LipidâPolymer Hybrid Nanoparticles for Oral Delivery of Tartary

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Food and Beverage Chemistry/Biochemistry

Lipid-polymer hybrid nanoparticles for oral delivery of Tartary buckwheat flavonoids Jinming Zhang, di wang, yihan wu, wei li, Yichen Hu, gang zhao, chaomei fu, shu fu, and Liang Zou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00714 • Publication Date (Web): 26 Apr 2018 Downloaded from http://pubs.acs.org on April 26, 2018

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Journal of Agricultural and Food Chemistry

Lipid-polymer hybrid nanoparticles for oral delivery of Tartary buckwheat flavonoids Jinming Zhang1†, Di Wang2,3†, Yihan Wu1, Wei Li3, Yichen Hu4, Gang Zhao4, Chaomei Fu1, Shu Fu1*, Liang Zou3* 1

School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu

611137, China 2

College of Pharmacy and Chemistry, Dali University, Dali 671000, Yunnan, China

3

School of Medicine, Chengdu University, Chengdu 610106, China

4

Key laboratory of coarse grain processing, Ministry of Agriculture, School of pharmacy

and bioengineering, Chengdu University, Chengdu 610106, China † These authors contributed equally to this work. * Corresponding Authors: Dr. Shu Fu, Tel: +86 02861800231; Fax: +86 02861800235; E-mail address: [email protected] Prof. Liang Zou, Tel: +86 02884617082; Fax: +86 02884617086; E-mail address: [email protected]

ACS Paragon Plus Environment


1

Abstract: Flavonoids rich in Tartary buckwheat (TBFs) are the acknowledged

2

health-promoting substances, while with the low oral bioavailability due to its chemical

3

instability in gastrointestinal tract and poor intestinal absorption. To obtain the enhanced

4

oral delivery, TBFs, obtained by an environmentally-friendly extraction strategy in advance

5

with the amount of 7.66 ± 0.47mg rutin/g, was incorporated in biocompatible lipid-polymer

6

hybrid nanoparticles (LPNs). Its high encapsulation efficiency of 96.4% ± 1.1%, narrow

7

size distribution of 61.25±1.83 nm with spherical shape, and good storage stability were

8

observed. Compared to free TBFs, TBFs/LPNs exhibited higher antioxidant activity and

9

significant suppression on the pro-inflammatory cytokine secretion in RAW 264.7

10

macrophage. Moreover, the enhanced delivery of TBFs/LPNs was also embodied in the

11

improved transmembrane transport in Caco-2 monolayer, suggesting its better intestinal

12

absorption, and significantly immune-enhancing efficacy in immunosuppressed mice.

13

These results demonstrated the new perspectives of Tartary buckwheat flavonoids-loaded

14

nano-system for pharmaceutical and nutraceutical applications.

15

Keywords: Tartary buckwheat, flavonoids, lipid-polymer nanoparticle, antioxidant,

16

immune-enhancing.

17 18 19 20 21 22 23 24 25 26 27 28 29


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■ INTRODUCTION

31

Tartary buckwheat [Fagopyrum tataricum (L.) Gaertn] is a well-known functional food,

32

mainly grown in southwest China, northern India, Bhutan and Nepal. Specifically, Tartary

33

buckwheat gains the increasing research interest for its abundant nutrients, including high

34

quality protein, abundant flavonoids, well-balanced essential amino acids and minerals, as

35

well as its health benefits.1, 2 Various Tartary buckwheat commercial health products, in

36

the forms of tea, vinegar, noodles, porridge, biscuits, cakes, and sprouts, are greatly

37

popular in China.3, 4 The epidemiological study revealed that people in the Liangshan

38

region of Sichuan Province, China, have a rather low morbidity of chronic diseases such

39

as diabetes and hypertension. It would be related to the long-term tartary diet containing

40

Tartary buckwheat.2 Previous studies also reported its various beneficial effects, such as

41

antioxidant, anti-diabetes, anti-inflammatory, insulin resistance, cholesterol-lowering and

42

so on.5-9 These diverse bioactivities were attributed to the presence of multiple flavonoids,

43

in which rutin and quercetin, as the aglycon form of rutin, are the major components.10 All

44

the previous studies indicate that Tartary buckwheat flavonoid is the acknowledged

45

bioactive fraction.11 Despite the promising results in preclinical and epidemiological

46

studies as chemo-preventive agent, the extensive application of Tartary buckwheat

47

flavonoids has met limited success, because of poor aqueous solubility, poor chemical

48

stability in gastroenteric environments, including change in pH and enzymolysis and the

49

limited oral bioavailability.12, 13

50

Recently, nano-scaled carriers have been deemed to the useful tool to improve the

51

oral delivery for natural compounds or extracts, with various advantages on increasing

52

gastrointestinal stability, bioavailability and transmembrane absorption.14 Among diverse

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nano-vehicles, liposomes are ideal artificial carriers with superior biocompatibility, formed

54

by one or more concentric lipid bilayers, to encapsulate hydrophobic, hydrophilic and

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amphiphilic compounds. Manconi et al incorporated grape pomace extract with high

56

phenolic content in liposomes, to prevent degradation in gastric environment and protect

57

Caco-2 cells against oxidative stress.15 Similarly, Orthosiphon stamineus ethanolic extract

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rich in flavones was loaded in nano liposomes in unpurified soybean phospholipids to

59

improve its solubility and permeability.16 However, the low encapsulation efficiency (EE) is



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a limiting factor in upscale production of liposomes. For example, the average EEs of

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grape pomace extract and Orthosiphon stamineus extract are only 53~91%, and 66.2%,

62

respectively. Furthermore, liposomes suffer from drawbacks of lack of structural integrity,

63

leading to the loaded cargo leakage and storage instability.17 In view of the

64

above-mentioned facts, lipid-polymer hybrid nanoparticles (LPNs) have been developed

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to combine the characteristics of both polymeric nanoparticles (NPs) and liposomes, in

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which a polymer core was used to replace the liposomal cavity, in order to strengthen the

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drug loading structure.18,

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advantages of LPNs for anticancer drug delivery by intravenous administration.

69

Nevertheless, few investigations have been generated to evaluate the potential of LPNs

70

as oral delivery systems. And there are no publications on Tartary buckwheat flavonoids

71

encapsulation into NPs.

19

Several previous studies have confirmed the potential

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In the present work, Tartary buckwheat flavonoids were primarily obtained by an

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environmentally-friendly extraction approach with optimized parameters by orthogonal

74

experimental design. And then, the flavonoid fraction was loaded in fabricated LPNs, in

75

order to improve the solubility and permeability of flavonoids, as major factors for

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improving oral bioavailability. It was characterized for size distribution, stability, drug

77

release,

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immune-regulation effect of Tartary buckwheat extract LPNs was investigated in RAW

79

264.7 cells and the immunosuppressed mice model.

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■ MATERIALS AND METHODS

FT-IR

spectra,

DSC

method,

antioxidant

activity

and

so

on.

The

81

Materials. Tartary buckwheat seeds were obtained from the Key Laboratory of Grain

82

Processing, Ministry of Agriculture, China. Tartary buckwheat plants were harvested from

83

the experimental farm of Chengdu University, Chengdu, Sichuan, China, in November

84

2016. The species identification was authenticated by Pro. Gang Zhao (Chengdu

85

University).

86

(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy

87

glycol)-2000]) was purchased from Avanti Polar Lipids, Inc (Alabaster, USA). PLGA

88

(D,L-lactide-co-glycolide, MW=5000, lactide: glycolide (50:50)), Egg lecithin (EPC),

89

Cholesterol (CHOL), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)

mPEG2000-DSPE


(polyethylene

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90

were obtained from Sigma-Aldrich (St. Louis, MO). Cyclophosphamide (CTX),

91

2,2-diphenyl-1-picrylhydrazine

92

2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) were obtained by Yan Yi

93

Biotechnology Co., Ltd (Shanghai, China). Penicillin-streptomycin (PS), fetal bovine

94

serum (FBS), trypsin without EDTA, phosphate-buffered saline (PBS), 0.25% trypsin (w/v)

95

with 1 mM EDTA, and DMEM culture medium were purchased from Gibco (Grand Island,

96

NY, USA). All other chemicals were of analytical grade and used as received.

97

(DPPH),

and

RAW 264.7 cells, obtained from American Type Culture Collection (Manassas, VA,

98

USA), were cultured in DMEM medium supplemented by 10% heat-inactivated FBS and 1%

99

PS at 37 °C in a 5% CO2 humidified incubator. Six-week-old female Kunming mice

100

weighing 18-22 g were purchased from Dashuo Laboratory Animal Reproduction Center

101

(Chengdu, China). All mice were kept under pathogen-free environment and allowed free

102

access to the diet and water. All methods were carried out in accordance with relevant

103

guidelines and regulations and procedures involving mice were approved by the animal

104

care center of Chengdu University.

105

Extraction of Tartary buckwheat flavonoids. The raw Tartary buckwheat seeds

106

including husks were powdered and sieved over a 60-mesh screen. Total flavonoids were

107

obtained by ethanol hot refluxing extraction optimized by orthogonal design (L9(3)4).20

108

Briefly, Tartary buckwheat powders were mixed with a certain amount of ethanol solution,

109

and the mixture kept micro-boiling via ethanol refluxing for a certain time-period. Based on

110

the initial study, extraction was implemented via two repeated times in the present study.

111

Originally, to evaluate the orthogonal design tests, the effect of ethanol concentration, the

112

extraction duration, and the ratio of sample to extraction solvent on extraction yield was

113

investigated. Levels for ethanol concentration (A) factor 1, 2, 3 representative of 50, 75,

114

90%, extraction duration (B) factor 1, 2, 3 representative of 0.5, 1, 2 h, and ratio of sample

115

to extraction solvent (C) factor 1, 2, 3 representative of 1:10, 1:20, 1:40, respectively. The

116

extracts were suction filtered to discard the residue and the filtrates were then filtered

117

through a 0.45 µm filter. The extraction solution was concentrated under reduced pressure

118

and the flavonoid extract was collected by dried at 50 °C under vacuum. The yield of

119

flavonoid (%) in Tartary buckwheat was calculated by the ratio of weight of dried flavonoid



120

extract to that of Tartary buckwheat sample.

121

The quantitative analysis of total flavonoids was performed by aluminum chloride

122

colorimetric method. In brief, 6 mL of Tartary buckwheat extracts prepared at a series of

123

concentrations dissolved in 60% ethanol were added in 25 mL volumetric flask. 1 mL of 5%

124

sodium nitrite (NaNO2), 1 mL of 10% aluminium nitrate (Al(NO3)3), and 10 mL of 4%

125

sodium hydroxide (NaOH) were reacted in turn for 10 min at room temperature. Finally,

126

the absorbance of the mixture was measured at 510 nm by ultraviolet and visible

127

spectrophotometer (UV5800PC, Yuan Xi instrument Co., Ltd, Shanghai, China). Rutin

128

was used as the standard, with the equation of linear regression Y=0.0129X+0.0023

129

(r=0.9995).

130

Rutin, as the standard substance, in flavonoids was determined by ultra-performance

131

liquid chromatography (UPLC, Waters. Co., Ltd). The UPLC determination was carried out

132

with ACQUITY BEH C18 chromatographic column (4.6mm×250mm, 5 µm) and mobile

133

phase consisting of methanol (A)-0.1% phosphoric acid (B) aqueous solution to determine

134

at a flow rate of 0.3 mL/min. The composition of mobile phase was 10%~36% (A) for

135

0~2min, 36%~48% (A) for 2~5min, 48%~65% (A) for 5~8min, 65%~10% (A) for 8-10min.

136

The column temperature and detection wavelength were set at 30°C and 260 nm,

137

respectively. Rutin standard compound was accurately weighed and dissolved in

138

methanol to prepare stock solution at a concentration of 528 µg/mL. Rutin stock solution

139

was serially diluted to construct calibration curves. The diluted concentrations of rutin

140

were plotted against the peak area on the calibration curves and the linearity was

141

measured from the correlation coefficient.

142

Preparation of Tartary buckwheat flavonoids-loading LPNs (TBFs/LPNs).

143

Tartary buckwheat flavonoids (TBFs) were encapsulated in LPNs via a single-step solvent

144

evaporation method with assistance of ultra-sound as reported studies.18, 21 Some key

145

preparation parameters were optimized by orthogonal design tests with encapsulation

146

efficiency (EE) as an index. Specifically, a lipid monolayer was formed by EPC and

147

mPEG-DSPE in different molar ratios (1:2, 1:1, 2:1), which were dissolved in anhydrous

148

ethanol and dropped in deionized water at 65 °C to keep the dissolution situation of lipids.

149

Additionally, a certain ratio of TBFs to PLGA (1:2, 1:1, 2:1) were dissolved in acetonitrile.


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The total amount of lipids used was 30% w/w of the amount of PLGA polymer used17. The

151

volume ratio of oil phase to lipid water phase was set to 1:8, 1:15 and 1:20, respectively.

152

The oil polymer solution containing drugs was added, mixed via ultrasonic emulsification

153

for 10 min at ice-bath, and subsequently kept stirring for another 2 h at 25 °C. Finally,

154

organic solvent residue in the LPNs solution was removed by vacuum rotary evaporation.

155

The hybrid NPs with light blue opalescence were obtained by removing unloaded drugs

156

using 0.45 µm filter filtration.

157

Characterization of TBFs/LPNs. The particle size and zeta potential of TBFs/LPNs

158

samples were diluted properly with Milli-Q water at the polymer concentration of 1mg/mL,

159

and determined by a Nano-series Zetasizer (Nano-ZS, Malvern Instruments Ltd.,

160

Worcestershire, UK). The surface morphology for prepared TBFs/LPNs was determined

161

by a Tecnai G20 transmission electron microscope (TEM, FEI, Hillsboro, OR). In detail,

162

one drop of suspension was put on a paraffin sheet successively followed by covering with

163

a copper grid, keeping for one minute, and negatively stained by an aqueous solution of

164

phosphotungstic acid. After clearing the remaining solution, samples were air dried and

165

observed by TEM. The storage stability of TBFs/LPNs at 4 °C during 7 d was evaluated by

166

the particle size change and drug leakage rate.

167

The EE% and loading efficiency (LE)% of LPNs were evaluated in terms of rutin’s

168

content. An aliquot of 1.0 mg of freeze-dried LPNs was dissolved in 200 µL of acetonitrile.

169

After ultrasound for 5 min and centrifugation (10000rpm, 10min), the supernatant was

170

analyzed by sodium nitrite-sodium nitrate-hydroxide colorimetric method and the UPLC

171

method previously reported. The EE and LE were calculated using the following equations,

172

respectively:

173

EE(%)=

174

amount of drug loaded ×100% amount of drug added amount of drug loaded LE(%)= ×100% amount of drug loaded+polymer

175

The chemical composition of TBFs, empty LPNs, TBFs/LPNs and blends of TBFs and

176

LPNs (weight ratio of 1:10) was analyzed by FT-IR spectra at a resolution of 4 cm-1 in KBr

177

pellets, in the range 400~4000 cm-1. Moreover, these samples were analyzed by

178

differential scanning calorimetry (DSC) study. Briefly, 5 mg sample was put on standard



179

aluminum pan, crimped and heated (20 °C~200 °C at a heating rate of 5 °C/min) with

180

continuous purging of nitrogen in a proper speed of 20 mL/min. All samples were

181

evaluated in triplicate along with a standard taken as an empty sealed pan.

182

In vitro antioxidant activity. Antioxidant activity was evaluated by in vitro DPPH

183

radical scavenging assay and ABTS radical scavenging assay.22 A volume of 2 mL of

184

different concentration of TBFs and TBFs/LPNs solution were added to the freshly

185

prepared 2 mL of DPPH solution (0.2 mM). Similarly, control group was acted by the

186

equivalent approach with 2mL of ethanol without TBFs containing. After incubation of 30

187

min in the dark place, absorbance was taken in 517 nm using UV-spectrophotometer. The

188

DPPH scavenging rate was calculated by the following formula. DPPH scavenging rate (%)

189

= (Absorbance of control - Absorbance of samples) / Absorbance of control × 100%

190

In addition, the ABTS cation solution (ABTS+) was prepared by mixing of 5 mL ABTS

191

(7 mM) with 100 µL of potassium persulfate (2.45 mM) for 16 h incubation. 2 mL of

192

different concentrations of TBFs and TBFs/LPNs were thoroughly mixed with 2 mL of

193

ABTS+ working solution, and kept in the dark for 10 min. Similarly, control group was acted

194

by the equivalent approach with 2 mL of ethanol without TBFs containing. The absorbance

195

of all samples were measured at 734 nm. The percentages of inhibition were calculated by

196

the following formula. ABTS scavenging rate (%) = (Absorbance of control- Absorbance of

197

samples) / Absorbance of control×100%.

198

Anti-inflammatory effect on RAW 264.7 cells in vitro. RAW 264.7 macrophage

199

cells were plated at a density of 1×104 cells/well in 96-well plates, in DMEM medium

200

supplemented with 10% FBS. After treatment of a series of amounts of TBFs and

201

TBFs/LPNs (equivalent to rutin amount of 0, 40, 80, 160 µg/mL) for 24 h, the assay liquid

202

in each well was replaced with fresh medium containing MTT solution (5 mg/mL) for

203

another 4 h incubation in the dark. Subsequently, the liquid was discarded and DMSO was

204

added to dissolve the formed crystal. The absorbance values of 96-well plates were

205

detected at 570 nm using a microplate reader.

206

Moreover, NO release by RAW 264.7 cells were determined by nitrite levels by mixing

207

with Griess reagent.23 Similarly, RAW 264.7 cells with a density of 5×105 cells/well were

208

plated in 24-well plates, pretreatment with various TBFs formulations with a series of


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equivalent to rutin amounts (0, 40, 80, 160 µg/mL), and stimulated with or without 0.1

210

µg/mL LPS for 24 h. 50µL of culture medium was pipetted out and mixed with 50µL of

211

Griess reagent with 10 min reaction. The absorbance was recorded at 540 nm and a

212

standard curve was established using NaNO2 to calculate the sodium nitrite

213

concentrations in the samples. The pro-inflammatory cytokines including TNF-α, IL-1β,

214

IL-6, and PGE2 were determined by ELISA kits based on the manufacturer’s protocols.

215

Cellular transport study in Caco-2 monolayer. Caco-2 cells were seeded on

216

Transwell inserts (6 well plates, Corning, USA) with a cell density of 5 × 105 cells per well.

217

Culture medium was replaced every 2 days. After 21 days incubation when the

218

transepithelial electrical resistance value reached more than 300 Ωcm−2, cell

219

differentiation was allowed to generate cell monolayer. Sodium fluorescein and TEM were

220

used to check the integrity and morphology of Caco-2 cell monolayer. And then,

221

transepithelial transport experiments were implemented as previous description.24 After

222

cell monolayer being equilibrated using preheated transport HBSS medium for 30 min at

223

37 °C, 0.5 mL of HBSS suspension containing free TBFs and TBFs/LPNs were added

224

from apical side (AP) or basolateral (BL) chamber to evaluate the transport profiles of rutin

225

in various TBFs formulations into cells in the presence or absence of a transporter inhibitor,

226

verapamil (ver) and cyclosporine (cyc). 0.2 mL of samples in BL or AP side were collected

227

at predetermined time intervals (0.5, 1, 2, and 4 h). Rutin concentration in samples was

228

determined by HPLC analysis as above-mentioned condition. The cellular protein

229

concentrations were determined by Bradford method with bovine serum albumin as the

230

standard to normalize the intracellular drug concentration between experiments. The

231

apparent permeability coefficient (Papp) was calculated by the equation Papp (cm/s) =

232

dQ/(dt×A×C0), in which dQ/dt was the amount of permeated rutin every second. A was

233

1.12cm2, C0 was initial concentration of rutin.

234

Immuno-potentiation effect in vivo. The immunosuppression mice model was

235

obtained based on previous report by intraperitoneally injecting with 80mg/kg of CTX once

236

per day, with the successive administration for 5 days.25 Female KM mice were

237

randomized into six groups (n=10), i.e. no treatment and five immunosuppression group

238

mice, gavage treatment with saline, levamisole hydrochloride 30mg/kg, empty LPNs,



239

TBFs and TBFs/LPNs, respectively, for successively 14 days. The dosages of TBFs and

240

TBFs/LPNs were calculated by the amount of rutin contained with 10mg/kg per day. At the

241

end of the experiment, 10mL/kg of India ink was injected via tail-vein. At the 5 min (t1) and

242

15 min (t2) post-injection, the eye balls of mice under anesthesia were removed to collect

243

blood samples. 20µL of blood sample was mixed in 2mL of Na2CO3 (0.1%). After 10min,

244

the absorbance of samples was determined at 650 nm, with Na2CO3 solution as control.

245

Two important indexes, carbon clearance index (K) and phagocytic index (α), were

246

calculated by the following formulas to indicate the immunity activity.

247

K= (LogA1-LogA2) / (t2-t1). Note: A1 and A2 was the absorbance of blood samples collected

248

at t1 and t2 time-point.

249

α = K1/3 × body weight / (liver weight+ spleen weight).

250

And then, mice were sacrificed by CO2 inhalation and weighted. The spleen and

251

thymus organs from all mice were removed, weighted and calculate the spleen and

252

thymus indices. Spleen and thymus indexes were obtained by the weight ratio of organs to

253

mice body.

254

Statistical analysis. All experiment results were expressed as the mean ± standard

255

deviation. Student's t-test was used for single variable comparisons, and a P value < 0.05

256

was considered statistically signiﬁcant. Statistical analyses were performed by Prism 6.0

257

software (GraphPad Software Inc., La Jolla, CA, USA).

258

■ RESULTS

259

Preparation and Characterization of TBFs. Total flavonoids in Tartary buckwheat

260

seeds were extracted by ethanol hot refluxing. The involved extraction parameters

261

(ethanol concentration, extraction time, and the ratio of Tartary buckwheat-to-liquid) were

262

selected as the main factors on the basis of their impact on the extraction process

263

efficiency and single factor experiments. The values of these extraction factors were

264

optimized by orthogonal design. For three factors at three levels each, the orthogonal test

265

design required 9 experiments, and the results and variance analysis were shown in Table

266

1 and Table 2. According to results, the contribution of solid-liquid ratio for the extraction

267

yield of TBFs are significant, whereas other indexes are not significant factors. Based on

268

the extreme difference analysis, the optimum extraction condition of TBFs was deduced


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as: ethanol concentration of 75%, extraction time of 60 min, solid-liquid ratio of 1:40

270

(g/mL). The higher extraction yield (9.011%±0.029%) was obtained via these optimized

271

parameters, in comparison to that in other orthogonal tests. So, this deduced condition

272

was rationally confirmed to be the best extraction process.

273

The chemical constitutes in total flavonoids of Tartary buckwheat seeds were

274

identified by a comparison of their retention times with those of standards at UV

275

absorption spectra of 260 nm. There was no chromatogram peak in Figure 1A as the

276

negative sample at the corresponding retention time with that of rutin standard substance

277

(Figure 1B). Figure 1C showed the representative chromatogram of a rutin standard

278

substance and TBFs, respectively. As illustrated, the peak detected at 4.2 min in TBFs

279

corresponds to rutin. As expected, TBFs was rich in rutin, with predominantly amount of

280

7.66 ± 0.47mg/g.

281

Preparation and Characterization of TBFs/LPNs. In present study, TBFs was

282

encapsulated into LPNs by a nanoprecipitation technique combining ultrasonic

283

emulsification and solvent evaporation. In view of the complex factors contributing to

284

loading efficiency, the encapsulation process was also optimized by orthogonal tests.

285

Primarily, owing to the LPNs’ structure and drug loading efficiency, the lipid/polymer ratio,

286

phase/volume ratio of organic to aqueous phase, and ratio of drug feeding to PLGA

287

polymer were currently considered as the critical factors.19, 26, 27 Based on the three factors

288

on three levels, different EE% values could be observed in Supplementary materials

289

(Table S1). According to the extreme difference analysis, the contribution of these three

290

factors to TBFs loading was as following: factor C ˃ factor B ˃ factor A. Especially, factor

291

C indicated the significant contribution by variance analysis result Supplementary

292

materials (Table S2). The optimum preparation parameters were deduced as: the weight

293

ratio of EPC/mPEG-DSPE of 1:1, volume ratio of organic/aqueous phase of 1:20, and

294

weight ratio of TBFs/PLGA of 1:1.

295

Based on the repeated triple tests, TBFs/LPNs were characterized by a mean particle

296

size of 61.25±1.83 nm with a narrow distribution (PDI 0.22±0.005), and the zeta potential

297

of -26.0±0.7 mV (Figure 2A), which indicated a good storage stability. The drug loading

298

LPNs suspension exhibited the homogeneous translucent colloid appearance with



299

opalescence. The TEM images (Figure 2B) provided the reliable evidence on the narrow

300

particle size distribution of TBFs/LPNs, showing that LPNs emerged the uniform particle

301

size, smooth surface and spherical shape. Importantly, taking rutin as the evaluation index,

302

the EE% and LE% were 96.4% ± 1.1%, and 80.1% ± 1.5%, respectively. UPLC

303

chromatogram of TBFs/LPNs were exhibited in Figure 2C, showing the similar chemical

304

constitutes with free TBFs in Figure 1C. It indicated that the encapsulation process

305

basically retained these flavonoids in TBFs, which was in accordance with the high EE

306

value of rutin. Furthermore, the storage stability was evaluated by the leakage rate of drug

307

loaded and particle size change during a week storage at 4 °C. As shown in Figure 2D,

308

after 7d, there was scarcely change on encapsulation rate with the leakage rate lower

309

than 2%. Accordingly, the particle size still maintained stable during the storage 7d. Thus,

310

based on the optimized preparation process, TBFs/LPNs were fabricated with high drug

311

loading capacity, good size particle, morphology and stability.

312

The encapsulation of TBFs into LPNs was investigated by FT-IR and DSC

313

spectroscopy. As shown in Figure 3A, the empty LPNs exhibited weak stretching vibration

314

peaks. The bands at 2950~2800 cm-1 and 3600~3300 cm-1 were assigned to C-H2 and

315

O-H stretching in PEG layer. Other stretching vibrations derived from chemical groups

316

such as C-O, C-O-C in PLGA and EPC in core of LPNs were covered. Due to the

317

abundant hydroxyl and glucoside, free TBFs displayed strong bands. In view of the

318

highest amount of rutin in TBFs, TBFs exhibited the similar FT-IR spectrum with that of

319

rutin as previously reported.28 Accordingly, the mixture of empty LPNs and free TBFs

320

showed the basically similar vibration bands as free TBFs, due to the scarce shield of

321

empty LPNs. However, the most intensive differences in positions, intensity and width of

322

the ranges: 500~800 cm-1 derived from wagging vibration of hydroxyl bond at the sugar

323

ring, and about 2800 cm-1 were recorded for TBFs/LPNs in comparison to free TBFs and

324

the mixture. These shifted peaks indicated that the encapsulation contains the

325

intermolecular and the hydrogen-bonding interactions between polymers and TBFs.29

326

Meanwhile, the FT-IR spectra of TBFs and LPNs mixture did not shown the significant

327

shift, suggesting that the mentioned chemical interactions occurred in TBFs loaded in

328

LPNs during nanoprecipitation, instead of physical mixture.


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Furthermore, DSC measurement of empty LPNs, TBFs, physical mixture, and

330

TBFs/LPNs have been depicted in Figure 3B. The endothermic peaks near 50 °C and

331

140 °C in empty LPNs were contributed from dehydration process and the crystalline

332

nature shift of lipid and PLGA polymers, respectively. DSC curves of TBFs shows the

333

sharp endothermic transition at 122 °C which corresponds to the integrated melting point

334

temperature of flavonoids. The melting points of quercetin and rutin, as the main

335

compounds in TBFs, were 129 °C and 190 °C, respectively.30, 31 Similarly, the physical

336

mixture showed the broadening endothermic band at 120 °C~150 °C, displaying the

337

characteristic peaks of TBFs and LPNs. However, these peaks were absent in TBFs/LPNs.

338

No obvious peaks of TBFs and LPNs were visible in TBFs/LPNs. This finding suggested

339

that TBFs are molecularly dispersed within LPNs as the amorphous nature. This result

340

further authenticates the entrapment of TBFs.

341

DPPH and ABTS radical scavenging activity. The flavonoids rich in alcoholic

342

hydroxyl groups have aroused great interest due to their abilities to scavenge free radicals,

343

thereby inhibiting oxidative stress involving in several diseases. It also be widely regarded

344

as the healthy source of Tartary buckwheat products.32, 33 Both TBFs and TBFs/LPNs

345

exhibited remarkable DPPH radical scavenging activity with a dose-dependent manner

346

(Figure 4A), comparison to hardly any inhibition effect of empty LPNs. Nevertheless,

347

TBFs/LPNs showed higher DPPH scavenging ability than free TBFs. Taking the amount of

348

containing rutin as measurement, their inhibition IC50 values were 4.48 and 3.99 µg/mL,

349

respectively. To further prove its antioxidant activity, the results from ABTS radical

350

scavenging assays were presented in Figure 4B. Similarly, a significant difference

351

between TBFs and TBFs/LPNs on ABTS radical scavenging activity could be observed.

352

After loading in LPNs, TBFs showed much higher antioxidant effect at each tested drug

353

concentration, in comparison to free TBFs. Taking the amount of rutin as measurement,

354

their ABTS inhibition IC50 values were 21.94 and 16.93 µg/mL, respectively. Analogously,

355

it was previously reported that NPs encapsulating white tea extract could protect it from

356

degradation, showing higher DPPH scavenging effect.29 These results indicated that TBFs

357

maintained a good free radical scavenging activity, while the encapsulation in LPNs could

358

remarkably enhance its antioxidant effect in vitro.



359

Anti-inflammatory effect in vitro. To elucidate the anti-inflammatory effect and

360

underlying molecular mechanism of TBFs/LPNs, RAW 264.7 macrophage cells were used

361

in this study. Firstly, the potential cytotoxicity of TBFs formulations against RAW 264.7

362

cells was evaluated that after 24 h treatment, and cell viability was not affected by either

363

free TBFs or TBFs/LPNs with the equivalent rutin amount of up to 160 µg/mL (Figure 5).

364

Nitric oxide (NO), as the representative signaling molecule in inflammation process, was

365

subsequently determined in LPS-stimulated RAW 264.7 cells. As shown in Figure 5, the

366

NO production of LPS-pretreated RAW 264.7 was markedly increased to about 80 µM

367

when 0.1µg/mL LPS was added, compared with 10 µM of NO in untreated control cells.

368

The NO production amount decreased significantly in a dose-dependent manner after

369

treated with both TBFs and TBFs/LPNs. Even so, TBFs/LPNs exhibited higher capacity to

370

recede the raise NO amount than TBFs at each drug concentration. Furthermore, the

371

secretions of the proinflammatory cytokines in RAW 264.7 cells were determined (Figure

372

5). The levels of TNF-α, IL-β, IL-6, and PGE2 were significantly increased in the

373

LPS-treated cells when compared with the untreated cells. However, both TBFs and

374

TBFs/LPNs could remarkably reduce the levels of these proinflammatory cytokines in a

375

dose-dependent manner, in which similarly TBFs/LPNs displayed the higher capacity than

376

TBFs. These results demonstrated that in comparison of TBFs, the encapsulation in LPNs

377

could reinforce the anti-inflammatory effect.

378

Permeability profiles in Caco-2 monolayer. Intestinal absorption is one of the

379

important factors to influence the bioactivity efficiency. Herein, Caco-2, as a well

380

differentiated human intestinal epithelial cell line, was cultivated to generate monolayer

381

and evaluate the absorption of rutin in various TBFs formulations across the intestinal

382

epithelium. Primarily, the potential cytotoxicity of free TBFs and TBFs/LPNs on Caco-2

383

cells was tested in aim to evaluate whether the dosage of TBFs formulations used would

384

result in epithelial cell injury. After 12 h incubation, both samples could lead to reducing

385

cell viability less than 10%, suggesting the resulted cell death was negligible. And then,

386

the Papp of sodium fluorescein was lower than 10-7 cm/s, indicated the integrity of Caco-2

387

cell monolayer.24 Furthermore, both microvilli and tight junctions of Caco-2 monolayer

388

were observed by TEM. And then, transcellular permeability of rutin in TBFs formulations


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389

on Caco-2 monolayer in the presence or absence of the above-mentioned drug transport

390

inhibitors was monitored.

391

The different permeability values of free TBFs or TBFs/LPNs in Table 3 suggested

392

that rutin in TBFs/LPNs possessed the higher transport ability from AP to BL than that in

393

parent TBFs, with the higher value of Papp AP→BL. More importantly, as the representative

394

efflux, these transported rutin were greatly apt to be pumped out by efflux protein such as

395

P-gp and MDR. Nevertheless, TBFs/LPNs exhibited the lower Papp BL→AP value than TBFs,

396

indicated that the drug efflux of rutin was declined. The efflux rate (ER) of TBFs/LPNs was

397

much lower than TBFs, suggesting the intestinal absorption of rutin in TBFs/LPNs was

398

greatly improved. As shown in Figure 6A, the transport of rutin in either TBFs or

399

TBFs/LPNs was continuously cumulative with a time-dependent manner. However,

400

TBFs/LPNs exhibited much more permeability amounts than TBFs at each time-point.

401

After incubation with drug transport inhibitors, the transport of rutin to BL side was greatly

402

improved. It would result from that both ver and cyc could inhibit drug efflux of rutin. Figure

403

6B also depicted that after it encapsulated in LPNs, TBFs exhibited higher transport from

404

AP to BL due to penetration enhancing effect of nanoparticles. However, the transport

405

from BL to AP of TBFs/LPNs, representing the drug efflux, was significantly gotten

406

remission in comparison to parent TBFs. It would be for the reason that the

407

nano-encapsulation of polymers and lipids could alleviate the drug efflux of rutin by

408

transporter proteins. Figure 6C and 6D provided the steady evidence that the permeability

409

of TBFs/LPNs into Caco-2 cells was higher than that of TBFs, and the addition of drug

410

transporter inhibitors could remarkably promote the cellular drug concentration. Thus,

411

Caco-2 permeation results, with a good correlation to human oral absorption, indicated

412

that more flavones like rutin in TBFs/LPNs can be absorbed, compared with free TBFs. It

413

suggested that TBFs/LPNs would show the improved bioactivity due to the higher

414

intestinal absorption.

415

Immuno-enhancing effects in immunosuppressed mice model. To investigate

416

the immune-enhancing effect of TBFs/LPNs, an in vivo study was performed on the

417

immunosuppressed short-term mice model. Throughout the test, the food intake every

418

day of each group was not significantly changed, suggesting CTX did not result in



419

decrease of diet. However, compared to untreated control group, mice in model group with

420

CTX administration greatly decreased body weight, spleen weight and thymus weight. As

421

shown in Table 4, both K value and α value in model group were remarkably dropped in

422

comparison to control group, indicating that the weaken macrophage phagocytosis

423

capacity. Meanwhile, the main immune organs including spleen and thymus in model

424

group got atrophy, resulting in the decrease of spleen index and thymus index.

425

Levamisole, as the positive drug, has been previously reported to activate immune

426

response.34 Compared to these immunosuppressed mice with only saline administration,

427

after gavage administration of either levamisole or TBFs for 14 days, mice exhibited the

428

improved immunity as the higher macrophage phagocytosis capacity and immune organs.

429

It indicated that TBFs possess the immuno-enhancing effects. However, in view of the

430

scarce effect of empty LPNs, TBFs/LPNs displayed much higher immuno-enhancing

431

effects than free TBFs. Both macrophage phagocytosis indexes and immunity organs of

432

mice in TBFs/LPNs group were almost close to these normal mice. These results suggest

433

that after nano-encapsulation, TBFs/LPNs effectively encouraged immune-enhancing by

434

promoting the CTX-stimulated decrease of macrophage phagocytosis capacity and

435

immunity organ weight.

436 437

■ DISCUSSION

438

Buckwheat is well-recognized as a functional food and an important healthy source of high

439

quality protein, well-balanced essential amino acids and minerals, with a widely global

440

consumption. Compared to common buckwheat (Fagopyrum esculentum), Tartary

441

buckwheat (F. tataricum) exhibits higher bioactive efficacy such as antioxidant, antitumor

442

and hypoglycemic properties, for the prevention of various diseases. These differentiated

443

effects were attributed to 9~300 times higher amount of flavonoids, mainly rutin and

444

quercetin, in Tartary buckwheat than common buckwheat.35 Nam et al reported that

445

flavonoid-rich extract in Tartary buckwheat could markedly reduce LPS-induced cytokine

446

production, indicated its potential anti-inflammatory effect.36 Additionally, a recent study

447

reported that rutin-rich Tartary buckwheat could decrease body weight, body fat

448

percentage, and oxidative stress in a randomised double-blind parallel test.37 According to


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449

Figure 1, rutin, i.e. 3, 3', 4', 5, 7-pentahydroxy flavone-3-rutinoside, is the main component

450

in TBF fraction, and also widely found in many food items, fruits, vegetables, beverages

451

as well as herbs. Recently, as an important dietary flavonoid, various excellent

452

nutraceutical effect of rutin such as anti-inflammatory, antioxidant, anti-allergic,

453

gastroprotective, hepatoprotective, and anti-diabetic effects have been discovered.38, 39

454

Thus, Tartary buckwheat flavonoid fraction rich with rutin would be a potential bioactive

455

source, and it specific extract process and the molecular mechanisms were elucidated

456

herein.

457

Although various flavonoids like rutin, quercetin, epigallocatechin-3-gallate (EGCG)

458

possess the well-known bioactivity on preventive properties against chronic diseases,

459

their oral application was greatly limited, due to the inefficient systemic delivery and poor

460

oral bioavailability. Commonly, the poor solubility, instability in the gastrointestinal (GI)

461

tract (pH, enzymes, presence of other nutrients), insufficient gastric residence time, and

462

low permeability in the intestine account for the barriers of diet flavonoids. Rutin, as one of

463

flavonoid glycosides, cannot readily penetrate the intestinal membrane after oral

464

administration, because of its hydrophilicity. Conversely, quercetin, as the flavonoid

465

aglycone, could be highly permeable in Caco-2 monolayer cells and perfused rat intestinal

466

model. Moreover, the efflux transporters (e.g., Multidrug resistance associated protein2

467

(MRP2), P-glycoprotein (P-gp)) in intestinal lumen also attributed to the low oral

468

bioavailability of flavonoids.40 A variety of pharmaceutical approaches including

469

nanoparticles, self-microemulsions, and bioadhensive drug delivery systems have been

470

developed to overcome these barriers for oral drug delivery.41-43 Amongst them,

471

polymer-based delivery nanoscaled systems have been developed extensively for the oral

472

delivery of biomedical and functional ingredients to protect and transport them in intestine.

473

These biomacromolecular based nanoparticles enhance the absorption and bioavailability

474

of bioactive molecules mainly through the following pathways: (1) preventing the instability

475

of cargoes in the harsh environment of GI tract, (2) prolonging the residence time in the

476

gut by mucoadhesion, (3) facilitate the endocytosis by nano-scaled particles, (4)

477

suppressing the drug efflux.44 As shown in Figure 6, rutin in TBFs exhibited low

478

transmembrane transport in Caco-2 monolayer and strong drug efflux profiles. However,



479

after loading into lipid-polymer hybrid nanoparticles (LPNs), about 1.75-fold of rutin can

480

transport to BL side from AP chamber, in comparison with that in TBFs. Meanwhile, the

481

drug efflux rate of rutin in TBFs/LPNs was lower than that unloaded, indicated the

482

encapsulation of LPNs could significantly increase the oral bioavailability of TBFs.

483

In conclusion, the present study discloses the feasibility of an environment-friendly

484

extraction approach and the encapsulating in biocompatible lipid-polymer hybrid

485

nanoparticles for total flavonoids in Tartary buckwheat (TBFs/LPNs). With the high loading

486

efficiency, ideal particle size distribution and storage stability, TBFs/LPNs demonstrated

487

the significant enhancement of free radical scavenging effect, anti-inflammatory activity in

488

LPS-stimulated RAW 264.7 cell, and immuno-enhancing effects in immunosuppressed

489

mice model. It would be attributed to the improvement in aqueous solubility, stability in

490

gastrointestinal tract, intestinal permeability, and colloidal stability of LPNs. Therefore, the

491

proposed lipid-polymer nano-system may present a promising oral drug delivery approach

492

for flavonoids in Tartary buckwheat in pharmaceutical and nutraceutical fields.

493

■ ASSOCIATED CONTENT

494

Supporting Information

495

The Supporting Information is available free of charge on the ACS Publications website.

496

Orthogonal test design and results of TBFs loading in LPNs; Variance analysis of

497

orthogonal test.

498

■ AUTHOR INFORMATION

499

Conflict of interest

500

The authors declare no conflict of interest.

501

Funding

502

The present study was financially supported by Program of Application Foundation from

503

Department of Science and Technology Program of Sichuan Province, China (No.

504

2015JY0259), Scientific Research Innovation Group Foundation of Educational

505

Committee of Sichuan Province, China (No. 17TD0010), earmarked fund for China

506

Agriculture Research System (No. CARS-08-02A)

507

Author Contributions

508

Jinming Zhang, Chaomei Fu, Shu Fu, and Liang Zou designed the research; Jinming


Page 18 of 34

Page 19 of 34


509

Zhang, Di Wang, Yihan Wu, Wei Li, and Yichen Hu performed the research and analyzed

510

the data. Gang Zhao provided the quality and resource check for Tartary buckwheat seeds.

511

Jinming Zhang and Di Wang contributed equally to this work.

512

■ REFERENCES

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Figure captions

679

Figure 1. Representative UPLC chromatograms of negative sample (A), rutin standard

680

substance (B), and TBFs (C).

681

Figure 2. Characterization of TBFs/LPNs. (A) particle size distribution and zeta potential.

682

(B) TEM images with scale bar as 200nm and 100nm, respectively. (C) Representative

683

UPLC chromatograms of TBFs loaded in LPNs. (D) Leakage rate of TBFs loaded in LPNs

684

during 7 days storage.

685

Figure 3. FT-IR spectra (A) and DSC thermogram (B) of various TBFs formulations.

686

Figure 4. Comparison of DPPH (A) and ABTS (B) radical scavenging activities of TBFs

687

and TBFs/LPNs at different equivalent rutin concentrations.

688

Figure 5. Effects of TBFs/LPNs on cell viability of RAW 264.7 cells, NO production and

689

secretions of proinflammatory cytokines including TNF-α, IL-β, IL-6, and PGE2 in

690

LPS-stimulated RAW 264.7 cells.

691

Figure 6. Transport of rutin contained in TBFs or TBFs/LPNs through Caco-2 monolayers.

692

(A) Cumulative amount of transported rutin at different time in the presence or absence of

693

verapamil and cyclosporine. (B) Differences in transport of rutin in TBFs or TBFs/LPNs.

694

Transport of rutin in the AP to BL (C) and the BL to AP direction (D) direction in the

695

presence of different inhibitors.



Page 24 of 34

Tables Table 1. Orthogonal Test Design and Results of TBFs Extraction EXP

Factors

Extraction Yield (%)

A

B

C

1

1

1

1

6.520

2

1

2

2

6.708

3

1

3

3

8.105

4

2

1

2

7.979

5

2

2

3

9.014

6

2

3

1

7.447

7

3

1

3

8.549

8

3

2

1

6.417

9

3

3

2

7.918

K1

21.333

23.048

20.384

K2

24.44

22.139

22.605

K3

22.884

23.47

25.668

R

1.03566

0.44366

1.76133


Page 25 of 34


Table 2. Variance Analysis of Orthogonal Test Factors

Sum of square

Degree of freedom

Mean of square

F value

P value

Significant

A B C Error

1.60890 0.30843 4.69282 0.16723

2 2 2 2

0.80445 0.15421 2.34641 0.08361

9.62065 1.84432 28.06131 -

0.09415 0.35157 0.03441 -

* -

Note: *: P< 0.05



Page 26 of 34

Table 3. Permeability Values of Rutin in TBFs and TBFs/LPNs in Caco-2 Monolayer from AP to BL and from BL to AP Side (mean ± sd, n=3) Samples

Papp AP→BL

Papp BL→AP

(×10-6 cm/s)

(×10-6 cm/s)

TBFs

6.34±0.68

7.21±0.56

1.15

TBFs/LPNs

11.12±2.13

9.36±1.03

0.83


Efflux rate (ER)

Page 27 of 34


Table 4. Immuno-enhancing Effects of TBFs/LPNs in Immunosuppressed Mice Model after 14 Days Gavage Administration (n=10). Groups

Carbon clearance index (K)

Phagocytic index (α)

Spleen index (%)

Thymus index (%)

Control

0.029±0.011

5.274±1.264

5.972±1.157

2.451±0.865

#

#

#

Model

0.007±0.003

3.796±0.430

4.752±0.440

1.534±0.446#

Levamisole

0.012±0.008*

4.233±0.539*

5.932±0.954**

2.090±0.289*

Empty LPNs

0.007±0.004

3.985±0.921

4.875±1.315

1.465±0.637

TBFs

0.013±0.009*

4.177±1.387*

5.673±1.479**

1.889±0.454*

TBFs/LPNs

▲

0.022±0.015*

▲

5.100±0.906*

▲

5.816±1.207*

▲

2.377±0.662*

Note: VS negative control group, #P< 0.05; VS model group, *P< 0.05; TBFs/LPNs VS TBFs group, ▲P< 0.05.



Figures

Figure 1


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Figure 2



Figure 3


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Figure 4



Figure 5


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Figure 6



Table of Contents Graphic


Page 34 of 34

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