Administration of Piceatannol Complexed with Î±-Cyclodextrin

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Administration of Piceatannol Complexed with Alpha-Cyclodextrin Improves its Absorption in Rats Hiroyuki Inagaki, Ryouichi Ito, Yuko Setoguchi, Yukihiro Oritani, and Tatsuhiko Ito J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00398 • Publication Date (Web): 14 Apr 2016 Downloaded from http://pubs.acs.org on April 28, 2016

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Journal of Agricultural and Food Chemistry 1

Administration of Piceatannol Complexed with Alpha-Cyclodextrin Improves its Absorption in Rats

Hiroyuki Inagaki,* Ryouichi Ito, Yuko Setoguchi, Yukihiro Oritani, and Tatsuhiko Ito

Research Institute, Morinaga & CO., Ltd., 2-1-16 Shimosueyoshi, Tsurumi-ku, Yokohama, Kanagawa-prefecture, 230-8504, Japan

*Corresponding Author Phone: +81 45 571 2982; Fax: +81 45 791 6109; E-mail: [email protected]

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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1 Abstract 2 Piceatannol is polyphenolic antioxidant found in passion fruit (Passiflora edulis) seeds. The aim of 3 this study was to improve the absorption of piceatannol using alpha-cyclodextrin (αCD). The 4 solubility of piceatannol in neutral and acidic solutions increased in an αCD 5 concentration-dependent manner. The maximum plasma concentration of intact piceatannol and the 6 time-to-maximum plasma concentration of O-methylated piceatannol metabolites increased in rats 7 administered αCD-piceatannol inclusion complexes (PICs). Administering the αCD inclusion 8 complexes significantly increased the area under the concentration-time curve of total stilbene 9 derivatives (0–3 h), in terms of the total amount of intact piceatannol, O-methylated piceatannol, 10 conjugated piceatannol, and isorhapontigenin. Gastrointestinal ligation experiments demonstrated 11 that substantially higher levels of piceatannol metabolites were present in the lower intestine (the 12 ileum) at 1 h post-intragastric αCD-PICs administration, as compared to that observed following 13 piceatannol administration only. These results suggested that αCD enhanced piceatannol movement 14 and absorption in the small intestine. 15

16 Keywords: piceatannol, alpha-cyclodextrin, stilbene, absorption, passion fruit


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17 Introduction 18 Piceatannol (trans-3,3',4,5'-tetrahydroxystilbene) is a naturally occurring hydroxylated analog of 19 resveratrol (trans-3,5,4'-trihydroxystilbene). The only difference between resveratrol and piceatannol 20 is the presence of an extra hydroxyl group at the 3' carbon of an aromatic ring in piceatannol (Figure 21 S1). Previously, we reported that passion fruit (Passiflora edulis) seeds contain high quantities of 1

22 piceatannol . Piceatannol can promote various health benefits similar to those of resveratrol, such as 23 melanogenesis inhibition, collagen synthesis, vascular endothelial cell-dependent vasorelaxation, 24 induction of nitric oxide synthase in cultured human umbilical vein endothelial cells, and protecting 25 keratinocytes against ultraviolet B-irradiation

1-4

.

26 The health effects of polyphenols depend on the amount consumed and on their bioavailabilities. 27 Polyphenols in the human diet are not necessarily highly active within the body because they may be 28 poorly absorbed from the intestine. Previously, several human subjects were used to measure the 29 maximum plasma concentration (Cmax) after administering various classes of polyphenols. Mean 30 Cmax values were calculated for different polyphenols, and the data were converted to correspond to 31 a supply of 50 mg aglycone equivalent. For example, the mean Cmax of epigallocatechin gallate was 32 0.12 ± 0.03 µM, while that for caffeic acid was 0.96 ± 0.26 µM

5,6

. In comparison with those

33 compounds, the oral absorption of stilbene is relatively low. Almeida et al. reported a Cmax for 7

8

34 resveratrol of 0.03 µM following a single 50-mg dose of trans-resveratrol . In our recent study , the 35 Cmax of piceatannol was 2.6 times higher than that of resveratrol. However, to demonstrate its



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36 potential in physiological applications, further improvement to the oral absorbability is desirable. 37 In the case of poorly water-soluble polyphenolic compounds such as stilbenes, the bioavailability 38 can be influenced by their intrinsic solubilities and dissolution rates in the gastrointestinal tract. 8

39 Recently, we studied the absorption and metabolism of piceatannol versus resveratrol in rats . We 40 found that the area under the curve (AUC) value in plasma for intact piceatannol after intragastric 41 administration was 2.1 times greater than that of resveratrol. In addition, the metabolic pathway of 42 piceatannol is more complicated than that of resveratrol; piceatannol metabolism involves 43 conjugation, methylation, or both. Furthermore, rhapontigenin (3,3′,5-trihydroxy-4′ methoxystilbene) 44 and isorhapontigenin (3,4',5-trihydroxy-3'-methoxystilbene) were identified as O-methylated 45 piceatannol metabolites in rat plasma and urine. Piceatannol administered intravenously to rats was 9

46 metabolized into a glucuronide conjugate and excreted in the urine . In contrast to piceatannol, 47 methylated metabolites such as isorhapontigenin, isorhapontigenin conjugates, rhapontigenin 48 conjugates, and O-methyl piceatannol-monosulfate were not detected following resveratrol 8

49 administration . Piceatannol and its characteristic metabolites might serve as useful pharmacologic 50 molecules. However, more data are needed on their pharmacological properties, as well as for 51 enhancing their absorbability after intragastric administration. It is important to increase the 52 absorbability of piceatannol to improve its bioactivity. However, there is a possibility that bitter or 53 astringent tastes may interfere with the tastes of foods due to an increased amount of added 54 polyphenols. For this reason, it is important to improve the absorption rate of piceatannol (rather than


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55 to increase the amount present in food) for applications of piceatannol as a functional food. 56 There are various strategic options for improving the solubilities for applications involving foods 57 and drinks, such as salt formation, solubilization with lecithin 58 with cyclodextrin

10

, solid dispersions 11, complexation

12

, nanoparticle formulations 13, and lipid-based formulations 14. When these

59 methods are applied with foods, safer alternatives are generally found to be required. 60 Alpha-cyclodextrin (αCD) is a common food additive in Japan and does not pose a safety concern at 61 the proposed usage levels and predicted consumption levels as a food ingredient or food additive. 62 The acceptable daily intake (ADI) of αCD was determined as 'not specified' by The Joint FAO/WHO 63 Expert Committee on Food Additives. Numerous reviews have been published regarding the use of 64 cyclodextrins in oral-dosage forms, many of which have specifically addressed the effects of 65 cyclodextrins on oral absorption and/or bioavailability

15,16

. Cyclodextrins have been used in

66 pharmaceutical formulation to enhance the stability or to modulate the biological activity of drugs 67 over wide pH ranges

17

.

68 The use of cyclodextrin to increase the physical and chemical stability or compounds in solution 69 and in other forms has been well documented in the literature

18

. Cyclodextrins have also been used

70 to overcome other issues, such as a bitter taste in foods and the controlled release of drugs for oral 71 and parenteral delivery. The interaction between cyclodextrins, chemical compounds, and dosage 72 form influences the kinetics of key processes of oral drug delivery, including dissolution and 73 absorption

19,20

. However, the effects of αCD-piceatannol inclusion complexes (PICs) were on the



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74 absorption of piceatannol and its behavior in the intestinal tract were not fully investigated in 75 previously. 76 The aim of this study was to test the hypothesis that αCD treatment can influence the intestinal 77 absorption of piceatannol in rats. To evaluate the effect of αCD on piceatannol solubility, an in vitro 78 study of piceatannol solubility in an αCD-piceatannol inclusion complex and a control was 79 performed to compare the effects of the intragastric administration agent (0.5% carboxymethyl 80 cellulose; CMC) and artificial gastric juice. The AUC, Cmax, and the time-to-reach Cmax (Tmax) 81 for piceatannol and total stilbene derivatives were determined following the intragastric 82 administration of αCD-PICs or a control to rats. The Cmax and Tmax were compared to 83 characterize the rate of piceatannol absorption with different αCD-piceatannol formulations. 84 Furthermore, behaviors in the gastrointestinal tract of αCD-piceatannol inclusion complex- or 85 control-treated rats were investigated in gastrointestinal-ligation experiments. 86

87 Materials and methods 88 Reagents. Piceatannol (>98.0% purity), isorhapontigenin (>95.0% purity), and rhapontigenin 89 (>98.0% purity) were purchased from Tokyo Chemical Industry Co., Ltd. αCD was obtained from 90 Cyclochem Co., Ltd. CMC was purchased from Nacalai Tesque, Inc. All other reagents were 91 purchased from Wako Pure Chemical Industries, Ltd. 92


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93 Solubility of piceatannol in artificial gastric juice. The solubility of piceatannol in artificial gastric 94 juice in intragastric-administration samples was measured in duplicate, as described previously

21, 22

.

95 Artificial gastric juice (pH 1.2) was prepared according to the methods of the Japanese 96 pharmacopoeia

23

. Intragastric piceatannol-administration samples contained 90 mmol/L piceatannol

97 suspended in 0.5% w/v CMC and mixed to various concentrations (0, 45, 90, and 270 mmol/L) of 98 αCD suspended in 0.5% w/v CMC. Each sample was vortexed for 15 s and sonicated 3 times in a 99 37 °C water bath for 10 min. To investigate the effect of an inclusion complex, a water-soluble 100 starch (approximately 90 mmol/L) was added to the piceatannol suspension instead of αCD. A 101 100-µL aliquot of piceatannol-suspension sample was added to 400 µL of artificial gastric juice and 102 vortexed and then incubated at 37 °C for 1 h in a water bath, with shaking. The soluble fraction was 103 collected after centrifugation at 10,000 × g for 10 min at 37 °C. A 50-µL aliquot of the soluble 104 fraction was vortexed in 1,700 µL ice-cold acetonitrile and centrifuged at 10,000 × g for 5 min at 105 4 °C. The supernatant aliquot was diluted with initial mobile phase and used for HPLC analysis. 106

107 Time-dependent changes of piceatannol solubility in artificial gastric juice. The solubility of 108 piceatannol in artificial gastric juice in the intragastric-administration samples was measured in 109 duplicate as described above. Piceatannol intragastric-administration samples contained 90 mmol/L 110 of piceatannol suspended in 0.5% w/v CMC mixed to various concentrations (0, 45, 90, and 270 111 mmol/L) of αCD suspended in 0.5% w/v CMC. The αCD-PICs were prepared as described above. A



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112 100-µL aliquot of the suspension sample containing piceatannol was added to 400 µL of artificial 113 gastric juice and vortexed, after which it was incubated at 37 °C for 0, 0.25, and 1 h in a water bath 114 with shaking. The soluble fraction was collected after centrifugation at 10,000 × g for 10 min at 115 37 °C. An aliquot (50 µL) of the soluble fraction was vortexed in 1,700 µL ice-cold acetonitrile and 116 centrifuged at 10,000 × g for 5 min at 4 °C. A supernatant aliquot was diluted in the initial mobile 117 phase and used for HPLC analysis. 118

119 Animals and treatments. Male Sprague–Dawley outbred rats (for plasma concentration experiments: 120 8 weeks old; for gastrointestinal ligation experiment: 9–10 weeks old) were obtained from Japan 121 SLC, Inc. Rats were individually housed in resin cages (22.5 × 33.8 × 14.0 cm) on a stainless-steel 122 mesh floor under a controlled temperature (23.0 ± 2.0 °C) with a relative humidity of 50 ± 10% and 123 a 12 h light/12 h dark cycle (lights on between 0700 and 1900 h). Water and purified diet AIN-93G 124 (Oriental Yeast Co., Ltd.) were provided ad libitum, unless otherwise stated. 125 Blood samples were collected from catheter indwelled rats. Following a conditioning period that 126 lasted 2 or 3 days, rats were anesthetized by intraperitoneal injection with 0.15 mg/kg of 127 medetomidine hydrochloride (Nippon Zenyaku Kogyo Co., Ltd.), 2 mg/kg of midazolam (Astellas 128 Pharma Inc.), and 2.5 mg/kg of butorphanol tartrate (Meiji Seika Pharma Co., Ltd.) in physiological 129 saline, and the right jugular vein was catheterized with silicone tubing (0.5-mm inner diameter, 130 1.0-mm outer diameter). After the catheterization procedure, rats were allowed a 1-week


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131 post-operative recovery period. Rats were fasted overnight and then administered piceatannol or 132 αCD-PICs. Thereafter, blood samples were collected from the indwelling catheter. All animal 133 experiments were performed with strict compliance with the guidelines for proper conduct of animal 134 experiments by the Science Council of Japan. The experimental protocols and procedures were 135 approved

(Protocol

#166-01-002-01,

#166-02-002-01,

#166-02-004-01)

by

the

Animal

136 Experimental Committee of Morinaga & Co., Ltd. 137

138 HPLC analysis of stilbenes in plasma. Twenty-three catheter-indwelling rats were randomly 139 assigned to 4 groups (n = 5–6). Each group was administered 180 µmol/kg of piceatannol. The 140 intragastric-administration samples contained 180 µmol/kg of piceatannol suspended in 0.5% w/v 141 CMC (control) or 180 µmol/kg of piceatannol suspended in 90 (LαCD), 180 (MαCD), or 540 142 (HαCD) µmol/kg of αCD in 0.5% w/v CMC. Each sample was vortexed for 15 s and sonicated 3 143 times in a 37 °C water bath for 10 min. The administered doses of piceatannol were based on our 8

144 previous study . Rats were directly intubated into their stomachs with a volume of 2 mL/kg. Blood 145 samples (0.3 mL) were collected from each rat before administration and at 0.25, 0.5, 1, 2, 3, 4, 6, 146 and 8 h after administration. The blood samples were collected into heparinized tubes directly from 147 the jugular vein catheter, and cooled immediately on ice. Plasma samples were separated by 148 centrifugation (4 °C, 10 min, 5,000 × g), and stabilizing agents (0.4% ascorbic acid, 0.02% EDTA 149 disodium, 80 mM sodium phosphate buffer [pH 3.6]) were added at a 1:10 volume. The plasma



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150 samples were stored at -30 °C until use. Plasma preparation for HPLC analysis was performed as 8

151 described previously . Briefly, plasma-sample aliquots (50 µL) were extracted with 100 µL ice-cold 152 acetonitrile and centrifuged at 10,000 × g for 10 min at 4 °C. Supernatants were transferred to a 153 separate microcentrifuge tube, and the precipitates were washed with 100 µL ice-cold acetonitrile 154 and centrifuged. Thereafter, the supernatants were combined with the previous supernatants and 155 evaporated to dryness in vacuo. The residue was re-dissolved in 50 µL of the initial mobile phase 156 and used for HPLC analysis. Appropriate dilutions were performed when concentrations fell outside 157 the analytical range (0.2–10 µM). The mean recovery (at 0.2, 0.5, 1, 2, 5, 10, and 20 µM; n = 2) for 158 piceatannol was 93.3%. 159

160 Gastrointestinal ligation experiment. Gastrointestinal contents were collected from rats that were 161 not given a catheter-indwelling operation. Briefly, rats (n = 3) were fasted overnight and 162 intragastrically administered an HαCD-piceatannol inclusion complex sample or control sample, 163 followed by analysis of stilbenes in the plasma. At 1 or 4 h post-administration, rats were sacrificed 164 by pentobarbital-anesthesia and blood was collected by cardiac puncture. Following blood 165 collection, the stomach, duodenum, and small intestine were clamped and harvested promptly with 166 cooling in ice-cold saline. The intestine was divided into the upper section (ileum) and lower section 167 (jejunum). The gastrointestinal contents of each part were collected by intestinal lavage with 10 mL 168 of cold saline. An aliquot (500 µL) of soluble supernatant and insoluble sediments were separated


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169 by centrifugation at 2,500 × g for 10 min at 4 °C. The supernatant (50 µL) containing the 170 gastrointestinal contents was extracted using 200 µL ice-cold acetonitrile, vortexed for 15 s, and 171 centrifuged at 10,000 × g for 5 min at 4 °C. The supernatant was transferred to new centrifuge tube 172 and evaporated to dryness in vacuo. The residue was dissolved in 50 µL of the initial mobile phase 173 and used for HPLC analysis. The insoluble sediments were extracted with 2 mL ice-cold acetonitrile, 174 vortexed for 15 s, and a supernatant aliquot (500 µL) was collected after centrifugation at 2,500 × g 175 for 10 min at 4 °C. Extracts of insoluble sediments (50 µL) were deproteinized in 200 µL ice-cold 176 acetonitrile and centrifuged at 10,000 × g for 5 min at 4 °C. The supernatants were transferred to 177 new centrifuge tubes and evaporated to dryness in vacuo. The residues were dissolved in 50 µL of 178 the initial mobile phase and used for HPLC analysis. 179

180 HPLC analysis for stilbenes. Stilbenes were analyzed using a Prominence HPLC system (Shimadzu 181 Corporation), equipped with an SPD-20A photodiode array detector. The photodiode array data 182 were processed using LabSolutions Chromatography Workstation software (version 1.25, SP2; 183 Shimadzu Corporation). Chromatographic separations were performed on a Mightysil RP-18 GP 184 ODS column (150 × 4.6 mm inner diameter, 5 µm particle size; Kanto Chemical Co., Inc.) equipped 185 with a Mightysil RP-18 GP guard column (5 x 4.6 mm inner diameter, 5 µm particle size; Kanto 186 Chemical Co., Inc.) at 40 °C, with 0.1% (w/v) phosphoric acid/water as mobile phase A, and 0.1% 187 (w/v) phosphoric acid/ acetonitrile as mobile phase B (total flow rate: 1 mL/min). Sample aliquots



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188 (10 µL) were injected into the HPLC system and eluted under the following gradient conditions: 0– 189 2 min, 14% B; 2–30 min, 14–0% B. The eluate was monitored at 320 nm by the detector. 190 Calibration curves were prepared by supplementation with known concentrations (0.2, 0.5, 1, 2, 5, 191 and 10 µM) of piceatannol, isorhapontigenin, and rhapontigenin in the initial mobile phase. The 192 correlation coefficients were evaluated by linear-regression analysis and were 0.997 or above. 193

194 Data analysis. Experimental values are expressed as the mean ± SEMs. The AUC was calculated 195 using the linear-trapezoidal method. Statistical significance of the in vitro piceatannol solubility in 196 artificial gastric juice was analyzed by 1-way analysis of variance (ANOVA). For the plasma AUC, 197 Cmax, and Tmax values after intragastric administration, HαCD-PICs (n = 6), MαCD-PICs (n = 6), 198 LαCD-PICs (n = 6), or a vehicle control (n = 5) were analyzed by 1-way ANOVA and Tukey's 199 honest-significant difference (HSD) test. Student’s t test was used to evaluate differences in the 200 mean piceatannol contents in the gastrointestinal tract (stomach, duodenum, upper small intestine, 201 and lower small intestine) within 1 h after administration of αCD-piceatannol inclusion complex (n 202 = 3) and control sample (n = 3). All statistical analyses were performed using SPSS version 13.0 J 203 for Windows (IBM Japan Inc., Tokyo Japan). Differences were considered statistical significant 204 when p < 0.05. 205

206 Results


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207 Piceatannol solubility in artificial gastric juice. Based on results of in vitro phase-solubility study, 208 we investigated the solubility of piceatannol in acidic artificial gastric juice. The solubility of 209 piceatannol in acidic artificial gastric juice was evaluated after incubation at 37 ºC for 1 h in a water 210 bath, with shaking. The concentration of piceatannol in acidic artificial gastric juice was higher than 211 that in neutral 0.5% CMC solution. As a control, the solubility of piceatannol in artificial gastric 212 juice was found to be 38.0 ± 3.3 mM (Table S1). Piceatannol solubility significantly increased to 213 65.3 ± 1.3, 79.9 ± 0.7, and 76.1 ± 4.2 mM when the molar ratio of piceatannol to αCD in the 214 solution was 1:0.5, 1:1, and 1:3 respectively (Figure 1). The presence of water-soluble starch did not 215 significantly increase the solubility of piceatannol in artificial gastric juice, compared to the control. 216

217 Time-dependent changes in piceatannol solubility in artificial gastric juice. The piceatannol 218 concentration in the supernatants gradually increased in control and αCD-PICs immediately after 219 mixing and throughout the 1 h mix period. The piceatannol solubility was higher after addition of 220 the HαCD inclusion complex into the artificial gastric juice immediately after mixing, compared 221 with the control, reaching 35.0 ± 0.8 and 3.2 ± 0.1 mM, respectively (Table S1). Furthermore, the 222 solubility of piceatannol at 1 h after addition to artificial gastric juice increased in LαCD, MαCD, 223 HαCD-PICs, and the control, reaching 65.3 ± 1.3, 79.9 ± 0.7, 76.1 ± 4.2, and 38.0 ± 3.3 mM 224 (Table S1). All αCD inclusion complexes showed higher piceatannol solubilities compared to that of 225 the control.



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226

227 Quantitative analysis of plasma concentration of stilbenes. Time-dependent plasma concentration 228 profiles were investigated for piceatannol and its metabolites following administration with the 229 vehicle control or L, M, or HαCD-PICs (Figure 2 and Table 1). In the plasma, the piceatannol 230 concentration-time profile showed 2 peaks. The first peak reached a maximum level at 30 min 231 post-administration. The second peak increased at 4 h post-administration (Figure 2A). In contrast, 232 the Cmax for piceatannol significantly increased (p < 0.01) following administration with L, M, and 233 HαCD-PICs, compared to that of the control. The Cmax for the piceatannol conjugates and total 234 stilbenes significantly increased (p < 0.05) with the MαCD- and HαCD-PICs, compared to that of 235 the control (Figure 2C and 3E, Table 1). 236 The AUC0–3 h for piceatannol increased with the LαCD-, MαCD-, and HαCD-PICs, compared to 237 that of the control. The AUC0–3

h

for isorhapontigenin (Figure 2B and Table 1) significantly

238 increased with the L (p < 0.05), M (p < 0.01), and HαCD-piceatannol (p < 0.01) inclusion 239 complexes, compared to that of the control. The AUC0–3 h for the O-methyl conjugates and total 240 stilbenes (Figure 2D and 2E, Table 1) significantly increased (p < 0.01) with the L, M, and 241 HαCD-PICs, compared to that of the control. Furthermore, the AUC0–8

h

for O-methyl and

242 piceatannol significantly increased with the L (p < 0.01), M (p < 0.05), and HαCD-piceatannol (p < 243 0.05) inclusion complexes, compared to that of the control (Table 1). Regarding the Tmax for 244 O-methyl-conjugates, piceatannol showed a tendency to quicken with LαCD, MαCD, and HαCD


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245 inclusion complexes, compared to that of the control. 246

247 Piceatannol behavior in gastrointestinal fluids. The recovery values were determined as the 248 percentage of piceatannol or total stilbene derivatives recovered from the intestinal tract and were 249 calculated from the piceatannol doses used for intragastric administration. The recovery percentages 250 of piceatannol in the stomach are depicted in Figure 3A. The recovery percentages at 1 h 251 post-intragastric administration showed no significant difference with the HαCD-piceatannol 252 inclusion complex, compared to that of the control. The recovery percentages of total stilbene 253 derivatives in the duodenum and upper and lower small intestines are indicated in Figure 3B. The 254 recovery percentages of total stilbene derivatives in the lower small intestine at 1 h post-intragastric 255 administration significantly increased (p < 0.05) with the HαCD-piceatannol inclusion complex, 256 compared to that of the control. The recovery percentages of total stilbene derivatives in the 257 duodenum and upper small intestine at 1 h post-intragastric administration showed no significant 258 differences with the HαCD-piceatannol inclusion complex, compared to those of the control. 259

260 Discussion 261 The major purpose of our study was to clarify whether absorbability could be enhanced after 262 intragastric administration of αCD-PICs, compared to that of the control. In this study, the plasma 263 piceatannol concentrations of all αCD-PICs significantly increased in terms of the Cmax at the early



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264 phase after intragastric αCD-PICs administration, as compared to that observed following 265 administration of piceatannol only. 266 The water solubilities of numerous compounds may be potentially increased through 267 inclusion-complex formation with cyclodextrins. In an in vitro phase-solubility study, αCD 268 improved the solubility of piceatannol in neutral CMC solution and acidic artificial gastric juice. 269 Based on the shape of the generated phase-solubility relationships, several types of behaviors can be 270 identified

24

. Phase-solubility diagrams fall into 2 major types: A and B. Type-B phase solubility

271 profiles are indicative of the formation of complexes with limited water solubility and are 272 traditionally observed with naturally occurring CDs, especially β CD. Type-A phase-solubility 273 profiles are obtained when the solubility of the guest molecule increases with increasing 274 cyclodextrin concentrations. The phase-solubility diagram for αCD-PICs indicated that a type-A 275 profile was observed in the solubility study. Collectively, these isotherms indicate that water-soluble 276 complexes formed with solubilities higher than that of the un-complexed substrate. However, there 277 is still a need for structural analysis of inclusion bodies by X-ray crystallography and nuclear 278 magnetic resonance. 279 By measuring the aqueous solubility in artificial gastric juice, we confirmed that the solubility of 280 piceatannol increased in the solvent by the αCD inclusion process, which was beneficial for 281 intragastric-administration purposes. However, using piceatannol mixed with water-soluble starch, 282 the piceatannol concentration in artificial gastric juice was lower than that of all αCD-PICs studied.


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283 These results demonstrated that inclusion action of αCD played an important role in improving the 284 solubility of piceatannol in acidic artificial gastric juice. The PICs did not dissolve in artificial 285 gastric juice even after 1 h, and the improved absorption observed in rats may have been because 286 the solubility was 2-fold higher than that of the control. Results from a previous pharmacokinetics 287 study showed that the improvement in water solubility is an important factor that affects the Cmax 288

25

. The Cmax, Tmax, and AUC are important factors that influence the medicinal effects of oral

289 administered compounds. The plasma Cmax value of the piceatannol concentration observed 290 following administration of HαCD-PICs was 2.3-fold higher than that of the control. However, we 291 did not investigate how administering αCD-PICs can cause downstream biological effects to prevent 292 or ameliorate diseases. In many cases, an increase in polyphenol serum concentrations leads to 293 enhanced effects, but this is not a general rule

26, 27

. Thus, it will be necessary to study in detail the

294 biological effects of αCD-PICs in vivo and, potentially, in clinical trials. 295 There is widespread recognition that the gastric-emptying rate and transit time through the 296 intestinal tract are significantly altered by rate and extent of drug absorption

28,29

. The

297 gastric-emptying rate in liquids is shorter than in solids. For example, Quini et al. reported evaluated 298 the mean times-of-gastrointestinal-transit in liquid and solid meals in the same rats

30

. The mean

299 gastric-emptying and cecum-arrival times for a liquid meal were significantly shorter than those 300 observed for a solid meal. However, the mean gastric-emptying rates following administration of 301 αCD-PICs have not been determined. The reason for this is that the recovery percentages of



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302 piceatannol from the stomach at 1 h post-intragastric administration showed no significant 303 difference with HαCD-PICs, compared to that of a control. Furthermore, we examined the recovery 304 rates of total stilbene derivatives in the duodenum and upper and lower small intestine (Figure 3B). 305 In this case, clear differences in the recovery rates of total stilbene derivatives in the duodenum and 306 the upper part of the small intestine was not observed. The recovery percentages of total stilbene 307 derivatives from the lower small intestine at 1 h post-intragastric administration were significant 308 higher than that of the control. The reasons for this phenomenon may be that αCD can accentuate 309 piceatannol movement in the small intestine. Juan et al.

31

reported that trans-resveratrol enters the

310 enterocyte by passive diffusion, where it is highly metabolized, and its conjugates are secreted back 311 to the intestinal lumen. Furthermore, the absorption of trans-resveratrol was 16% higher in the 312 ileum than in the jejunum in rats. Considerable total stilbene derivatives were recovered from the 313 lower

small intestine, which is considered the main absorption site for piceatannol.

314 Piceatannol-related compounds observed in the lower small intestine were almost completely 315 comprised of piceatannol metabolites. The recovery rate of total stilbene derivatives observed in the 316 lower small intestine indicated that αCD-inclusion piceatannol was absorbed and metabolized in 317 intestinal epithelial cells or internal organs to a greater degree than that of the control at 1 h 318 post-administration. Therefore, αCD-PICs potentially contributed to piceatannol movement and 319 concentration in the small intestine at 1 h post-intragastric administration. Another possible 320 explanation for the observed Cmax increase is that piceatannol may have arrived at the small


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321 intestine before being digested, due to protection by αCD. The αCD moiety may act as a stabilizer 322 or control the release of piceatannol. The αCD-inclusion complexes can be potentially used for 323 immediate-release applications allowing the drug to dissolve in the gastrointestinal tract, without 324 delaying or prolonging the dissolution or absorption of drugs

32,33

. We did not measure piceatannol

325 stability in the small intestine; thus, it is necessary to examine the influence of αCD in improving 326 the stability in the intestinal tract more carefully. 327 Results from our recent study indicated that the metabolic pathway for piceatannol is more 8

328 complicated than that for resveratrol . The Tmax of O-methyl-conjugated piceatannol showed a 329 tendency to quicken with αCD-PICs, compared to that of the control. Although this phenomenon 330 was not observed in the current study, an important factor to consider is that piceatannol metabolism 331 is mediated in parallel by glucuronidation and methylation enzymes, which increase the absorption 332 rate. The mechanism underlying the interaction with the oral absorption rate and the associated 333 metabolic pathways were not fully investigated, and this will need to be more studied in detail in the 334 future. We observed substantial isorhapontigenin levels in the plasma that increased by αCD 335 inclusion formulations compared with that of the control. Isorhapontigenin has antioxidant, 336 anti-arteriosclerotic, anticancer effects, and induces SIRT1 expression in human monocytic cell 337 lines

34-36

. When administering αCD-PICs, it can be expected that new biological functions will be

338 unveiled because of the considerable production of methylation metabolites observed, such as 339 isorhapontigenin in plasma, which are not found following the administration of piceatannol alone.



Page 20 of 34 20

340 Oritani et al. examined the influence of piceatannol and isorhapontigenin on fasting blood-glucose 341 concentrations and glucose-tolerance test results by administering them intravascularly to freely 342 moving, healthy rats

37

. In their study, it was reported that intravascularly administered piceatannol

343 reduced both fasting blood-glucose concentrations and glucose tolerance, but that isorhapontigenin 344 did not. Thus, αCD-PICs administration was considered important for ascertaining its physiological 345 activity. In this study, we were concerned that the dose-dependency of αCD was uncertain due to a 346 disagreement between the in vivo absorption study and in vitro phase solubility study. A detailed 347 examination will be necessary to study the effects of αCD and various inclusion conditions for 348 piceatannol in order to increase the internal absorption more effectively. 349 In conclusion, our results revealed that the intragastric administration of αCD-inclusion complexes 350 with piceatannol in rats increased the Cmax of intact piceatannol and total stilbene derivatives, as 351 well as the Tmax of O-methyl-conjugated piceatannol. αCD markedly improved the solubility of 352 piceatannol in acidic artificial gastric juice and the recovery percentages of total stilbene derivatives 353 from the lower small intestine, suggesting that αCD-PICs may show enhanced therapeutic activities 354 in dietary supplements. This is because of the increased chance of piceatannol reaching therapeutic 355 concentrations in biological tissues and the considerable methylation metabolites observed, such as 356 isorhapontigenin in plasma, which are not observed following administration of piceatannol alone. 357

358 Abbreviations used


Page 21 of 34


359 αCD, alpha-cyclodextrin; AUC, area under the concentration-time curve; CD, cyclodextrin; Cmax, 360 maximum compound concentration; CMC, carboxymethyl cellulose sodium salt; HPLC, 361 high-performance

liquid chromatography; PICs, piceatannol inclusion complexes; Tmax,

362 time-to-maximum blood concentration 363

364 Acknowledgement 365 We would like to thank Editage (www.editage.jp) for English language editing. 366



Page 22 of 34 22

367 Supporting Information 368 Molecular structure of trans-resveratrol (A) and trans-piceatannol (B) (Figure S1). Aqueous 369 piceatannol solubility as a function of an increasing αCD molar ratio in 0.5% CMC at 25 ℃ (Figure 370 S2). Time-dependent changes in piceatannol solubility in artificial gastric juice (Table S1) 371

372 Conflict of Interest 373 The authors declare no competing financial interests. 374

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473 Note of acknowledgement for funding source 474

This work was supported by the Morinaga & CO., Ltd. research fund.

475



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476 Figure Legends 477

478 Figure 1 479 Piceatannol concentrations in the supernatants of artificial gastric juice, 1 h after mixing. The mean 480 ± SD is shown. Different superscripts indicate significant differences between the indicated columns. 481 Differences were considered significant at p < 0.05, as determined by Tukey’s honest significant 482 difference (HSD) method. 483

484 Figure 2 485 Plasma concentration profiles for piceatannol and related metabolites after intragastric piceatannol 486 administration (180 µmol/kg body weight). (A) Intact piceatannol, (B) isorhapontigenin, (C) 487 O-methyl piceatannol conjugates, (D) piceatannol conjugates, and (E) total stilbenes. Data are 488 presented as the mean ± SEM (control group, n = 5; αCD inclusion group, n = 6). 489

490 Figure 3 491 Recovery rate of stilbenes from the gastrointestinal tract. Piceatannol was recovered from the 492 stomach 1 h after administration (A). Piceatannol metabolites were recovered from the duodenum, 493 upper small intestine, or lower small intestine at 1 h after administration (B). Open columns indicate 494 the piceatannol recovery percentages of controls and closed columns indicate the piceatannol


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495 recovery percentages from HαCD inclusion samples. The values shown are the mean ± SEM (n = 3). 496 *p < 0.05, relative to the controls, Student’s t-test



Page 30 of 34 30

Table 1 Plasma AUC, Cmax, and Tmax Values for Piceatannol and its Metabolites After Intragastric Administration of the Indicated αCD-Piceatannol Preparations AUC0-3h piceatannol

isorhapontigenin

O-methyl-conjugates

(µmol/L) 2.5

(h) 0.50

7.1 ± 0.7

8.6 ± 0.9

5.8

0.25

MαCDd

7.0 ± 0.6

9.0 ± 0.7

4.8

0.50

d

LαCD

7.4 ± 0.6

10.1 ± 0.6

5.3

0.25

Control

0.8 ± 0.1

2.4 ± 0.2

0.9

0.25

1.3 ± 0.1

e

2.5 ± 0.2

1.0

0.25

1.4 ± 0.1

e

2.9 ± 0.2

1.1

0.25

f

LαCD

1.2 ± 0.1

2.6 ± 0.2

1.3

0.25

Control

10.8 ± 1.7

19.5 ± 0.6

4.8

1.00

HαCD

12.8 ± 1.1

18.2 ± 1.1

5.8

0.50

MαCD

12.9 ± 0.9

18.7 ± 0.6

6.8

1.00

LαCD

12.6 ± 1.6

20.7 ± 1.6

5.8

1.00

Control

6.9 ± 0.5

16.6 ± 1.1 e

11.1 ± 0.5

e

3.1

4.00

21.6 ± 0.9

f

4.5

1.00

f

5.3

1.00

MαCD

11.7 ± 0.6

21.2 ± 0.9

LαCD

11.0 ± 0.6e

22.3 ± 1.1e

4.5

1.00

Control

23.3 ± 1.5

47.7 ± 2.6

9.8

0.25

e

51.0 ± 2.2

16.3

0.25

e

51.8 ± 1.9

16.8

1.00

e

55.6 ± 2.7

15.4

0.25

HαCD MαCD LαCD

32.3 ± 1.3

33.0 ± 1.8

32.1 ± 1.2

a

The area under the plasma concentration-time curve from 0 to 3 h. The area under the plasma concentration-time curve from 0 to 8 h. c Data are expressed as the mean ± SEM (n = 5). b

d e

Time

HαCDd

HαCD

total stilbene

Cmax

(µmol · h/L) 4.8 ± 0.5

MαCD

(µmol · h/L) 9.1 ± 1.0

b

Controlc

HαCD

conjugates

AUC0-8h a

Data are expressed as the mean ± SEM (n = 6). p < 0.01; fp < 0.05, relative to the control group; Tukey's HSD.


Page 31 of 34


Figure 1



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Figure 2


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Figure 3



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Administration of Piceatannol Complexed with Î±-Cyclodextrin

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