cytotoxicity of CAPE can be much improved by

274

glycosylation.

275

276

Antitumor activity of CAPE glycosides

277

To investigate the effect of glycosylation on the anti-tumor activity of CAPE, we used

278

the MTT method, a common method for screening anti-tumor drugs, to conduct

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experiments in different tumor cell lines (A375, SMMC-7721, SGC-7901, and A549).

280

As shown in table 2, we measured the inhibition of cell growth by CAPE and its

281

glucosides (100 µM) after 48 h of incubation by MTT assay. We observed that

282

glycosides reduced the inhibition of tumor cells, and this effect was further increased

283

as the amount of sugar linkage increased. These data showed that glycosylation is not

284

conducive to its anticancer activity of CAPE.

285

286

Increased inflammation activity of CAPE by glycosylation

287

It has been demonstrated that CAPE can inhibit the activation of NF-κB and IRF-3

288

and thereby significantly decrease the production of inflammatory mediators. The

289

pro-inflammatory mediators IL-6, NO, and TNF-α are closely linked to the

290

development of inflammation-related diseases.10,25 It is generally accepted that IL-6,

291

NO, and TNF-α inhibitors offer potential opportunities to identify new methods for

292

the treatment of inflammatory diseases.26 Hence, in the preliminary anti-inflammatory

293

activity screening studies, the nitrite and nitrate assay and the ELISA were used to

294

screen the inhibition of three compounds toward LPS-induced NO and LPS-induced 23



295

TNF-α and IL-6 release in RAW 264.7 mouse macrophages, respectively. The ability (%

296

inhibition) of CAPE and the glucosides to reduce pro-inflammatory cytokines NO,

297

IL-6 and TNF-α was summarized in figure 4(B, C and D). Since CAPE showed

298

significant cytotoxicity at a concentration of 5 µM in the cytotoxicity experiment

299

(Figure 4A), it is likely that its inhibitory effect on inflammatory factors at a

300

concentration greater than 3.125 µM is caused by its cytotoxicity rather than by its

301

anti-inflammatory effects. Therefore, above 5 µM to 20 µM, only the inhibition of

302

G-CAPE and 2G-CAPE is indicated. Among them, the inhibition rate by G-CAPE of

303

NO is as high as 93.40%, and it also shows outstanding inhibitory effects on IL-6 and

304

TNF-α, i.e., 76.81% and 56.58%, respectively, at a concentration of 20 µM. Besides,

305

the figure S1 shows that the effect of different concentrations of CAPE, G-CAPE, and

306

2G-CAPE on the production of inflammatory factors, NO, IL-6 and TNF- α. When

307

the concentration of G-CAPE is from 1.25 µM to 20 µM, the inhibition rate of NO

308

was observed from 31.43% to 93.4%, and the inhibition rate of IL-6 and TNF-α also

309

showed enhanced in a dose-dependent manner. Similarly, the inhibitory effect of

310

CAPE and 2G-CAPE was enhanced with increasing dose. The other hand，at each of

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the same dosing concentrations, G-CAPE showed the best inhibitory effect on the

312

three factors, namely the optimal anti-inflammatory activity, expect that the inhibition

313

of TNF-a in 5 µm. At this point, CAPE seems to be tested slightly higher effect than

314

G-CAPE, which was due to the cytotoxicity of CAPE. Through comparative analysis,

315

it is obvious that G-CAPE has excellent anti-inflammatory activity, especially

316

inhibition of NO, at the safe max concentration of 20 µm, a relatively low

317

anti-inflammatory drug dose, and the inhibition rate was as high as 93.4%. For

318

2G-CAPE, achieving a comparable anti-inflammatory effect to that of the

319

mono-glycoside of CAPE required a significant increase in the amount administered,

320

which is not conducive to its application. The introduction of more glycosyl groups

321

may affect the binding of the active site to MD2,10 and thus the inhibitory effect of the

322

binding of LPS to MD2 is weakened. These investigations indicated that

323

glycosylation can effectively improve the anti-inflammatory activity of CAPE.

324

G-CAPE showed superior anti-inflammatory effects at relatively low drug

325

concentrations. This study is of great significance for development of the

326

anti-inflammatory drug.

25



327

328

Collectively, in the present study, we found that CAPE is a new receptor substrate for

329

mutant dextransucrase from L. mesenteroides 0326. Further, a promising CAPE

330

glycoside and a novel glycoside (G-CAPE and 2G-CAPE) were synthesized by the

331

acceptor reaction of dextransucrase from L. mesenteroides, with CAPE and sucrose.

332

The glycosides were separated, purified, and characterized. Several important

333

observations were made with regard to the biochemical properties of these products.

334

The glycosides possessed beneficial physical properties, such as higher water

335

solubility (which increased with the number of linked glycosyl groups), overcoming

336

the limitation of the poor water solubility of CAPE to some extent. In addition, the

337

cytotoxicity and the anti-tumor activity of CAPE, G-CAPE, and 2G-CAPE were

338

tested by an MTT assay and we found that the cytotoxicity of G-CAPE and 2G-CAPE

339

was significantly lower than that of CAPE. The highest non-toxic concentration

340

increased from 3.125 µM (CAPE) to 80 µM (2G-CAPE). The anti-inflammatory

341

activities of these three compounds were also evaluated in an LPS-induced

342

RAW264.7 cell model. Interestingly, compared to CAPE and 2G-CAPE, G-CAPE 26


Page 26 of 42

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343

was found to be more potent in suppressing NO, TNF-α, and IL-6 production in

344

LPS-induced RAW264.7 cells at the same dosage. Also, G-CAPE showed excellent

345

anti-inflammatory effects with relative lower concentrations (20 µM)，which is

346

suitable for clinical treatment. In summary, we have demonstrated that the enzymatic

347

glycosylation is an environmentally friendly and effective strategy to improve the

348

biological activity and physicochemical properties of a natural bioactive compound.

349

G-CAPE exerts stronger anti-inflammatory effects than CAPE and 2G-CAPE. Also,

350

G-CAPE possesses lower cytotoxicity and higher water solubility. As CAPE has been

351

the focus of great interest for its bioavailability, G-CAPE can be expected to

352

eventually be useful as a pro-drug for inflammatory diseases. Further investigations

353

can provide beneficial information for the development of new drugs to prevent

354

chronic inflammatory diseases. Thus, further studies of the mechanisms behind the

355

anti-inflammatory activity of the CAPE glycosides are in progress.

356

357

Abbreviations used

358

CAPE, caffeic acid phenethyl ester; G-CAPE, the mono-glucoside of caffeic acid

27



Page 28 of 42

359

phenethyl ester; 2G-CAPE, the di-glucoside of caffeic acid phenethyl ester; HPLC,

360

high-performance

361

time-of-flight mass spectrometry analysis； HMBC, heteronuclear multiple bond

362

correlation; NMR, nuclear magnetic resonance; DNS, 3,5-dinitrosalicylic acid; MTT,

363

3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromid; ELISA，enzyme-linked

364

immunosorbent assay; NO, nitric oxide; IL-6, interleukin-6; TNF-α, tumor necrosis

365

factor-α; NF-κB, Nuclear factor –κB; IRF-3, interferon- regulatory factor-3.

liquid

chromatography;

LC-MS ， liquid

chromatography

366

367

Notes

368

The authors declare no competing financial interest.

369

370

Supporting information description

371

1

372

The effect of different concentrations of CAPE, G-CAPE, and 2G-CAPE on the

373

production of inflammatory factors, NO, IL-6 and TNF- α are showed in figure S1 as

374

Supporting Information.

H-NMR spectrum of G-CAPE and 2G-CAPE supplied as Supporting Information.

28


Page 29 of 42


375

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Funding

454

This work was financially supported by the National Natural Science Foundation of

455

China (Grant No. 81573399).

456

34


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457

Figure captions

458

Figure 1. (A) Biotransformation process for the glucosides of CAPE. (B) TLC and

459

HPLC-UV chromatograms of G-CAPE and their glucosides in the fermentation

460

supernatants of mutant strains (TLC; lane 1, controls; lanes 2, 3, 4, biotransformation

461

samples). (C) MS analyses of purified products G-CAPE and 2G-CAPE; m/z = 445

462

[M-H]- and m/z = 607 [M-H]-, respectively.

463

Figure 2. NMR spectroscopy of G-CAPE. (A) The 13C of CAPE. (B) The HMBC of

464

G-CAPE. (C) The structure of G-CAPE.

465

Figure 3. NMR spectroscopy of 2G-CAPE. (A) The

466

HMBC of 2G-CAPE. (C) The structure of 2G-CAPE.

467

Figure 4. (A) The results of the MTT assay. (B), (C), and (D) The effect of different

468

concentrations of CAPE, G-CAPE, and 2G-CAPE on the production of inflammatory

469

factors, NO, TNF- α and IL-6. ###P < 0.001 compared with unstimulated cells, *P

Transglycosylation Improved Caffeic Acid Phenethyl Ester Anti

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