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Enzymatic synthesis of a novel kaempferol-3-O-#-D-glucopyranosyl-(1#4)O-#-D-glucopyranoside using cyclodextrin glucanotransferase, and its inhibitory effects on aldose reductase, inflammation, and oxidative stress Woo-Jae Choung, Seung Hwan Hwang, Dam-Seul Ko, Set Byeol Kim, Seo Hyun Kim, Sung Ho Jeon, Hee-Don Choi, Soon Sung Lim, and Jae-Hoon Shim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00501 • Publication Date (Web): 16 Mar 2017 Downloaded from http://pubs.acs.org on March 16, 2017

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Journal of Agricultural and Food Chemistry

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Enzymatic synthesis of a novel kaempferol-3-O-β-D-glucopyranosyl-(1→4)-O-α-D-

2

glucopyranoside using cyclodextrin glucanotransferase, and its inhibitory effects on

3

aldose reductase, inflammation, and oxidative stress

4

Running title: Development of astragalin transfer product

5

Woo-Jae Choung*,†,‡, Seung Hwan Hwang*,†, Dam-Seul Ko†,‡, Set Byeol Kim†, Seo Hyun

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Kim‡,§, Sung Ho Jeon‡,§, Hee-Don Choi‡, Soon Sung Lim**,† and Jae-Hoon Shim**,†,‡

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8

†

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Chuncheon, Gwangwon-do, 24252, South Korea

Department of Food Science and Nutrition, Hallym University, 1 Hallymdaehak-gil,

10

‡

11

Gwangwon-do, 24252, South Korea

12

§

13

Gwangwon-do, 24252, South Korea

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‡Division of Strategic Food Research, Korea Food Research Institute, Gyeonggi, 13539,

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South Korea

Center for Aging and Health Care, Hallym University, 1 Hallymdaehak-gil, Chuncheon,

Department of Life Science, Hallym University, 1 Hallymdaehak-gil, Chuncheon,

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* Woo-Jae Choung and Seung Hwan Hwang have contributed equally to this work.

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**

19

this work.

Jae-Hoon Shim and Soon Sung Lim were corresponding authors who contributed equally to

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ACS Paragon Plus Environment


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ABSTRACT: Kaempferol-3-O-β-D-glucopyranoside (astragalin, AS), a major flavonoid that

22

exists in various plants, exerts anti-oxidant, anti-tumor, anti-human immunodeficiency virus

23

(HIV), and anti-inflammatory effects. However, the low water solubility of AS limits its use.

24

In this study, we used cyclodextrin glucanotransferase (CGTase) with maltose (G2) as a

25

donor molecule to enzymatically modify AS to improve its water solubility and

26

physiochemical properties. We isolated the glycosylated astragalin (G1-AS) and identified

27

the

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glucopyranoside, where one glucose residue was transferred to AS. G1-AS retained the anti-

29

oxidative activity of the original AS compound; however, the solubility of G1-AS was 65-

30

fold higher than that of AS. In addition, G1-AS showed enhanced anti-inflammatory effects

31

and aldose reductase inhibitory activity compared to AS when applied to rat lenses.

structure

of

G1-AS

as

kaempferol-3-O-β-D-glucopyranosyl-(1→4)-O-α-D-

32 33

Keywords: glucanotransferase, transglycosylation, astragalin, solubility, aldose reductase,

34

anti-inflammatory

35 36


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INTRODUCTION

38

Flavonoids

39

antiallergenic, antibacterial, and anti-tumor activity.1, 2 Although flavonoids can have a large

40

impact on humans, the low bioavailability due to poor water solubility has made it difficult to

41

be used in industry. Thus, the flavonol glucoside form of flavonoids has recently garnered

42

attention because of its improved solubility, stability, and functionality.3

have

important

functions

including

antioxidative,

anti-inflammatory,

43

Glucanotransferases are enzymes that transfer glucosyl moieties derived from

44

sucrose, cyclodextrins, or starches to synthesize dextrans or glucans.3 In particular, one of the

45

glucanotransferases, cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19), belongs to

46

glycoside hydrolase (GH) family 13, a group of α-amylase enzymes that contains α-amylases,

47

isoamylases, amylopullulanases, pullulanases, neopullulanases, and branching enzymes.4

48

CGTase is mainly expressed in Bacillus sp. and employed to transglycosylate glucosyl

49

residues to sugars, glycosides, and non-sugar compounds such as sugar alcohols, polyols,

50

polyphenols, flavonoids, saponins, and vitamins.4, 5 CGTase has been reported to primarily

51

transglycosylate donor molecules to α(1→4)-glycosidic linkages and to α(1→6)-glycosidic

52

linkages.4 Enzymatic transglycosylation has been employed to modify the physiochemical

53

properties of various compounds in foods such as elongation of 13-O-glucosyl to improve the

54

sweetness of stevioside.6, 7 Mono- or di-saccharides are also transferred by the enzyme to

55

acceptor molecules such as ascorbic acid to form either α (1→3)-, α(1→4), or α(1→6)-

56

glycosidic linkages.8 Transglycosylation has also been used to modify the bioactivity of

57

substances to improve their functionality, including water solubility, hypo-cholesterolemic

58

properties, anti-tumor activity, oxidative stability, and for use in pharmaceutical industries.3

59

Kaempferol-3-O-β-D-glucopyranoside (astragalin, AS) is a major flavonoid found in

60

various plant species.3 Persimmon leaves, green tea seeds, traditional herbs, and medical

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plants contain AS and are known to exert anti-oxidant, anti-tumor, anti-human



anti-inflammatory effects.3,

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9-11

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immunodeficiency

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lipopolysaccharide (LPS)-induced responses such as nitric oxide (NO), prostaglandin E2, and

64

interleukin (IL)-6 production in Raw 264.7 cells.11 In addition, a recent study showed that

65

pretreatment with AS enhanced survival during lethal endotoxemia and attenuated

66

inflammatory responses in a murine model of LPS-induced acute lung injury.9

virus

(HIV),

and

AS inhibits

67

Aldose reductase (AR, EC 1.1.1.21) catalyzes the reduction of glucose to sorbitol,

68

which is metabolized to fructose by sorbitol dehydrogenase.12 However, in diabetes, cells

69

undergoing insulin-independent glucose uptake produce significant amounts of sorbitol

70

because sorbitol cannot penetrate cellular membranes and is metabolized by sorbitol

71

dehydrogenase. In addition, sorbitol and fructose produced from the polyol pathway are

72

important contributors to the formation of advanced glycation end products, which cause the

73

dysfunction of vascular wall components. The formation and accumulation of fructose and

74

sorbitol result in induction of cellular oxidative stress and inflammation with subsequent

75

deleterious effects on various cellular functions including diabetic complications.13

76

In this study, we synthesized G1-AS using CGTase, and identified its structure and

77

nuclear magnetic resonance (NMR) spectrum for the first time. Moreover, we compared the

78

physiochemical properties of G1-AS with AS, kaempferol (KP; a precursor of AS), and

79

kaempferol-3-O-glucuronide (K-O-G; a different type of sugar). In addition, we investigated

80

the inhibitory effect of G1-AS synthesized from AS on rat lens AR (rAR), inflammation, and

81

oxidative stress to evaluate its potential for treating diabetic complications.

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MATERIALS AND METHODS

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Materials. AS was purchased from Chengdu Biopurify (Sichuan, China). Maltose (G2),

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dimethyl-d6

87

ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS+), reduced nicotinamide

88

adenine dinucleotide phosphate (NADPH), sodium phosphate, quercetin trolox, Nω-nitro-L-

89

arginine methyl ester hydrochloride (L-NAME), LPS, DL-glyceraldehyde, KP, and K-O-R

90

were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). CGTase (Toruzyme) was

91

purchased from Novozymes (Bagsvaerd, Denmark). Acetonitrile (high performance liquid

92

chromatography (HPLC) grade) was purchased from Merck KGaA (Darmstadt, Deutschland).

93

Other reagents and chemicals were purchased from Sigma-Aldrich and Merck KGaA.

sulfoxide

(DMSO-d6),

trifluoroacetic

acid

(TFA),

2,2’-azinobis(3-

94 95

Optimization of the synthesis of transglycosylation reaction products. To optimize

96

transglycosylation, various substrates dissolved in water, including sucrose, G2, α-

97

cyclodextrin (α-CD), β-CD, and γ-CD, were used and experiments were performed using

98

various reaction times (1–12 h). The optimized reaction condition used G2 as a substrate with

99

a reaction time of 3 h. The glycosylation reaction was performed in 50 mM sodium acetate

100

buffer (pH 6.0) and 11 mM AS dissolved in DMSO, 29 mM G2, and Toruzyme (5 U), in a

101

heating block at 60°C for 3 h. After the enzyme reaction, the reaction mixture was boiled for

102

10 min to inactivate the enzyme.

103

104

Thin-layer chromatography analysis for transglycosylation product monitoring of AS.

105

Thin-layer chromatography (TLC) analysis was performed using the TLC Silica gel 60 F254

106

(Merck). To activate the TLC plate, it was heated at 110°C for 30 min. After the plate was

107

activated, the samples were spotted. The silica gel plate was developed using a solvent

108

mixture of chloroform/methanol/water/acetic acid (60:30:10:0.5, v/v/v/v). After developing,



109

the reaction products were confirmed using ultraviolet (UV) light at a wavelength of 254 nm.

110

Finally, the plate was dipped into a dipping solution containing 3 g of N-(1-naphthyl)-

111

ethylenediamine in 475 mL of methanol and 50 mL of H2SO4 in 1 L of methanol. The spotted

112

plate was dried and baked in an oven at 110°C for 10 min.

113

114

HPLC for transglycosylation product analysis of AS. HPLC analysis was performed on an

115

UltiMate 3000 Standard LC System (Thermo Fisher Scientific, Waltham, MA, USA).

116

Separation was achieved using a Triart C18 column (150 × 4.6 mm, 3 µm, YMC Korea)

117

coupled with a guard column at 40°C. Samples (20 µL) were injected into the system and

118

were eluted with acidified water (0.1% TFA, A) and acetonitrile (0.1% TFA, B) at a flow rate

119

of 1.0 mL/min. The optimized gradient chromatographic conditions were as follows: 20–35%

120

B at 0–10 min and 35–100% B at 10–15 min. The detector monitored the eluent at a

121

wavelength of 254 nm.

122

123

Purification of G1-AS. G1-AS (80.8 mg) obtained from AS transglycosylation was purified

124

using the LC-Forte/R preparative HPLC system with a Triart C18 column (250 × 20 mm, 5

125

µm, YMC Korea), isocratic 30% acetonitrile solvent as the eluent at a flow rate of 12.0

126

mL/min, and UV absorbance at 254 nm to obtain eight pooled fractions. Among the fractions,

127

G1-AS was directly obtained from the fraction 3 to the fraction 5. The combined filtrates

128

were concentrated, dried in vacuo at 50°C, and freeze-dried to yield G1-AS (11.4 mg).

129

130

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-


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TOF MS) analysis. The molecular mass of G1-AS was measured using the Voyager DE STR

132

MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA, USA). The samples

133

were dissolved in water and mixed with a matrix, α-cyano-4-hydroxycinnamic acid (CHCA).

134

The ratio of the amount of the sample and matrix was 1:1 (v/v). The mixture was spotted on a

135

stainless steel plate and dried at room temperature. After the water vaporized, MALDI-TOF

136

analysis was performed with an accelerating voltage of 20 kV.

137 138

NMR analysis. Approximately 11.0 mg of G1-AS was dissolved in 600 µL of DMSO-d6 and

139

distributed to 3-mm NMR tubes and the structure was analyzed using a Bruker Avance II 600

140

FT-NMR 600 MHz spectrometer (Bruker, Madison, WI, USA). NMR analysis was

141

performed at 600 MHz for 1H and 13C at room temperature. Linkages of purified G1-AS were

142

identified using heteronuclear single quantum coherence (HSQC) and heteronuclear multiple

143

bond correlation (HMBC).

144

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Preparation of aldose reductase. Crude rAR was prepared as previously described.14-17 Rat

146

lenses were removed from eyes of Sprague-Dawley rats weighing 250–280 g and frozen at –

147

70°C until use. The rat lenses were homogenized in 10 volumes of 100 mM phosphate buffer

148

saline (pH 6.2) and centrifuged at 6,000 ×g for 20 min at 4°C. The supernatant was used as a

149

crude enzyme. A crude rAR with a specific activity of 6.5 U/mg was employed for the rAR

150

inhibitory assay.

151

152

rAR inhibitory activity assay. The activity of rAR was analyzed using a microplate

153

photometer (Thermo Fisher Scientific), which measured the decrease in the absorption of



154

NADPH at 340 nm over a 3-min period using

155

mL cuvette contained one unit of the enzyme, 100 mM potassium phosphate buffer (pH 6.2),

156

1.6 mM NADPH, and 25 mM

157

inhibition rate was calculated as follows: inhibition rate = [1 − (△Abs sample/min) – (△Abs

158

blank/min) / (△Abs control/min) - (△Abs blank/min)] × 100%.

DL-glyceraldehyde

DL-glyceraldehyde

as the substrate. Each 1.0-

as the substrate and inhibitor.16, 18, 19 The

159 160

ABTS+ assay. The radical scavenging activities of KP, K-O-G, AS, and G1-AS were

161

measured using an ABTS+ radical scavenging assay as previously described with slight

162

modifications.16, 20 ABTS diammonium salt (2 mM) and potassium persulfate (3.5 mM) were

163

mixed and diluted with distilled water. The solution was stored at room temperature in the

164

dark for 24 h before use. The ABTS+ solution was reacted with 10 µL of the sample at 750

165

nm and the activity was recorded after 10 min. Trolox was used as a positive control.

166 167

RAW 264.7 cell culture. The macrophage cell line RAW 264.7 was obtained from the

168

American Type Culture Collection (Manassas, VA, USA) and grown to confluence at 37°C

169

under a humidified 5% CO2 atmosphere in Dulbecco’s Modified Eagle’s Medium (DMEM,

170

Gibco, Waltham, MA, USA) containing 10% bovine calf serum (GenDEPOT, Katy, TX,

171

USA) and 100 U/mL penicillin-streptomycin (Gibco).

172

173

Cell viability. The cytotoxic effects of four compounds on RAW 264.7 cells were examined

174

using the 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-

175

tetrazolium, inner salt (MTS) assay kit (Promega, Madison, WI, USA). Cells (5 × 103

176

cells/well) were cultured in 96-well plates and treated with samples of the four compounds

177

(10, 50, and 100 µg/mL) for 12, 24, 48, and 72 h. After incubation, 20 µL of MTS solution


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178

was added to each well and incubated for 90 min at 37°C in a humidified 5% CO2

179

atmosphere. The optical density was measured at 490 nm for three times using an EL-800

180

Universal microplate reader. The untreated group was considered 100% viable.21

181

182

NO determination. RAW 264.7 cells were seeded into 12-well plates at a density of 4 × 105

183

cells/well and subsequently incubated with LPS (1 µg/mL) and various concentrations of

184

samples of the four compounds for 24 h. The concentration of NO in the medium was

185

measured using the Griess reagent system (Promega, Madison, WI, USA) following the

186

manufacturer’s instructions.22 L-NAME was used as a positive control. The production of NO

187

was measured at 570 nm using an EL-800 Universal microplate reader and was compared to

188

a sodium nitrite standard calibration curve. The inhibition percentage was calculated as

189

follows: inhibition percentage = [1 – (Csample – Cblank / Ccontrol – Cblank)] × 100%, where Ccontrol

190

is the NO concentration of cells with LPS, Cblank is the NO concentration of cells without LPS,

191

and Csample is the NO concentration of cells with LPS and sample. The results were also

192

expressed as the IC50 of NO inhibition, where the IC50 indicates the concentration of extract

193

necessary to decrease the NO generation of RAW 264.7 cells by 50%.

194 195

Solubility analysis. KP, K-O-G, AS, and G1-AS were dissolved in distilled water and

196

incubated at 37°C with sonication for 1 h to maximize solubility. After sonication,

197

undissolved samples were eliminated by centrifugation (7,000 ×g, 37°C, 5 min). The

198

solutions were diluted in methanol and filtered through a 0.45-µm disposable syringe filter

199

(Advantec, Dublin, CA, USA) to analyze the concentration of the samples using HPLC

200

analysis.



201 202

Statistical analysis. All of the experiments were performed in triplicate and values were

203

expressed as the mean ± the standard deviation (SD). The results were analyzed using

204

Duncan’s multiple range test using the SPSS statistical software ver. 21 (IBM, Armonk, NY,

205

USA). A value of p < 0.05 was considered to indicate statistical significance.

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RESULTS

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Optimization of the transglycosylation reaction for the synthesis of G1-AS.

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Transglycosylation products were obtained using several substrates including 29 mM sucrose,

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29 mM G2, 10 mM α-CD, 8.8 mM β-CD, and 7.7 mM γ-CD, all of which dissolved in water

211

(Table S1 in supplementary information). The yield of G1-AS transglycosylation products

212

ranged from 0.5 to 8.6%. The lowest yield was obtained using sucrose as the donor, while the

213

highest was obtained using γ-CD; however, further experiments were performed using G2 as

214

the donor, which resulted in a yield of 7.7%. After determining that G2 was the proper

215

substrate, we performed experiments to determine an appropriate reaction time. A variety of

216

reaction times were tested, ranging from 1 to 12 h (Table S2 in supplementary information).

217

The highest yield of G1-AS (7.7%) was achieved with a reaction time of 3 h and the lowest

218

yield (5.9%) was achieved after 12 h. Each reaction product was identified using TLC.

219 220

Enzymatic synthesis of G1-AS. Enzymatic transglycosylation using 1% (w/v) AS in DMSO

221

and 1% (w/v) G2 in water was performed using CGTase (Figure 1). The reaction products

222

that resulted from the transfer of glucosyl residues to AS were analyzed using TLC. Two

223

transglycosylated AS products are shown in supplementary data (Figure S1 in supplementary

224

information); these results demonstrate that CGTase successfully produced AS transfer

225

products. Thus, we hypothesized that glucosyl residues released from G2 were transferred to

226

AS. To separate G1-AS from the transglycosylated AS reaction products, we used preparative

227

HPLC with a Triart C18 column. In addition, the transglycosylated AS reaction products

228

were detected at 254 nm using HPLC analysis (Figure S2A in supplementary information).

229

Pure G1-AS with >99% purity was obtained from the preparative HPLC fractions and then

230

freeze-dried as a yellow powder (Figure S2B in supplementary information). It was identified

231

by comparing the 1H and 13C NMR spectra and the correlation NMR spectra with previously



232

reported data and MALDI-TOF MS. Thus, to our knowledge, the α-1,4-glycosidic linkage of

233

AS was synthesized for the first time.

234 235

Structure identification of G1-AS. We analyzed the molecular structure and weight of the

236

purified G1-AS using 1H,

237

spectroscopy (COSY), HMBC, and HMQC with MALDI-TOF MS. The molecular weight of

238

G1-AS was larger than that of AS and two peaks were found at m/z 633 (M+Na)+ and m/z

239

658 (M+2Na)+, which corresponded to the molecular mass of the sodium adducts of G1-AS

240

(Figure S3 in supplementary information). In the

241

flavone peaks were observed (99.17–177.87 ppm) and the peaks that resonated at 101.14 ppm

242

and 101.22 ppm were characterized as C-1'' and C-1''' glucose residues with β and α forms,

243

respectively. The two anomeric protons (H-1'' of glucose at δ 5.48 ppm and H-1''' of glucose

244

at δ 5.04 ppm) were both doublets (J = 7.62 Hz and J = 3.60 Hz, respectively) with β and α

245

configurations, respectively. The detailed data and linkage of G1-AS were analyzed using

246

NMR (Table 1) including 1H, 13C, HSQC, and HMBC correlation data. As shown in Table 1,

247

a large downfield shift was observed at C-4'' in the glucose moiety of AS from 70.26 to 79.53

248

ppm, indicating that the transferred glucose was combined to C-4''. The HMBC correlation

249

data proved that the glucose molecule in AS was combined with an α-1,4-glycosidic linkage,

250

between the anomeric proton of the 1''' position of α-glucose and the C-4'' position of β-

251

glucose. In addition, we treated G1-AS with isoamylase and α-amylase from porcine

252

pancreas, which specifically hydrolyze α-1,6- and α-1,4-glycosidic linkages, to demonstrate

253

that the linkage between AS and the transferred glucose was an α-1,4-glycosidic linkage.

254

After treatment with isoamylase and α-amylase, only α-amylase resulted in the hydrolysis of

255

G1-AS to AS (data not shown).

13

C NMR, and correlation 2D NMR spectra including correlation

13

C NMR spectrum of G1-AS, the typical

256


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Determination of water solubility. We measured the water solubility of G1-AS and

258

compared it with the solubilities of KP, K-O-G, and G1-AS (Table 2). The solubilities of KP,

259

K-O-G, and AS in water were 0.30, 3.33 and 0.56 mM, respectively, while that of G1-AS

260

was 36.19 mM. The solubility of G1-AS was 121-, 11-, and 65-fold higher than those of KP,

261

K-O-G, and AS, respectively.

262 263

Effect of G1-AS on rAR inhibition and ABTS+ radical scavenging activity. We compared

264

the abilities of KP, K-O-G, AS and G1-AS to inhibit rAR activity (Table 3). Among the four

265

compounds, G1-AS showed the strongest inhibitory activity on rAR with an IC50 value of

266

0.32 µM, which was 5.5-fold higher than that of KP (IC50 = 1.76 µM). However, the

267

inhibitory activity of G1-AS on rAR was 3.2-fold lower than that of the positive control

268

(quercetin, IC50 = 0.10 µM). On the other hand, K-O-G and AS showed lower inhibitory

269

activities against rAR, with inhibition of 46.35 and 29.31%, respectively, at a concentration

270

of 10 µg/mL. In addition, the antioxidant activities of the four compounds were evaluated in

271

vitro by examining their ABTS+ radical scavenging activity (Table 4). As shown in Table 4,

272

only KP showed mid-level ABTS+ radical scavenging activity (45.28%), whereas K-O-G, AS,

273

and G1-AS showed low activities ranging from 20.74 to 23.84% at a concentration of 33.3

274

µg/mL. Based on these results, there was no significant relationship between a compound’s

275

structure and its inhibitory activity.

276 277

Anti-inflammatory effect of G1-AS. We also investigated the effects of the four compounds

278

on LPS-induced inflammation in RAW 264.7 cells, and used NO concentration as a

279

biomarker of cellular inflammation.22 As shown in supplementary information (Figure S4 in

280

supplementary information), the four compounds did not affect cell viability; however, the



281

concentration of NO supernatant increased after LPS treatment. The four compounds at a

282

concentration of 5–250 µg/mL showed non-cytotoxic effects after 24 h of treatment.

283

Moreover, treatment with each of the four compounds resulted in concentration-dependent

284

inhibition of NO in LPS-induced RAW 264.7 cells (Figure 2). The rates of NO inhibition of

285

KP, K-O-G, AS, and G1-AS were 36.84, 88.13, 53.12, and 96.88%, respectively, and the IC50

286

values of the four compounds were 15, 8.68, 13.21, and 8.38 µM, respectively.

287

288 289 290


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291

DISCUSSION

292

To date, many studies investigating AS have reported that it has anti-oxidant, anti-tumor, anti-

293

HIV, and anti-inflammatory activities, and that it exerts inhibitory effects on diabetes and

294

cancer.3, 10, 23, 24 In 2012, Kim et al. reported an α(1→6)-glucosyl transfer product prepared

295

using dextransucrase. This AS transfer product showed increased inhibitory activity toward

296

melanin synthesis and matrix metalloproteinase-1 (MMP-1) expression, which could be

297

applicable to the cosmetic industry.3 However, the water solubility of this product was not

298

reported. In our study, G1-AS was transglycosylated with a glucosyl residue in an α(1→4)-

299

glycosyl transfer reaction using CGTase, and the water solubility of G1-AS increased

300

significantly (Table 2). The newly formed α(1→4) glycosidic linkage of our transfer product

301

can be hydrolyzed in humans by α-amylase and α-glucosidases from various microorganisms

302

present in the intestine.25, 26 Therefore, G1-AS produced in our study not only has enhanced

303

water solubility, but it could also have the same functionality as AS in the human body.

304

Indeed, G1-AS retained its anti-oxidative effect (Table 4) and showed enhanced anti-

305

inflammatory and inhibitory effects on rAR (Figure 2 and Table 3). AR is a monomeric

306

enzyme that belongs to the aldo-keto reductase superfamily. AR-derived polyols such as

307

sorbitol accumulate in the diabetic ocular lens, which results in osmotic swelling, ionic

308

imbalances, and protein insolubilization leading to cataractogenesis.16 G1-AS was more

309

effective than KP, K-O-G, and AS at inhibiting rAR, suggesting that G1-AS could be effective

310

in the prevention of diabetes complications.

311

The application of flavonoids in medicine is often limited by their low solubility.

312

Therefore, many researchers have made an effort to search for new methods of improving the

313

physical- and physiological activities of flavonoids through structural modifications.27-29 In a

314

previous study, Mok and Lee reported that KP showed inhibitory effects on rAR30; however,

315

in our study, glycosylation at the C-3 position of KP resulting in AS led to significantly



316

decreased inhibition against AR (Table 3). This phenomenon, in which glycosylation

317

decreases bioactivity, is common for flavonoid compounds. For example, the rAR inhibitory

318

activity of quercetin derivatives decreased with 3-diglycosylation (resulting in rutin) and 3-

319

monoglycosylation (resulting in hyperoside and isoquercitrin).31 As shown in Table 3,

320

monoglycosylation at the C-3 position of KP diminished rAR inhibitory activity. However,

321

diglycosylation at the same position elevated its inhibitory potency significantly, suggesting

322

that the rAR inhibitory activity of KP is strongly related to the number of sugar moieties.

323

In general, the O-glycosylation of flavonoids significantly reduces their inhibitory

324

potential against NO production.32, 33 Deglycosylation results in increased anti-inflammatory

325

activity of flavonoid compounds.32, 34 However, it has also been reported that glycosylation

326

can enhance the absorption of flavonoids in the human gut.35 Pharmacokinetic parameter

327

analysis using puerarin and hesperidin revealed that glycosylated compounds were absorbed

328

more efficiently and promptly than their original non-glycosylated structures.36, 37 Recently, it

329

was reported that glycosylated baicalein uptake in its intact form in RAW 264.7 cells

330

suppressed NO production more effectively than baicalein.38 Similarly, in this study, G1-AS

331

(diglycosylated KP) showed better inhibition of NO production than KP or AS

332

(monoglycosylated KP) in LPS-treated cells (Figure 2). Generally, flavonoids show low

333

stability in aqueous solutions since they are easily degraded by chemical and enzymatic

334

oxidation.39-41 Therefore, it seems that glycosylation increases the stability of KP by

335

protecting it from chemical and enzymatic oxidation in cells, which may enhance the

336

inhibition of NO production. In our study, the more glycosylated KP compound showed the

337

most effective inhibition of NO production, which supports our hypothesis (Figure 2).

338

In conclusion, we prepared AS transfer products using CGTase. G1-AS showed

339

enhanced water solubility, inhibition of NO production, and rAR inhibition compared to AS.

340

By comparing G1-AS with di-, mono-, and non-glycosylated KP, we propose that


Page 16 of 33

Page 17 of 33


341

glycosylation may increase the bioavailability of KP by stabilizing it in cells. This study

342

provides an alternative method for the preparation of KP-glycosylated products and extends

343

the industrial applicability and structural diversity of KP.

344

345



Page 18 of 33

346

347

Table 1. 1H and 13C NMR data (ppm) of kaempferol-3-O-glucoside (AS) and kaempferol-3-

348

O-β-D-glucopyranosyl-(1→4)-O-α-D-glucopyranoside (G1-AS) AS

Carbon no.

H (δH)

a

G1-AS 13

1

C (δC)

H (δH)

a

13

C (δC)

2

-

156.61

-

156.79

3

-

133.55

-

133.58

4

-

177.82

-

177.87

5

-

161.58

-

161.68

6

6.15 (d, 1H, J = 1.27 Hz)

99.07

6.15 (d, 1H, J = 1.27 Hz)

99.17

7

-

164.58

-

164.63

8

6.43 (d, 1H, J = 1.26 Hz)

94.03

6.43 (d, 1H, J = 1.26 Hz)

94.14

9

-

156.75

-

156.85

10

-

104.35

-

104.46

1'

-

121.27

-

121.30

2' 6'

8.04 (d, 2H, J = 8.67 Hz)

4'

-

3' 5'

6.86 (d, 2H, J = 8.64 Hz)

1''

5.48 (d, 1H, J = 7.62 Hz)

101.13

5.48 (d, 1H, J = 7.62 Hz)

101.14

2''

-

74.58

-

72.87

β-D-Glucose

3''

-

76.78

-

73.92

(1→3)

4''

3.60–3.03 (m, 6H)

70.26

3.60–3.03 (m, 6H)

79.53

5''

-

77.85

-

76.58

6''

-

61.21

-

61.23

1'''

5.04 (d, 1H, J = 3.60 Hz)

101.22

2'''

-

70.25

-

73.71

3.60-3.03 (m, 6H)

70.34

5'''

-

76.23

6'''

-

60.82

Kaempferol

349

1

α-D-Glucose

3'''

(1→4)

4'''

a)

-

131.25 (overlap)

8.04 (d, 2H, J = 8.64 Hz)

160.31 115.47 (overlap)

-

6.89 (d, 2H, J = 8.70 Hz)

δ ppm in DMSO;150 MHz for 13C; 600 MHz for 1H.

350

351


131.37 (overlap) 160.45 115.60 (overlap)

Page 19 of 33


352

Table 2. Solubility of kaempferol (KP), kaempferol-3-O-glucuronide (K-O-G), kaempferol-

353

3-O-glucoside (AS), and kaempferol-3-O-β-D-glucopyranosyl-(1→4)-O-α-D-glucopyranoside

354

(G1-AS) Water solubility (mM, 37°C)

Relative solubility

Kaempferol (KP)

0.30 ± 0.00

1

Kaempferol-3-O-glucuronide (K-O-G)

3.33 ± 0.00

1.1 × 10

Kaempferol-3-O-glucoside (AS)

0.56 ± 0.00

1.9

36.19 ± 0.08

1.2 × 102

Compound

Kaempferol-3-O-β-D-glucopyranosyl-(1→4)-Oα-D-glucopyranoside (G1-AS) 355

356



Page 20 of 33

357 358

Table 3. Inhibitory effects of kaempferol (KP), kaempferol-3-O-glucuronide (K-O-G),

359

kaempferol-3-O-glucoside (AS), and kaempferol-3-O-β-D-glucopyranosyl-(1→4)-O-α-D-

360

glucopyranoside (G1-AS) on rAR Concentration Inhibition (µg/mL) (%) 10 84.02 ± 4.28

Compound

Kaempferol (KP)

IC50 (µg/mL)

IC50 (µM)a)

5.05 ± 0.21

1.76

5

51.57 ± 2.70

1

20.33 ±0.47

10

46.35 ± 2.97

n.d.b)

10

29.31 ± 1.09

n.d.

Kaempferol-3-O-β-D-

5

75.13 ± 3.71

glucopyranosyl-(1→4)-O-α-D-

1

35.89 ± 1.10

0.5

10.86 ± 0.57

1

88.73 ± 4.97

0.5

54.16 ± 3.14

0.1

17.47 ± 1.51

Kaempferol-3-O-glucuronide (K-O-G) Kaempferol-3-O-glucoside (astragalin, AS)

glucopyranoside (G1-AS)

Quercetinc)

361 362 363

a)

1.91 ± 0.09

0.32

0.32 ± 0.01

0.10

The IC50 value was defined as the half-maximal inhibitory concentration. The values presented are the mean of three experiments. b)Not determined c)Quercetin was used as a positive control for rAR.

364

365


Page 21 of 33


366 367

Table 4. Inhibitory activities of kaempferol (KP), kaempferol-3-O-glucuronide (K-O-G),

368


369

glucopyranoside (G1-AS) on ABTS+ free radical scavenging

Concentration (µg/mL)

Inhibition (%)

Kaempferol (KP)

33.3

45.28 ± 2.84

Kaempferol-3-O-glucuronide (K-O-G)

33.3

22.75 ± 1.71

Kaempferol-3-O-glucoside (AS)

33.3

23.84 ± 2.95

33.3

20.74 ± 1.31

8.33

61.40 ± 2.17

3.33

14.58 ± 0.83

1.66

1.48 ± 0.08

Compound

Kaempferol-3-O-β-D-glucopyranosyl(1→4)-O-α-D-glucopyranoside (G1-AS)

Trolox*

370

*

Trolox was used as a positive control for the ABTS+ assay.

371 372



373

AUTHOR INFORMATION

374

Corresponding Authors

375

**

376

Hallymdaehak-gil, Chuncheon, Gwangwon-do, 24252, South Korea, Tel: +82-33-248-2137;

377

Fax:+82-33-248-2146, E-mail: [email protected]; **Soon Sung Lim, Department of Food

378

Science and Nutrition, Hallym University, 1 Hallymdeahak-gil, Chuncheon, Gwangwon-do,

379

24252, South Korea. E-mail: [email protected].

380

Funding

381

This study was supported in part by the Basic Science Research Program (NRF-2014-

382

R1A1A2057436) of the National Research Foundation, funded by the Korean Government

383

and Main Research Program (E0164800-02) of the Korea Food Research Institute (KFRI)

384

funded by the Ministry of Science, ICT & Future Planning. The authors appreciate the

385

technical support provided by YMC Korea.

386

Notes

387

The authors declare that they have no conflicts of interest.

Jae-Hoon Shim, Department of Food Science and Nutrition, Hallym University, 1

388

389


Page 22 of 33

Page 23 of 33


390

ABBREVIATIONS USED

391

ABTS+, 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt; AR, aldose

392

reductase; AS, astragalin, kaempferol-3-O-β-D-glucopyranoside; α-CD, α-cyclodextrin;

393

CGTase, cyclodextrin glucanotransferase; CHCA, cyano-4-hydroxycinnamic acid; COSY,

394

correlation spectroscopy; CGTase, cyclodextrin glucanotransferase; DMSO-d6, dimethyl-d6

395

sulfoxide; G1-AS, kaempferol-3-O-β-D-glucopyranosyl-(1→4)-O-α-D-glucopyranoside; G2,

396

maltose;

397

kaempferol-3-O-β-D-glucopyranosyl-(1→4)-O-α-D-maltotrioside; G4-AS, kaempferol-3-O-β-

398

D-glucopyranosyl-(1→4)-O-α-D-maltotetraoside;

399

immunodeficiency virus; HMBC, heteronuclear multiple bond correlation; HPLC, high

400

performance liquid chromatography; HSQC, heteronuclear single quantum coherence; IL,

401

interleukin; K-O-G kaempferol-3-O-glucuronide; KP, kaempferol; L-NAME, Nω-nitro-L-

402

arginine methyl ester hydrochloride; LPS, lipopolysaccharide; MALDI-TOF MS, matrix-

403

assisted laser desorption/ionization time-of-flight mass spectrometry; MMP-1, matrix

404

metalloproteinase-1; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NMR,

405

nuclear magnetic resonance; NO, nitric oxide; rAR, rat lens AR; SD, standard deviation; TFA,

406

trifluoroacetic acid; TLC, thin-layer chromatography; UV, ultraviolet.

G2-AS,

kaempferol-3-O-β-D-glucopyranosyl-(1→4)-O-α-D-maltoside;

G3-AS,

GH, glycoside hydrolase; HIV, human

407



408

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544

Figure captions

545

Figure 1. Mechanism of the transglycosylation reaction for the production of the kaempferol-

546

3-O-glucoside (astragalin, AS) transfer product (kaempferol-3-O-β-D-glucopyranosyl-(1→4)-

547

O-α-D-glucopyranoside, G1-AS).

548 549

Figure 2. Inhibitory effects of kaempferol (KP), kaempferol-3-O-glucuronide (K-O-G),

550


551

glucopyranoside (G1-AS) on NO production. The data presented are the mean ± the standard

552

error of the mean (SEM) (n = 3). *p < 0.05 and **p < 0.01 vs. the negative control.

553


Page 30 of 33

Page 31 of 33


Figure 1. Mechanism of the transglycosylation reaction for the production of the kaempferol-3-O-glucoside (astragalin, AS) transfer product (kaempferol-3-O-β-D-glucopyranosyl-(1→4)-O-α-D-glucopyranoside, G1AS). 168x149mm (150 x 150 DPI)



Figure 2. Inhibitory effects of kaempferol (KP), kaempferol-3-O-glucuronide (K-O-G), kaempferol-3-Oglucoside (AS), and kaempferol-3-O-β-D-glucopyranosyl-(1→4)-O-α-D-glucopyranoside (G1-AS) on NO production. The data presented are the mean ± the standard error of the mean (SEM) (n = 3). *p < 0.05 and **p < 0.01 vs. the negative control. 198x109mm (150 x 150 DPI)


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TOC 210x183mm (150 x 150 DPI)


Enzymatic Synthesis of a Novel Kaempferol-3 - ACS Publications

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