Extraction optimization, antidiabetic and antiglycation potentials of aqueous glycerol extract from rice (Oryza sativa L.) bran

Accepted Manuscript Extraction optimization, antidiabetic and antiglycation potentials of aqueous glycerol extract from rice (Oryza sativa L.) bran Halah Aalim, Tarun Belwal, Lei Jiang, Hao Huang, Xianghe Meng, Zisheng Luo PII:

S0023-6438(19)30007-6

DOI:

https://doi.org/10.1016/j.lwt.2019.01.006

Reference:

YFSTL 7748

To appear in:

LWT - Food Science and Technology

Received Date: 22 August 2018 Revised Date:

4 January 2019

Accepted Date: 7 January 2019

Please cite this article as: Aalim, H., Belwal, T., Jiang, L., Huang, H., Meng, X., Luo, Z., Extraction optimization, antidiabetic and antiglycation potentials of aqueous glycerol extract from rice (Oryza sativa L.) bran, LWT - Food Science and Technology (2019), doi: https://doi.org/10.1016/j.lwt.2019.01.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

TITLE

2

Extraction optimization, antidiabetic and antiglycation potentials of

3

aqueous glycerol extract from rice (Oryza sativa L.) bran

4

AUTHORS

RI PT

1

Halah Aalima, Tarun Belwala, Lei Jianga, Hao Huanga, Xianghe Meng b, Zisheng Luoa*

6 7 8

Halah Aalim, [email protected]; Tarun Belwal, [email protected]; Lei Jiang, [email protected]; Hao Huang, [email protected]; Xianghe Meng, [email protected]; and Zisheng Luo, [email protected].

9

AFFILIATIONS

M AN U

SC

5

10

a

11

Laboratory for Agri-Food Processing, Key Laboratory of Agro-Products Postharvest Handling of

12

Ministry of Agriculture and Rural Affairs, Hangzhou, 310058, People’s Republic of China.

13

b

14

China.

15

*Corresponding author

16

Professor Zisheng Luo

17

College of Biosystems Engineering and Food Science, Zhejiang University

18

Hangzhou 310058, People’s Republic of China

19

E-mail: [email protected]

20

Phone: +86-571-88982175

TE D

Zhejiang University, College of Biosystems Engineering and Food Science, Zhejiang Key

AC C

EP

Zhejiang University of Technology, Ocean college, Hangzhou, 310018, People’s Republic of

21 1

ACCEPTED MANUSCRIPT

Abstract

23

The purpose of this investigation was to explore aqueous glycerol as food grade solvent for rice

24

bran polyphenolic antioxidants extraction and investigate the antidiabetic and antiglycation

25

potential of the extract and its major polyphenols. Response surface methodology (Box-Behnken

26

design) was implemented to optimize experimental conditions for rice bran polyphenols and

27

antioxidants extraction. The optimum conditions provided a very satisfactory outcome in total

28

polyphenols (549.6±8.05 mg gallic acid equivalent per 100 g dry weight) and DPPH scavenging

29

activity (57.83±1.8%). The chromatographic separation of the extract showed higher

30

concentrations of ferulic acid, ρ-coumaric acid and chlorogenic acid followed by caffeic acid,

31

syringic acid and rutin. Moreover, the extract inhibited α‐amylase, α‐glucosidase, and lipase

32

activity as 34.2±3.2%, 29.9±1.0%, and 12.4±2.3%, respectively. Furthermore, fructose readily

33

forms Advanced Glycation End Products (AGEs) fluorescence with basic amino acids in low

34

concentration in simulated intestinal digestion conditions. The extract inhibited AGEs formation

35

by 52.6% at 20 µL /mL. Our study indicated that the antidiabetic and antiglycation potential of

36

rice bran extract was mainly driven by the extract polyphenols. Rice bran glycerol extract could

37

be employed as a natural antidiabetic and antiglycation remedy, and as economically and eco-

38

friendly source of potent bioactive compounds with satisfactory outcome.

39

Keywords: Rice bran; Polyphenol; Antioxidant activity; Enzyme inhibition; Glycation.

40

Abbreviations

41

AGEs: Advanced Glycation End Products

42

RB: Rice bran extract

43

FA: Ferulic acid

AC C

EP

TE D

M AN U

SC

RI PT

22

2

ACCEPTED MANUSCRIPT

ρ-CA: ρ-Coumaric acid

45

AC: Acarbose

46

OR: Orlistat

47

AMG: Aminoguanidine

48

TPC: Total phenolic contents

49

DPPH: 2, 2-diphenyl-1-picrylhydrazyl free radical

M AN U

50 51 52

57 58 59

EP

56

AC C

55

TE D

53 54

SC

RI PT

44

60 61

3

ACCEPTED MANUSCRIPT

1. Introduction

63

The alarming estimation of 554 million people around the globe suffering from the epidemic of

64

diabetes mellitus (DM) and prediabetes is cause for concern (Cefalu et al., 2016). Hyperglycemia

65

associated with DM cause high oxidative stress and formation of stable covalent adducts

66

between the amino group and the carbonyl group of reducing sugars, commonly known as

67

Advanced Glycation End Products (AGEs), which contribute to the debilitating diabetes

68

complications (Crascì, Lauro, Puglisi, & Panico, 2018). Dietary polyphenols exhibit strong

69

efficacy to manage glucose intolerance and oxidative stress in diabetes both in the in-vitro and

70

in-vivo models, without having the side effects of synthetic pharmaceutical drugs (Tanveer,

71

Akram, Farooq, Hayat, & Shafi, 2017).

72

Rice (Oryza sativa L.) bran is one of the most abundant agricultural by-products of rice polishing.

73

It was estimated that over 29.3 million tons of rice bran produced annually and often used for

74

rice bran oil production or utilized for animal feed (Sharif, Butt, Anjum, & Khan, 2014).

75

Previous studies indicate that rice bran polyphenols have potential health benefits in ameliorating

76

the development of DM (Premakumara, Abeysekera, Ratnasooriya, Chandrasekharan, & Bentota,

77

2013), reduce oxidative stress and protect blood vessels (Perez-Ternero et al., 2017). Although

78

rice bran contains a wide spectrum of potent nutraceuticals, its poor digestibility and storage

79

stability hinder its potential utilization as a functional food. Thus, exploring green extraction

80

techniques for rice bran bioactive compounds would be an appropriate alternative to benefit from

81

its full potentials.

82

Conventional organic solvents traditionally used for solid-liquid extraction of rice bran

83

polyphenol were having volatile, flammable and toxic qualities, which can cause deleterious

84

health and environmental effects (Pourali, Asghari, & Yoshida, 2010). With more than 1500 safe

AC C

EP

TE D

M AN U

SC

RI PT

62

4

ACCEPTED MANUSCRIPT

application of glycerol, it has been widely used in the oral pharmacological formulations and

86

food products (Tan, Abdul Aziz, & Aroua, 2013). Glycerol has been proposed as a valuable

87

green solvent that combines the advantages of water and ionic liquids (Jessop, 2016). Recently,

88

glycerol was successfully used to recover phenolic antioxidants from various plant tissues such

89

as eggplant peels (Philippi, Tsamandouras, Grigorakis, & Makris, 2016), olive leaves

90

(Mourtzinos et al., 2016), and coffee (Michail, Sigala, Grigorakis, & Makris, 2016). Furthermore,

91

studies of oral supplements with glycerol showed beneficial health effects, such as attenuates

92

liver cancer (Capiglioni et al., 2018), and improve metabolism during physical exercise (Andrade

93

et al., 2014), with no reported toxicity (Tamta, Chaudhary, & Sehgal, 2009).

94

Therefore, the purpose of this study was to explore the potential of glycerol as an alternative

95

green solvent. Response surface methodology (RSM) was employed to optimize extraction

96

process variables, such as glycerol concentration, solvent to solid ratio and extraction

97

temperature. Also, investigate the ability of AGEs formation in simulated intestine conditions.

98

Furthermore, antidiabetic and antiglycation activities of the extract and its polyphenol were

99

determined and compared to the standard drugs.

EP

TE D

M AN U

SC

RI PT

85

2. Materials and methods

101

2.1. Materials

102

Rice bran (Oryza sativa L. cv. Huazhan) was acquired commercially from the local market at

103

Hangzhou, Zhejiang province, China. Glycerol (>99%), 2, 2-diphenyl-1-picrylhydrazyl free

104

radical (DPPH), aminoguanidine hydrochloride (AMG, ≥98%), potato starch, acarbose orlistat,

105

p-nitrophenyl-α-d-glucopyranoside (α-pNPG), and nitrophenyl acetate (pNPA) were obtained

106

from Aladdin Industrial Co. (Shanghai, China). α-Glucosidase from Saccharomyces cerevisiae,

AC C

100

5

ACCEPTED MANUSCRIPT

α-amylase (type VI-B; from porcine pancreas) and lipase (type II; from porcine pancreas) was

108

obtained from Sigma Chemical Co. (St. Louis, MO, USA). All chemicals were analytical grade

109

and chromatographic chemicals were of HPLC grade.

110

2.2. Extraction

111

Rice bran (1.0 g) was shaken on an immersion oscillator (140 rpm) with pre-estimated glycerol

112

concentration, volume and extraction temperature according to the experimental design (Table 1).

113

After the extraction, rice bran extract (RB) was centrifuged at 11000 g for 15 min at 4 ‐ and the

114

supernatants were collected and stored at 4 ˚C until further analysis.

115

2.3. Response surface experimental design

116

Rice bran was extracted using aqueous glycerol and the extraction parameters were optimized

117

using Box-Behnken design (BBD). Glycerol concentration (X1, 5 - 30 %, w/v), liquid/solid ratio

118

(X2, 20 - 50 mL g-1) and extraction temperature (X3, 30 – 90 ‐) were chosen as independent

119

variables, while TPC (Y1, mg GAE/100 g) and DPPH scavenging activity (Y2, %) were selected

120

as the response variables. Each variable was coded at three levels -1, 0 and 1 and a total of

121

fifteen randomized experimental runs were conducted including three replicates at the center

122

point.

123

The experimental data was fitted to a second-order polynomial model Eq. (1), and the multiple

124

regression coefficients were calculated using the least squares technique.

125

Y = β 0 + ∑ β i X i + ∑ β ii X ii2 + ∑∑ β ij X i X j

AC C

EP

TE D

M AN U

SC

RI PT

107

K

K

K −1 K

i =1

i =1

i =1 j =1

(1)

6

ACCEPTED MANUSCRIPT

Where, Y is the response variable, β0, βi, βii, βij are regression coefficients for intercept, linear,

127

quadratic and interaction terms, respectively. Xi and Xj are the coded value of the independent

128

variables while k equals the number of the tested factors (k = 3). Analysis of variance (ANOVA)

129

and the significance of the model's terms was reported at p < 0.05, 0.01 or 0.001.

130

2.4 Determination of total phenolic content (TPC)

131

TPC was determined as described by Singleton, Orthofer, & Lamuela-Raventós, (1999) with

132

modifications. Briefly, 0.5 mL of RB mixed with 2.5 mL of 10 % Folin Ciocalteu reagent (v/v).

133

The mixture was kept for 5 min at room temperature, then 2.5 mL of 7.5% sodium carbonate

134

(w/v) was added, mixed and allowed to stand for 45 min at room temperature in dark. Finally,

135

the absorbance was measured at 765 nm against a blank, using a UV–visible spectrophotometer

136

(Shimadzu, Japan). A calibration curve was prepared using gallic acid and results were expressed

137

as mg gallic acid equivalent per 100 g dry weight (mg GAE/ 100 g).

138

2.5 Determination of DPPH radical scavenging activity

139

DPPH scavenging activity assay was described by Butsat & Siriamornpun, (2010) with

140

modifications. Briefly, RB (0.2 mL) was mixed with 3.8 mL of 0.1 mM of DPPH in ethanol. The

141

mixture was shaken vigorously and incubated for 30 min in the dark at room temperature. The

142

absorbance was registered at 517 nm against a blank, using UV–Vis spectrophotometer. Results

143

were expressed as scavenging percentage (%).

144

2.6 Determination of individual phenolic composition

145

Quantification of polyphenols was carried out by Shimadzu HPLC system (LC-20AD, Japan)

146

equipped with SPDM20A PDA detector and using Luna C18 column (5 µm, 250 × 4.6 mm,

147

Phenomenex). The mobile phase was composed of a 0.4% acetic acid (v/v) (solution A) and

AC C

EP

TE D

M AN U

SC

RI PT

126

7

ACCEPTED MANUSCRIPT

acetonitrile (solution B) and the flow rate at 1.0 mL/ min and follow the gradient program

149

reported previously (H. Ti et al., 2014): 0–40 min, 2–25% of solution B; 40–45 min, 25–35% of

150

solution B; and 45–50 min, 35–50% of solution B. The injection volume was 20 µL and the

151

samples were filtered through a 0.22 µm membrane filter prior to analysis. The identification and

152

quantification of each compound was based on known standard compound retention time and

153

linear equation, respectively.

154

2.7 Digestive enzymes inhibition assays

155

The assays were adopted from our previous report (Aalim, Belwal, Wang, Luo, & Hu, 2018).

156

For α-amylase inhibition assay, 20 µL of RB or control and 40 µL of 0.1 % starch (w/v) were

157

combined with 20 µL of α-amylase (0.1 mg mL−1) in 0.1 M phosphate buffer (pH 6.9). The

158

mixture was incubated at 37 °C for 20 min, and the reaction was stopped by the addition of 80 µL

159

of 0.4 M HCl followed by 100 µL of 5 mM iodine (in 5 mM KI). The absorbance was measured

160

at 630 nm against blank and the results were expressed as inhibition present (%). For α-

161

glucosidase inhibition assay, 50 µL of the RB or control was combined with 50 µL of α-

162

glucosidase enzyme solution (0.3 U mL−1) and 50 µL of 5 mM α-pNPG solution all in 0.1 M

163

phosphate buffer (pH 6.9) in a 96-well plate. The mixture was incubated at 37 °C for 20 min in

164

the dark. The absorbance was recorded at 405 nm against blank and the results were expressed as

165

percentage inhibition (%). AC, as well as FA and ρ-CA, were used as positive control and their

166

IC50 values were calculated. For lipase inhibition assay, 40 µL of the RB or control was mixed

167

with 20 µL of substrate solution (2.5 mM of p-NPA in ethanol) and 40 µL of the lipase ( 0.1

168

mg mL−1 in 0.1 mM phosphate buffer, pH 8.0). After incubation for 20 min at 37 °C, absorbance

AC C

EP

TE D

M AN U

SC

RI PT

148

8

ACCEPTED MANUSCRIPT

was read at 405 nm and the results were expressed as percentage inhibition (%). OR, as well as

170

FA and ρ-CA, were used as positive control and their IC50 values were calculated.

171

2.8 AGE formation and inhibition

172

The assay was performed as proposed previously (Bains & Gugliucci, 2017) with some

173

modifications. Briefly, AGEs was investigated through fluorescent formation of basic and

174

neutral amino acids (arginine, lysine , histidine and glycine at concentration of 50 mM) when

175

incubated with different dietary carbohydrate (fructose, sucrose, xylose or glucose at

176

concentration of 50 mM) in simulated intestinal fluid (SIF) described by Minekus et al., (2014),

177

which consist of 6.8 mM KCL, 0.8 mM KH2PO4, 85 mM NaHCO3, 38.4 mM NaCl, 0.33 mM

178

MgCl2(H2O)6 and 0.6 mM CaCl2(H2O)2 at pH 7 (adjusted with standard HCl) and incubated at

179

37 °C for up to 23 h in the presence of FeCl3 and NaN3. Further, AGEs formation was evaluated

180

between amino acids (50 mM) and fructose at concentrations ranging from 10 mM to 50 mM and

181

time ranging from 1 h to 23 h. Fluorescence spectrophotometer (Cary Eclipse, USA) was used to

182

record fluorescence at excitation wavelength λ370 nm and emission λ440 nm against blank. RB

183

as well as FA, ρ-CA, and AMG were employed as inhibitors and their IC50 values were

184

calculated.

185

2.9 Statistical analysis

186

Design-Expert software version 10.0.7 (Stat-Ease Inc., and Minneapolis, USA) was used to

187

perform RSM. One way (ANOVA) and Duncan’s Multiple Range Test (p ≤ 0.05) were used

188

to evaluate the statistical difference. Data analysis was performed using SPSS version 20.0

AC C

EP

TE D

M AN U

SC

RI PT

169

9

ACCEPTED MANUSCRIPT

(SPSS Inc., Chicago, Illinois, USA) software. All determinations were conducted in triplicate.

190

The data were presented as mean values ± standard deviation.

191

3. Results

192

3.1 Response surface analysis of TPC and DPPH scavenging activity

193

Experimental and predicted data of TPC and DPPH are presented in (Table 1). The regression

194

coefficients of the predicted second-order polynomial model for TPC and DPPH are presented

195

by Eq. (2) and (3), respectively. The polynomial equations showed adequacy to fit the data (p

196

<0.001). The absence of lack of fit (p > 0.05) of the models further strengthened the reliability of

197

both models to sufficiently predict the responses.

198

TPC (mg GAE/100 g) = 496.63 + 44.74X2 + 35.17 X3 - 15.54 X21 - 20.46 X22 + 22.73 X23 (2)

199

DPPH (% inhibition) = 54.41 -15.68 X2 - 3.40 X21 (3)

200

Where, X1, X2 and X3 are glycerol concentration (%), liquid/solid ratio (mL g-1) and temperature

201

(‐), respectively.

202

Regression analysis results indicated that TPC was highly affected by liquid/solid ratio and

203

extraction temperature (p < 0.0001). Also, glycerol concentration and liquid/solid ratio both had

204

significant negative quadratic effects (p < 0.05) and (p < 0.01), respectively, indicating that TPC

205

at certain glycerol concentration and liquid/solid ratio reached its highest value, and then begins

206

to decline. The positive significant linear and quadratic effect obtained for extraction temperature,

207

indicating that TPC required more energetic conditions to be liberated from the plant matrix. The

208

relationship between RB TPC and process variables is shown in Figure 1. The TPC increased

209

consistently with increasing solvent volume from 44–50 mL g-1 and extraction temperature from

AC C

EP

TE D

M AN U

SC

RI PT

189

10

ACCEPTED MANUSCRIPT

84–90 °C (Fig. 1C). On the other hand, the interaction between extraction temperature and

211

glycerol concentration (Fig. 1B) did not play any significant contribution towards TPC.

212

Regression analysis indicated that the liquid/solid ratio (p < 0.0001) was the main extraction

213

parameter influences DPPH scavenging activity, while temperature showed only negligible

214

impact (Table 2). The significant quadratic effect of glycerol concentration confirms its role in

215

influencing DPPH scavenging activity. The relationship between the antioxidant activity and

216

process variables is depicted in Figure 2. As shown, the DPPH scavenging activity decreased

217

with the increase in liquid/solid ratio (Fig. 2A and C). The highest activity was noticed from the

218

range of 64.4 – 66.8 % at the liquid/solid ratio between 20 – 26 mL g-1 at 60 ‐. However, the

219

interaction between glycerol concentration and extraction temperature showed a negligible effect

220

on the DPPH scavenging activity (Fig. 2B).

221

3.2 Optimization of extraction parameters and model validation

222

Ideally, the optimum extract should have the maximum concentration of phenolic compounds

223

and the highest DPPH scavenging activity, but experiments have shown that more favourable

224

extraction parameters for polyphenols result in low DPPH scavenging activity. Hence, the

225

desirability function was not appropriate to be used in this situation. Thereby, a joint design point

226

was chosen to determine the optimum condition for RB TPC and DPPH scavenging activity, the

227

optimum conditions calculated as 15.9% glycerol concentration, 31.6 mL g-1 liquid/solid ratio

228

and an extraction temperature of 90 ‐. The experimental TPC under this condition was

229

determined as 579.11 ± 2.4 mg GAE/100 g, and DPPH scavenging activity as 57.83±1.8%. No

230

significant difference between the experimental and the theoretical values were obtained which

AC C

EP

TE D

M AN U

SC

RI PT

210

11

ACCEPTED MANUSCRIPT

indicated the reliability of the model. Additionally, the extracts were prepared under optimized

232

conditions for further experimental analysis.

233

3.3 Characterization of the extract

234

3.3.1 Polyphenol composition

235

The major polyphenols were identified in the extract (Fig. 3) such as chlorogenic acid (27 ±

236

0.09), caffeic acid (3.5 ± 0.1), syringic acid (7.1 ± 0.1), ρ-coumaric (39.5 ± 0.2), ferulic acid

237

(67.8 ± 1.1), and rutin (1.7 ± 0.08) mg/100g dry weight. Furthermore, ferulic acid, p-coumaric

238

were the most dominant polyphenols in the extract.

239

3.3.2 Digestive enzymes inhibition

240

The inhibitory effects of RB on pancreatic α-amylase, yeast α- glucosidase and on pancreatic

241

lipase enzymes, and its dominant individual polyphenol are shown in Fig. 4. The positive control

242

AC exhibited the most potent inhibition against α-amylase (98.6 %) with an IC50 value of 0.07

243

mg/mL, followed by ρ-CA (66.8%) with an IC50 value of 0.02 mg/mL (Fig. 4 A). RB had

244

moderate inhibition activity (34.2%) on α-amylase and FA displayed only week inhibitory

245

activity (9.9%) towards α-amylase with an IC50 value of (> 5 mg/mL).

246

As shown in Fig. 4 B, ρ-CA showed the most potent inhibition against α- glucosidase (98.8 %)

247

with an IC50 value of 0.6 mg/mL. The positive control AC exhibited lower inhibition activity

248

against α- glucosidase (62.5 %) with an IC50 value of 2.5 mg/mL. Finally, FA and RB displayed

249

moderate inhibitory activity of 32.1 % (IC50 of 7.1 mg/mL) and 29.9%, respectively.

250

As presented in Fig. 4 C. The positive control OR exhibited 65.1 % inhibition with an IC50 of

251

0.016 mg/mL. FA and ρ-CA were much potent inhibitors against lipase with inhibitory activity

AC C

EP

TE D

M AN U

SC

RI PT

231

12

ACCEPTED MANUSCRIPT

of 82.8 % (IC50 of 0.019 mg/mL) and 74.0% (IC50 of 0.014 mg/mL), respectively. However,

253

RB showed week inhibitory effect (12.4%) on the lipase activity.

254

3.3.3 In vitro anti-glycation inhibition

255

Incubation of amino acids with different sugars leads to the formation of AGE fluorescence

256

under simulated intestinal conditions. Fructose exhibited the most potent activity (p > 0.05) when

257

compared with xylose, sucrose and glucose (Supplementary Fig. S1 A). Fructose was chosen for

258

further investigations. AGE fluorescence formation was found to be dependent upon fructose

259

concentration and time (Supplementary Fig. S1 B and C). Furthermore, AGE fluorescence was

260

evident after only 1 h incubation and in presence of low fructose concentration in the simulated

261

intestinal digestive conditions but not the gastric conditions (data not shown). As shown in Fig.

262

4D, RB (52.6% at 20 µL/mL) was found to be as equally potent inhibitor as the standard

263

inhibitor AMG (50.7 %), with an IC50 of 4.80 mg/mL. Standard polyphenols, FA and ρ-CA

264

were found to be more potent fructose-AGE inhibitor than the standard drug AMG, with an

265

inhibition ratio of 82.8% (IC50 of 0.36 mg/mL) and 60.2 % (IC50 of 2.14 mg/mL), respectively.

266

4. Discussion

267

The scope of this work was to optimized extraction conditions for polyphenol antioxidant from

268

rice bran and investigate its mechanism of inhibition for digestive enzymes and AGEs formation.

269

In the extraction process, selection of adequate extraction techniques, solvent type, optimal

270

temperatures and extraction time is of extreme importance (Chemat et al., 2017). Our findings

271

suggest that a substantial amount of polyphenols has been liberated from rice bran upon exposure

272

to high temperature and increasing glycerol volume. High temperature with a combination of the

273

high liquid/solid ratio is crucial to achieving a high solubility and diffusion rate of polyphenols

AC C

EP

TE D

M AN U

SC

RI PT

252

13

ACCEPTED MANUSCRIPT

(Cacace & Mazza, 2003). Glycerol increases water polarity; and therefore enhance the solubility

275

of more polar polyphenols (Feng, Luo, Tao, & Chen, 2015). Previous reports have also

276

demonstrated that glycerol function differently upon various plant materials and its optimal

277

concentration varied from 90 % (w/v) for eggplant peels (Philippi, Tsamandouras, Grigorakis, &

278

Makris, 2016) to 9.3% (w/v) for olive leaves and 3.6% (w/v) for filter coffee residues (Michail et

279

al., 2016). However, glycerol behaviour upon rice bran polyphenol extraction was comparable to

280

olive leaves polyphenol (Apostolakis, Grigorakis, & Makris, 2014), as the TPC start to decline

281

after certain glycerol concentration.

282

DPPH scavenging activity of rice bran mainly influenced by the phenolic content, extraction

283

solvents and rice cultivars (Bhat & Riar, 2017). Also, increasing solvent polarity promote

284

antioxidant extraction (Iqbal, Bhanger, & Anwar, 2005). In the present study, DPPH scavenging

285

activity of glycerol extract was significantly higher than acidified methanolic extract (Iqbal et al.,

286

2005). In agreement with our findings, the lower liquid/solid ratio was also reported to promote

287

higher DPPH scavenging activity (Zhang et al., 2018). Likewise, the optimal liquid/solid ratio

288

for rice bran antioxidants extracted with aqueous ethanol was also reported to be lower as 20 mL

289

g−1 (Tabaraki & Nateghi, 2011). In the present study, the DPPH scavenging activity was

290

promoted by lower extraction temperature as compared to TPC. It could be assumed that DPPH

291

scavenging activity was greatly influenced by RB flavonoids, as flavonoids are potent DPPH free

292

radical scavengers (Hyun et al., 2010), and the thermal degradation of flavonoids starts at 60 ‐

293

(Silva, Rogez, & Larondelle, 2007).

294

Additionally, the extract was dominated by FA and ρ-CA. In line with our finding, FA and ρ-CA

295

were found as dominant phenolic compounds in most rice samples (H. H. Ti et al., 2014; Zhao et

296

al., 2018). Moreover, Philippi, Tsamandouras, Grigorakis, & Makris, (2016), reported that

AC C

EP

TE D

M AN U

SC

RI PT

274

14

ACCEPTED MANUSCRIPT

glycerol extract resulted in virtually comparable TPC and polyphenols profile, when compared to

298

aqueous ethanol. Critical comparison with previous studies on rice bran polyphenols, ρ-CA and

299

FA concentration of glycerol extract were higher than acetone extract reported by Stefanello et

300

al., (2018). Interestingly, in our previous finding, the increase in glycerol concentration leads to

301

significantly increase in ρ-CA concentration (Aalim et al., 2018).

302

In addition, we investigated the RB and its dominant polyphenols for their possible significance

303

in the control of DM. This investigation had revealed that ρ-CA exhibited a higher inhibitory

304

effect on α-glucosidase than the standard drug AC. On the other hand, FA was week inhibitor for

305

carbohydrate hydrolyzing enzymes, which may be due to its limited water solubility, since the

306

enzymes (α-amylase and α-glucosidase) are functioning in the aqueous phase. This suggested

307

that RB effect was mainly influenced by the presence of ρ-CA. However, RB was found to be

308

less effective than the standard drug AC in inhibiting α-amylase and α-glucosidase activity.

309

Similarly, (Premakumara et al., 2013) observed significantly lower α-amylase inhibitory activity

310

of rice bran ethanol extract.

311

Accumulating evidence suggests that OR can reduce the absorption of the fat-soluble vitamins A,

312

D, E and K (R., A., L., I., & A., 2002), and increase the risk of colon cancer (Garcia et al., 2006).

313

Our finding demonstrates that FA and ρ-CA were much more effective than OR (p < 0.05) as

314

lipase inhibitors, and therefore could provide a plausible alternative to avoid orlistat adverse

315

effects. Additionally, we demonstrated that the increase of ρ-CA and decline of FA significantly

316

impact RB antidiabetic properties (Aalim et al., 2018). Likewise, the combined effect of the

317

extract polyphenols displays much potent enzyme inhibitory activity than individual polyphenols

318

(Villa-Rodriguez et al., 2018).

AC C

EP

TE D

M AN U

SC

RI PT

297

15

ACCEPTED MANUSCRIPT

Moreover, our investigation provides an evidence of employing standard SIF to test AGE

320

formation mechanism in simulated human lumen conditions. The biological buffers such as

321

bicarbonate and phosphate can influence the glycation reaction rate and specificity (Johansen,

322

Kiemer, & Brunak, 2006). Generally, the typical western meal combines processed meat,

323

carbohydrates, and fructose sweeten drinks. This combination is thought to slow the digestion

324

rate, delay gastric motility and increase the intestinal residence time, which in turn inducing

325

AGEs formation. Additionally, AGEs occur in the side chains of lysine, arginine, and histidine

326

(Ansari, Moinuddin, & Ali, 2011). This study confirmed that fructose can readily form more

327

intense fluorescent AGEs with basic amino acids, than other tested dietary carbohydrates. Also,

328

the tested concentrations can be easily attained in the small intestine (Bains & Gugliucci, 2017).

329

There was no detectable formation of fluorescent AGEs in the acidic simulated gastric fluids

330

described by Minekus et al., (2014). This could be attributed to the inability of the carboxyl

331

group to dissociate in the gastric acidic conditions (Johansen et al., 2006).

332

Our results show that the formation of fructose-AGEs fluorescence was inhibited by RB and its

333

main polyphenols, more effectively than the standard anti-glycating agent AMG. It is worth to

334

note that glycerol extract displayed significantly higher anti-glycation activity that the ethanolic

335

extract reported by Premakumara et al., (2013). Furthermore, AGE inhibitors are chelators and

336

antioxidants at concentrations commonly found in food (Crascì et al., 2018). Previous reports

337

demonstrate the ability of caffeic acid and chlorogenic acid to be potent glycation inhibitors

338

(Gugliucci, Bastos, Schulze, & Souza, 2009). Further, FA and ρ-CA show AGEs inhibition

339

activity in a concentration-dependent manner (Chen, Virk, & Chen, 2016).

340

5. Conclusion

AC C

EP

TE D

M AN U

SC

RI PT

319

16

ACCEPTED MANUSCRIPT

Glycerol is a natural substance has wider applications in medicine, pharmaceutical, cosmetic and

342

food industry. Its physiochemical qualities and absence of toxicity make it a suitable candidate

343

for separation of nutraceuticals from food matrixes. In this study, glycerol was validated for its

344

efficiency to recover polyphenolic antioxidants from rice bran. Furthermore, this study has

345

bridged the knowledge gap on the antidiabetic potentials of rice bran and its polyphenols. Our

346

results showed that RB dominant polyphenols FA and ρ-CA exhibited anti-diabetic and

347

antiglycation potentials more than the commercial AC, OR and AMG drugs. Crude aqueous

348

glycerol extract of rice bran is a good candidate to be utilized in antidiabetic and antiglycation

349

formulations, and as a valuable source to separate and purify potent antidiabetic polyphenols.

350

Further, in vitro, in vivo and clinical studies are required to verify the antioxidant, antidiabetic

351

and antiglycation potentials of the extract and its polyphenols.

352

Acknowledgments

353

This work was supported by the Key Research and Development Program of Zhejiang Province

354

[2018C02049]; and the National Key Research and Development Program of China

355

[2016YFD0401201].

356

Conflict of interest

357

The authors declare no conflicts of interest.

358

Reference

359 360 361 362 363 364

Aalim, H., Belwal, T., Wang, Y., Luo, Z., & Hu, J. (2018). Purification and identification of rice bran (Oryza sativa L.) phenolic compounds with in-vitro antioxidant and antidiabetic activity using macroporous resins. International Journal of Food Science & Technology, 0(0). doi:doi:10.1111/ijfs.13985 Andrade, E. F., Lobato, R. V., Araújo, T. V., Orlando, D. R., Gomes, N. F., Alvarenga, R. R., . . . Pereira, L. J. (2014). Metabolic effects of glycerol supplementation and aerobic physical training on Wistar

AC C

EP

TE D

M AN U

SC

RI PT

341

17

ACCEPTED MANUSCRIPT

EP

TE D

M AN U

SC

RI PT

rats. Canadian Journal of Physiology and Pharmacology, 92(9), 744-751. doi:10.1139/cjpp-20140187 Ansari, N. A., Moinuddin, & Ali, R. (2011). Glycated lysine residues: A marker for non-enzymatic protein glycation in age-related diseases. Disease Markers, 30(6), 317-324. doi:10.3233/DMA-2011-0791 Apostolakis, A., Grigorakis, S., & Makris, D. P. (2014). Optimisation and comparative kinetics study of polyphenol extraction from olive leaves (Olea europaea) using heated water/glycerol mixtures. Separation and Purification Technology, 128(Supplement C), 89-95. doi:https://doi.org/10.1016/j.seppur.2014.03.010 Bains, Y., & Gugliucci, A. (2017). Ilex paraguariensis and its main component chlorogenic acid inhibit fructose formation of advanced glycation endproducts with amino acids at conditions compatible with those in the digestive system. Fitoterapia, 117, 6-10. doi:https://doi.org/10.1016/j.fitote.2016.12.006 Bhat, F. M., & Riar, C. S. (2017). Extraction, identification and assessment of antioxidative compounds of bran extracts of traditional rice cultivars: An analytical approach. Food Chemistry, 237, 264-274. doi:https://doi.org/10.1016/j.foodchem.2017.05.113 Butsat, S., & Siriamornpun, S. (2010). Antioxidant capacities and phenolic compounds of the husk, bran and endosperm of Thai rice. Food Chemistry, 119(2), 606-613. doi:https://doi.org/10.1016/j.foodchem.2009.07.001 Cacace, J. E., & Mazza, G. (2003). Mass transfer process during extraction of phenolic compounds from milled berries. Journal of Food Engineering, 59(4), 379-389. doi:https://doi.org/10.1016/S02608774(02)00497-1 Capiglioni, A. M., Lorenzetti, F., Quiroga, A. D., Parody, J. P., Ronco, M. T., Pisani, G. B., . . . Alvarez, M. d. L. (2018). Attenuation of liver cancer development by oral glycerol supplementation in the rat. European Journal of Nutrition, 57(3), 1215-1224. doi:10.1007/s00394-017-1404-4 Cefalu, W. T., Buse, J. B., Tuomilehto, J., Fleming, G. A., Ferrannini, E., Gerstein, H. C., . . . Kahn, S. E. (2016). Update and Next Steps for Real-World Translation of Interventions for Type 2 Diabetes Prevention: Reflections From a Diabetes Care Editors’ Expert Forum. Diabetes Care, 39(7), 11861201. doi:10.2337/dc16-0873 Chemat, F., Rombaut, N., Meullemiestre, A., Turk, M., Perino, S., Fabiano-Tixier, A.-S., & Abert-Vian, M. (2017). Review of Green Food Processing techniques. Preservation, transformation, and extraction. Innovative Food Science & Emerging Technologies, 41, 357-377. doi:https://doi.org/10.1016/j.ifset.2017.04.016 Chen, H., Virk, M. S., & Chen, F. (2016). Phenolic acids inhibit the formation of advanced glycation end products in food simulation systems depending on their reducing powers and structures. International Journal of Food Sciences and Nutrition, 67(4), 400-411. doi:10.3109/09637486.2016.1166187 Crascì, L., Lauro, M. R., Puglisi, G., & Panico, A. (2018). Natural antioxidant polyphenols on inflammation management: Anti-glycation activity vs metalloproteinases inhibition. Critical Reviews in Food Science and Nutrition, 58(6), 893-904. doi:10.1080/10408398.2016.1229657 Feng, S., Luo, Z., Tao, B., & Chen, C. (2015). Ultrasonic-assisted extraction and purification of phenolic compounds from sugarcane (Saccharum officinarum L.) rinds. LWT - Food Science and Technology, 60(2, Part 1), 970-976. doi:https://doi.org/10.1016/j.lwt.2014.09.066 Garcia, S. B., da Costa Barros, L. T., Turatti, A., Martinello, F., Modiano, P., Ribeiro-Silva, A., . . . Uyemura, S. A. (2006). The anti-obesity agent Orlistat is associated to increase in colonic preneoplastic markers in rats treated with a chemical carcinogen. Cancer Letters, 240(2), 221-224. doi:https://doi.org/10.1016/j.canlet.2005.09.011

AC C

365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410

18

ACCEPTED MANUSCRIPT

EP

TE D

M AN U

SC

RI PT

Gugliucci, A., Bastos, D. H. M., Schulze, J., & Souza, M. F. F. (2009). Caffeic and chlorogenic acids in Ilex paraguariensis extracts are the main inhibitors of AGE generation by methylglyoxal in model proteins. Fitoterapia, 80(6), 339-344. doi:https://doi.org/10.1016/j.fitote.2009.04.007 Hyun, J., Woo, Y., Hwang, D.-s., Jo, G., Eom, S., Lee, Y., . . . Lim, Y. (2010). Relationships between structures of hydroxyflavones and their antioxidative effects. Bioorganic & Medicinal Chemistry Letters, 20(18), 5510-5513. doi:https://doi.org/10.1016/j.bmcl.2010.07.068 Iqbal, S., Bhanger, M. I., & Anwar, F. (2005). Antioxidant properties and components of some commercially available varieties of rice bran in Pakistan. Food Chemistry, 93(2), 265-272. doi:10.1016/j.foodchem.2004.09.024 Jessop, P. G. (2016). The use of auxiliary substances (: E.g. solvents, separation agents) should be made unnecessary wherever possible and innocuous when used. Green Chemistry, 18(9), 2577-2578. doi:10.1039/c6gc90039a Johansen, M. B., Kiemer, L., & Brunak, S. (2006). Analysis and prediction of mammalian protein glycation. Glycobiology, 16(9), 844-853. doi:10.1093/glycob/cwl009 Michail, A., Sigala, P., Grigorakis, S., & Makris, D. P. (2016). Kinetics of Ultrasound-Assisted Polyphenol Extraction from Spent Filter Coffee Using Aqueous Glycerol. Chemical Engineering Communications, 203(3), 407-413. doi:10.1080/00986445.2015.1004667 Minekus, M., Alminger, M., Alvito, P., Ballance, S., Bohn, T., Bourlieu, C., . . . Brodkorb, A. (2014). A standardised static in vitro digestion method suitable for food - an international consensus. Food & Function, 5(6), 1113-1124. doi:10.1039/C3FO60702J Mourtzinos, I., Anastasopoulou, E., Petrou, A., Grigorakis, S., Makris, D., & Biliaderis, C. G. (2016). Optimization of a green extraction method for the recovery of polyphenols from olive leaf using cyclodextrins and glycerin as co-solvents. Journal of Food Science and Technology-Mysore, 53(11), 3939-3947. doi:10.1007/s13197-016-2381-y Perez-Ternero, C., Werner, C. M., Nickel, A. G., Herrera, M. D., Motilva, M.-J., Böhm, M., . . . Laufs, U. (2017). Ferulic acid, a bioactive component of rice bran, improves oxidative stress and mitochondrial biogenesis and dynamics in mice and in human mononuclear cells. The Journal of Nutritional Biochemistry, 48, 51-61. doi:https://doi.org/10.1016/j.jnutbio.2017.06.011 Philippi, K., Tsamandouras, N., Grigorakis, S., & Makris, D. P. (2016). Ultrasound-Assisted Green Extraction of Eggplant Peel (Solanum melongena) Polyphenols Using Aqueous Mixtures of Glycerol and Ethanol: Optimisation and Kinetics. Environmental Processes, 3(2), 369-386. doi:10.1007/s40710-016-0140-8 Pourali, O., Asghari, F. S., & Yoshida, H. (2010). Production of phenolic compounds from rice bran biomass under subcritical water conditions. Chemical Engineering Journal, 160(1), 259-266. doi:https://doi.org/10.1016/j.cej.2010.02.057 Premakumara, G. A. S., Abeysekera, W. K. S. M., Ratnasooriya, W. D., Chandrasekharan, N. V., & Bentota, A. P. (2013). Antioxidant, anti-amylase and anti-glycation potential of brans of some Sri Lankan traditional and improved rice (Oryza sativa L.) varieties. Journal of Cereal Science, 58(3), 451-456. doi:https://doi.org/10.1016/j.jcs.2013.09.004 R., M. J., A., C. K., L., B. S., I., U. G., & A., Y. J. (2002). Effects of Orlistat on Fat‐Soluble Vitamins in Obese Adolescents. Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy, 22(7), 814-822. doi:doi:10.1592/phco.22.11.814.33627 Sharif, M. K., Butt, M. S., Anjum, F. M., & Khan, S. H. (2014). Rice Bran: A Novel Functional Ingredient. Critical Reviews in Food Science and Nutrition, 54(6), 807-816. doi:10.1080/10408398.2011.608586 Silva, E. M., Rogez, H., & Larondelle, Y. (2007). Optimization of extraction of phenolics from Inga edulis leaves using response surface methodology. Separation and Purification Technology, 55(3), 381387. doi:https://doi.org/10.1016/j.seppur.2007.01.008

AC C

411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458

19

ACCEPTED MANUSCRIPT

493 494

RI PT

SC

M AN U

TE D

EP

492

Singleton, V. L., Orthofer, R., & Lamuela-Raventós, R. M. (1999). [14] Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. Methods in Enzymology, 299, 152-178. doi:http://dx.doi.org/10.1016/S0076-6879(99)99017-1 Stefanello, F. S., dos Santos, C. O., Bochi, V. C., Fruet, A. P. B., Soquetta, M. B., Dörr, A. C., & Nörnberg, J. L. (2018). Analysis of polyphenols in brewer’s spent grain and its comparison with corn silage and cereal brans commonly used for animal nutrition. Food Chemistry, 239, 385-401. doi:http://dx.doi.org/10.1016/j.foodchem.2017.06.130 Tabaraki, R., & Nateghi, A. (2011). Optimization of ultrasonic-assisted extraction of natural antioxidants from rice bran using response surface methodology. Ultrasonics Sonochemistry, 18(6), 12791286. doi:https://doi.org/10.1016/j.ultsonch.2011.05.004 Tamta, A., Chaudhary, M., & Sehgal, R. (2009). A 28-days sub-acute toxicity study in Swiss albino mice to evaluate toxicity profile of neurotol plus (Mannitol and Glycerol combination). International Journal of Biomedical Science, 5(4), 428-433. Tan, H. W., Abdul Aziz, A. R., & Aroua, M. K. (2013). Glycerol production and its applications as a raw material: A review. Renewable and Sustainable Energy Reviews, 27, 118-127. doi:https://doi.org/10.1016/j.rser.2013.06.035 Tanveer, A., Akram, K., Farooq, U., Hayat, Z., & Shafi, A. (2017). Management of diabetic complications through fruit flavonoids as a natural remedy. Critical Reviews in Food Science and Nutrition, 57(7), 1411-1422. doi:10.1080/10408398.2014.1000482 Ti, H. H., Zhang, R. F., Zhang, M. W., Li, Q., Wei, Z. C., Zhang, Y., . . . Ma, Y. X. (2014). Dynamic changes in the free and bound phenolic compounds and antioxidant activity of brown rice at different germination stages. Food Chem., 161, 337. doi:https://doi.org/10.1016/j.foodchem.2014.04.024 Villa-Rodriguez, J. A., Kerimi, A., Abranko, L., Tumova, S., Ford, L., Blackburn, R. S., . . . Williamson, G. (2018). Acute metabolic actions of the major polyphenols in chamomile: an in vitro mechanistic study on their potential to attenuate postprandial hyperglycaemia. Scientific Reports, 8(1), 5471. doi:10.1038/s41598-018-23736-1 Zhang, H., Birch, J., Xie, C., Yang, H., Dias, G., Kong, L., & Bekhit, A. E.-D. (2018). Optimization of extraction parameters of antioxidant activity of extracts from New Zealand and Chinese Asparagus officinalis L root cultivars. Industrial Crops and Products, 119, 191-200. doi:https://doi.org/10.1016/j.indcrop.2018.03.066 Zhao, G., Zhang, R., Dong, L., Huang, F., Tang, X., Wei, Z., & Zhang, M. (2018). Particle size of insoluble dietary fiber from rice bran affects its phenolic profile, bioaccessibility and functional properties. LWT - Food Science and Technology, 87, 450-456. doi:https://doi.org/10.1016/j.lwt.2017.09.016

AC C

459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491

495 496 497 20

ACCEPTED MANUSCRIPT

498

Table Captions

500

Table 1. Total phenolic contents and DPPH radical scavenging activity of the rice bran extract

501

obtained under different conditions based on a Box-Behnken design for response surface analysis.

502

Values are reported as mean ± SD (n = 3).

503

Table 2. Regression coefficients of predicted models for the total polyphenol content and DPPH

504

radical scavenging activity of rice bran extracts and the independent effects of factors.

505

Figure Captions

506

Figure 1. Response surface 3D plots for the combined effect of (a) liquid/solid ratio and glycerol

507

concentration (b) temperature and glycerol concentration and (c) temperature and liquid/solid

508

ratio on the total polyphenol content (TPC, mg GAE/ 100g).

509

Figure 2. Response surface 3D plots for the combined effect of (a) glycerol concentration and

510

liquid/ solid ratio (b) temperature and glycerol concentration and (c) temperature and liquid/solid

511

ratio on DPPH scavenging activity (inhibition %).

512

Figure 3. HPLC-PDA chromatograms obtained at the detection wavelength of 320 nm for rice

513

bran polyphenols. The numbers refer to the peak appeared in the chromatogram: (1) chlorogenic

514

acid, (2) caffeic acid, (3) syringic acid, (4) ρ-coumaric acid, (5) ferulic acid, and (6) rutin.

515

Figure 4. Inhibition of α-amylase (a), α-glucosidase (b), lipase (c), and AGEs formation (d) by

516

rice bran extract (RB), ferulic acid (FA), ρ-coumaric acid (ρ-CA), and the standard drugs. Values

517

are reported as mean ± SD (n = 3). Different letters indicate significant differences by Duncan's

518

multiple range test (p < 0.05).

AC C

EP

TE D

M AN U

SC

RI PT

499

21

ACCEPTED MANUSCRIPT

Fig. S1: Formation of fructose-AGE fluorescent adducts with fructose, glucose, sucrose, and

520

xylose (a), Concentration-dependent fructose the AGE formation (b), and Formation of fructose-

521

AGE fluorescent adducts over time (c). Values are reported as mean ± SD (n = 3). Different

522

letters indicate significant differences by Duncan's multiple range test (p < 0.05).

RI PT

519

523

SC

524

AC C

EP

TE D

M AN U

525

22

ACCEPTED MANUSCRIPT

1

Table 1. Total phenolic contents and DPPH radical scavenging activity of the rice bran extract

2

obtained under different conditions based on a Box-Behnken design for response surface analysis.

3

Values are reported as mean ± SD (n = 3).

X1, %

X2, ml g−1

1

-1 (5)

2

TPC (mg GAE/ 100 g)

Measured

-1 (20)

0 (60)

412.0 ± 7.8

1 (15)

-1 (20)

0 (60)

419.3 ± 9.0

3

-1(5)

1 (50)

0 (60)

499.7 ± 11.3

4

1 (15)

1 (50)

0 (60)

5

-1 (5)

0 (35)

-1 (30)

6

1 (30)

0 (35)

-1 (30)

7

-1 (5)

0 (35)

1 (90)

8

1 (30)

0 (35)

1 (90)

9

0 (17.5)

-1 (20)

-1 (30)

10

0 (17.5)

1 (50)

11

0 (17.5)

12

Predicted 415.5

Measured

64.4 ± 1.9

Predicted 64.6

416.3

66.8 ± 1.8

64.9

502.7

31.0 ± 0.4

32.9

M AN U

X3,

DPPH (% inhibition)

RI PT

Independent variables

SC

Design point

496.6

34.2 ± 0.8

34.0

471.4 ± 6.2

466.6

54.8 ± 1.7

53.4

469.0 ± 7.3

470.7

50.1 ± 1.9

50.8

539.7 ± 17.9

538.0

50.9 ± 0.4

50.2

535.2 ± 13.7

540.0

52.8 ± 1.1

54.2

419.4 ± 11.1

420.7

68.6 ± 1.9

69.8

-1 (30)

505.0 ± 12.9

506.8

38.1 ± 0.1

37.5

-1 (20)

1 (90)

489.4 ± 14.9

487.6

68.4 ± 1.1

68.9

0 (17.5)

1 (50)

1 (90)

581.8 ± 16.8

580.5

39.6 ± 1.5

38.5

13

0 (17.5)

0 (35)

0 (60)

484.0 ± 12.8

496.6

52.7 ± 0.5

54.4

14

0 (17.5)

0 (35)

0 (60)

494.1 ± 15.4

496.6

53.7 ± 1.6

54.4

15

0 (17.5)

0 (35)

0 (60)

511.7 ± 15.7

508.1

56.9 ± 1.0

54.4

AC C

EP

TE D

511.6 ± 12.2

Abbreviations: X1= glycerol concentration, X2= liquid/solid ratio, X3= extraction temperature, TPC= Total phenol content, DPPH= DPPH radical scavenging activity. (n=3) 4

5

6

ACCEPTED MANUSCRIPT

Table 2. Regression coefficients of predicted models for the total polyphenol content and DPPH

8

radical scavenging activity of rice bran extracts and the independent effects of factors. Coefficient

Response DPPH 54.41***

TPC 496.63***

β1

+1.54

0.35

β2

+44.74***

-15.68***

β3

+35.17***

0.038

β12

+1.16

0.20

β13

-0.53

1.65

β23

+1.71

0.45

β11

-15.54*

-3.40*

β22

-20.46**

-1.9

β33

+22.73**

1.15

Lack of fit

ns

ns

R2

0.9840

0.9878

M AN U

TE D

ns, not significant (p > 0.05). *

significant at p ≤ 0.05.

**

significant at p ≤ 0.01.

AC C

9

significant at p ≤0.001.

EP

***

SC

β0 Intercept

RI PT

7

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

Highlights

• RSM has been used to optimize glycerol extraction of polyphenolic antioxidants.

RI PT

• ρ-coumaric and ferulic acid were the predominant polyphenols in rice bran extract.

• The extract shows moderate inhibitory effects on carbohydrate hydrolyzing enzymes.

SC

• The extract shows antiglycation inhibitory effect comparable to aminoguanidine.

AC C

EP

TE D

M AN U

• Likewise, in vitro studies show potent antidiabetic activity for ρ-coumaric acid.