Complexation of Tocotrienol with \gamma-Cyclodextrin ... - J-Stage

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oral administration of vitamin E.20) This indicates that the plasma vitamin E of the Triton-treated rats after oral administration is nearly equivalent to the amount.

Biosci. Biotechnol. Biochem., 74 (7), 1452–1457, 2010

Complexation of Tocotrienol with -Cyclodextrin Enhances Intestinal Absorption of Tocotrienol in Rats Saiko I KEDA,1; y Tomono U CHIDA,1 Tomio I CHIKAWA,1 Takashi WATANABE,2 Yukiko U EKAJI,3 Daisuke N AKATA,3 Keiji T ERAO,3 and Tomohiro Y ANO4 1

Department of Nutritional Sciences, Nagoya University of Arts and Sciences, Nissin 470-0196, Japan Oryza Oil & Fat Chemical Co., Ltd., Ichinomiya 493-8001, Japan 3 CycloChem Co., Ltd., KIBC, Kobe 650-0047, Japan 4 Project for Complementary Medicine, National Institute of Health and Nutrition, Tokyo 162-8636, Japan 2

Received February 24, 2010; Accepted April 20, 2010; Online Publication, July 7, 2010 [doi:10.1271/bbb.100137]

To determine the bioavailability of tocotrienol complex with -cyclodextrin, the effects of tocotrienol/

-cyclodextrin complex on tocotrienol concentration in rat plasma and tissues were studied. Rats were administered by oral gavage an emulsion containing tocotrienol, tocotrienol with -cyclodextrin, or tocotrienol/

-cyclodextrin complex. At 3 h after administration, the plasma -tocotrienol concentration of the rats administered tocotrienol/ -cyclodextrin complex was higher than that of the rats administered tocotrienol and

-cyclodextrin. In order to determine the effect of complexation on tocotrienol absorption, rats were injected with Triton WR1339, which prevents the catabolism of triacylglycerol-rich lipoprotein by lipoprotein lipase, and then administered by oral gavage an emulsion containing tocotrienol, tocotrienol with cyclodextrin, or tocotrienol/ -cyclodextrin complex. The plasma -tocotrienol concentration of the Tritontreated rats administered tocotrienol/ -cyclodextrin complex was higher than that of the other Tritontreated rats. These results suggest that complexation of tocotrienol with -cyclodextrin elevates plasma and tissue tocotrienol concentrations by enhancing intestinal absorption. Key words:

cyclodextrin; tocotrienol; vitamin E

Vitamin E is a potent fat-soluble antioxidant that inhibits lipid peroxidation in biological membranes. In nature, compounds with vitamin E activity are -, -, -, and -tocopherol, which have a saturated phytyl chain, and -, -, -, and -tocotrienol, which have an unsaturated phytyl chain. Tocotrienol has been suggested to have some beneficial biological effects, including antioxidative,1,2) anticancer,3,4) antiatherosclerotic,5,6) and neuroprotective,7,8) but the tocotrienol concentration in the plasma and various tissues is extremely low compared with -tocopherol. We have reported that - and -tocotrienol levels fell below measurable limits in the plasma and some tissues of rats fed a diet containing 50 mg of -tocopherol, 78 mg of -tocotrienol, and 109 mg of -tocotrienol for 8 weeks, in contrast to high levels of -tocopherol.9)

The reasons for the low levels of tocotrienol include the following: First, tocotrienol is present only in limited plant sources, palm oil and rice bran, and daily foods are low in it.10) Sookwong et al.11) reported recently that the daily intake of tocotrienol of the Japanese population is estimated to be 2 mg/d, rather low compared with the intake of -tocopherol. Secondly, the affinity of tocotrienol for -tocopherol transfer protein (-TTP) is lower than that of -tocopherol.12) -TTP has a critical role in the maintenance of vitamin E levels in the blood and tissues through hepatic intracellular transport of vitamin E.13,14) Thus, among vitamin E isoforms, tocopherol is preferentially transported to VLDL from the liver and subsequently transported to the various tissues. Thirdly, the affinity of tocotrienol for cytochrome P450 (CYP) 4F2 is higher than that of -tocopherol.15) CYP4F2 is known as leukotriene B4 !-hydroxylase,16) and catalyzes vitamin E !-hydroxylation, the first and late-limiting step of vitamin E catabolism to carboxyethylhydroxychroman (CEHC), a phytyl chain-shortened metabolite.17) Thus tocotrienol is preferentially catabolized to CEHC in the liver and is secreted into the bile and urine. Finally, tocotrienol is likely to be poorly absorbed in the intestine as compared with -tocopherol. We have reported that Triton WR1339 (Triton), an inhibitor of the catabolism of triacylglycerol-rich lipoprotein by lipoprotein lipase,18,19) completely inhibited vitamin E transport to the liver and caused its accumulation in the plasma of rats after oral administration of vitamin E.20) This indicates that the plasma vitamin E of the Triton-treated rats after oral administration is nearly equivalent to the amount absorbed in vivo. The plasma - and -tocotrienol concentrations of the Triton-treated rats after tocotrienol administration were much lower than the -tocopherol concentration after -tocopherol administration,20) suggesting poor absorption of tocotrienol. -, -, and -cyclodextrin, constituted of 6, 7, and 8 glucopyranoside units, respectively, can form an inclusion complex with hydrophobic molecules, and is widely used in the pharmaceutical and chemical industries. The formation of the inclusion compounds greatly modifies the physical and chemical properties of the

y To whom correspondence should be addressed. Fax: +81-561-73-8539; E-mail: [email protected] Abbreviations: CEHC, carboxyethylhydroxychroman; CYP, cytochrome P450; Triton, Triton WR1339; -TTP, -tocopherol transfer protein

Bioavailability of Tocotrienol/-Cyclodextrin Complex Table 1. Vitamin E Isoform Compositions of the Tocotrienol Complex and the Tocotrienol Mixture Used for Complexation Vitamin E isoform

Tocotrienol/-Cyclodextrin complex

-Tocopherol -Tocopherol -Tocopherol -Tocopherol -Tocotrienol -Tocotrienol -Tocotrienol -Tocotrienol

0.09 0.05 0.55 0.09 0.30 0.07 12.04 1.32

Table 2. Compositions of Diets Component

Tocotrienol mixture

% 0.5 0.4 2.8 0.5 1.8 0.4 65.0 4.6

guest molecule, and in particular water solubility. In contrast to - and -cyclodextrin, -cyclodextrin is completely digested by salivary and pancreatic amylase.21) Terao et al.22) reported recently that complexation of coenzyme Q10 with -cyclodextrin elevated the plasma coenzyme Q10 concentration in healthy adults after a single dose of a capsule containing coenzyme Q10. This suggests that complexation of fat-soluble vitamin with -cyclodextrin improves its bioavailability. The aim of the present study was to clarify the effects of complexation of tocotrienol with -cyclodextrin on tocotrienol concentrations in rat plasma and tissues. The enhancing effect of complexation with -cyclodextrin on the in vivo absorption of tocotrienol was also studied using Triton-treated rats.

Materials and Methods Preparation of tocotrienol/-cyclodextrin complex. The tocotrienol mixture extracted from rice bran was used for complexation with -cyclodextrin. The tocotrienol mixture (96 g) was put in a beaker, and 680 ml of deionized water and 383.5 g of -cyclodextrin (CAVAMAX W8 Food, Wacker Chemical, Adrian, MI, USA) were added. The suspension was mixed using a disperser (T25 Digital UltraTurrax , IKA Japan, Nara, Japan) at 6,000 rpm for 10 min, followed by 12,000 rpm for 5 min, and then was incubated at room temperature for 18 h under a nitrogen atmosphere in the dark. After freezing overnight, the emulsified paste was lyophilized with a Freeze Dryer (EYELA FD-1000, Tokyo Rikakikai Co., Tokyo, Japan) and used for this study as tocotrienol complex with -cyclodextrin (tocotrienol/ -cyclodextrin complex). The vitamin E isoform composition of both the tocotrienol mixture and the tocotrienol/-cyclodextrin complex is shown in Table 1. The percentages of total vitamin E content in the tocotrienol mixture and of tocotrienol/-cyclodextrin complex were 76.0% and 14.5% respectively, and the major vitamin E isoform was -tocotrienol.

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Casein1 L-Cystine Mineral mixture2 Vitamin mixture3 Choline Corn oil4 Cellulose powder Sucrose Cornstarch Tocotrienol mixture -Cyclodextrin Tocotrienol complex

Vitamin E free

T3

200.0 3.0 35.0 10.0 2.5 70.0 50.0 100.0 529.5

200.0 3.0 35.0 10.0 2.5 70.0 50.0 100.0 529.3

— — —

154 — —

T3 þ CD g/kg 200.0 3.0 35.0 10.0 2.5 70.0 50.0 100.0 528.6

mg/kg 154 711 —

Complex

200.0 3.0 35.0 10.0 2.5 70.0 50.0 100.0 528.7 — — 831

1

Vitamin-free casein (Wako Pure Chemicals, Osaka, Japan) AIN93-MX23) 3 Vitamin E-free AIN93-VX23) 4 Vitamin E-stripped corn oil (Funabashi Farm, Chiba, Japan) 2

contents in the emulsions of the T3 þ CD and complex groups were equal. All the emulsions also contained 200 mg of triolein, 200 mg of sodium taurocholate, and 50 mg of albumin, and were freshly prepared just before administration. The rats were deprived of food for 12 h until oral administration of the emulsion. At 1 and 3 h after oral administration, eight rats of each group were anesthetized with diethyl ether, and blood was drawn from the heart using a heparinized needle and syringe. The upper one-third section (about 25 cm) of the small intestine between the stomach and cecum was taken, and the upper 5 cm was cut off. The remaining section was sampled as jejunum and washed twice with 10 ml of saline using a glass syringe. The fluid was removed from the jejunum with paper and stored at 80  C. Experiment 2. Rats (mean weight 278 g) were administered an oral gavage of 1 ml of emulsion containing 13.9 mg of tocotrienol mixture (T3 group, n ¼ 5), 13.9 mg of tocotrienol mixture and 62.2 mg of -cyclodextrin (T3 þ CD group, n ¼ 5), or 72.8 mg of tocotrienol/ -cyclodextrin complex (complex group, n ¼ 5). The tocotrienol contents of all the emulsions were equal, and the -cyclodextrin contents in the emulsions of the T3 þ CD and the complex group were equal. All the emulsions also contained 200 mg of triolein, 200 mg of sodium taurocholate, and 50 mg of albumin. Another group of rats (mean weight 278 g) was anesthetized with diethyl ether and injected with Triton (0.2 g/kg of body weight, Sigma-Aldrich Japan, Tokyo, Japan) into a caudal vein and, after 10 min, administered an oral gavage of the emulsion containing the tocotrienol mixture, the tocotrienol mixture and -cyclodextrin, or the tocotrienol/-cyclodextrin complex (n ¼ 7 for each group). The rats were deprived of food for 12 h until oral administration of the emulsion. At 6 h after oral administration of tocotrienol, the rats were anesthetized with diethyl ether, and blood was drawn from the heart using a heparinized needle and syringe.

Animals and diets. Male Wistar rats (6 weeks old) were purchased from Japan SLC (Shizuoka, Japan) and maintained at 24  C with a 12-h light dark cycle (lights on from 08:00 to 20:00). Before the start of experiments 1 and 2, the rats were fed a vitamin E-free diet (Table 2) for 4 weeks to deplete tissue -tocopherol stores, because it is difficult to elevate the tocotrienol concentration in -tocopherol-rich tissues. This study was approved by the Laboratory Animal Care Committee of Nagoya University of Arts and Sciences, and all procedures were performed in accordance with the Animal Experimentation Guidelines of Nagoya University of Arts and Sciences.

Experiment 3. Rats were fed a diet containing 154 mg of tocotrienol mixture/kg (T3 group, n ¼ 8), 154 mg of tocotrienol mixture/kg and 711 mg of -cyclodextrin/kg (T3 þ CD group, n ¼ 8), or 831 mg of tocotrienol/-cyclodextrin complex/kg (complex group, n ¼ 8) for 6 weeks. The compositions of the diets are shown in Table 2. The tocotrienol contents in all the diets were equal, and the -cyclodextrin contents in the diet of the T3 þ CD and complex groups were equal. After deprivation for 12 h, the rats were killed by decapitation and tissues were sampled and handled as described for experiment 1.

Experiment 1. Rats (mean weight 255 g) were administered an oral gavage of 1 ml of emulsion containing 13.9 mg of tocotrienol mixture (T3 group, n ¼ 16), 13.9 mg of tocotrienol mixture and 62.2 mg of -cyclodextrin (T3 þ CD group, n ¼ 16), or 72.8 mg of tocotrienol/ -cyclodextrin complex (complex group, n ¼ 16). The tocotrienol contents in all the emulsions were equal, and the -cyclodextrin

Tocotrienol concentration. Tissue homogenate (0.5 ml) was inserted into a test tube, and 0.5 ml of ethanol containing 60 g/l of pyrogallol and 0.45 mg of 2,2,5,7,8-pentamethyl-6-chroman as an internal standard were added. Then 0.1 ml of 600 g/l potassium hydroxide was added and this was saponified at 70  C for 30 min. After the addition of 2.25 ml of 20 g/l of sodium chloride, tocotrienol was extracted with

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Tocotrienol complex 0 (min) 60

γ -Cyclodextrin 0

60

H 2O

H2O+TG

Emulsion

Fig. 1. Emulsification Test of the Tocotrienol Complex. Each solution in a test tube was mixed using a Vortex mixer for 1 min and left for 60 min at room temperature. H2 O, distilled water containing 72.8 mg/ml of tocotrienol/-cyclodextrin complex or 62.2 mg/ml of -cyclodextrin; H2 O þ TG, distilled water containing 200 mg/ml of triolein with 72.8 mg/ml of tocotrienol/-cyclodextrin complex or 62.2 mg/ml of -cyclodextrin; Emulsion, distilled water containing 200 mg/ml of triolein, 200 mg/ml of sodium taurocholate, and 50 mg/ml of albumin with 72.8 mg/ml of tocotrienol/-cyclodextrin complex or 62.2 mg/ml of -cyclodextrin. 0.5 ml of hexane containing 10% v/v ethyl acetate. Serum (75 ml) was inserted into a test tube, and 90 ng of 2,2,5,7,8-pentamethyl-6-chroman was added as an internal standard. After the addition of 0.5 ml of water and 1.0 ml of ethanol, tocotrienol was extracted with 5 ml of hexane. The tocotrienol concentration was determined by HPLC.24) The instrumentation used for HPLC was a Shimadzu LC-10AD (Shimadzu, Kyoto, Japan) with a Shimadzu RF-10AXL fluorescence detector (excitation 298 nm, emission 325 nm). The analytical column used was a Wakosil 5SIL (4:6  250 mm, Wako Pure Chemical Industries, Osaka, Japan). The mobile phase was hexane containing 1% v/v dioxane and 0.2% v/v isopropyl alcohol at a flow rate of 1 ml/min. Triacylglycerol concentration. The plasma triacylglycerol concentration was measured with Triglyceride E-Test Wako (Wako Pure Chemical Industries, Osaka, Japan). Statistical analysis. Data are presented as means  SEM, n ¼ 8 (experiments 1 and 3), 5, or 7 (experiment 2). They were analyzed by one-way ANOVA (experiments 1 and 3) or two-way ANOVA (experiment 2) by Tukey’s post-hoc test (Graph Pad Prism for Windows version 4.0, GraphPad Software, La Jolla, CA, USA). Differences were regarded as significant at p < 0:05.

Results Tocotrienol/-cyclodextrin complex is a white powder although tocotrienol is an oily liquid. Tocotrienol/ -cyclodextrin complex dispersed in water after mixing for 1 min using a Vortex mixer, and was poorly soluble in water (Fig. 1). The addition of triolein, sodium taurocholate, and albumin emulsified the complex solution, and the emulsion was stable and homogeneous for more than 1 h. The emulsion was used for oral administration to the complex group in experiments 1 and 2. Emulsion for oral administration to the T3 þ CD group in experiments 1 and 2 was similar in appearance to the emulsion containing -cyclodextrin (Fig. 1), because tocotrienol was soluble in triolein. The emulsified solution of T3 þ CD was homogenous just after mixing but divided into lipid and aqueous layers at 60 min.

The -tocotrienol concentration in the jejunum and plasma of the complex group at 3 h after oral administration was lower than that at 1 h, and that in the liver at 3 h was higher than that at 1 h in experiment 1 (Fig. 2A). At 3 h, the -tocotrienol concentrations in the tissues and plasma of the complex group were higher than that of the T3 group, although the concentrations in the jejunum, plasma, liver, spleen, and heart of the T3 and T3 þ CD groups did not differ (Fig. 2B). The -tocotrienol concentration in the plasma and heart of the complex group was higher than that of the T3 þ CD group. To determine the effects of complexation of tocotrienol with -cyclodextrin on intestinal absorption of -tocotrienol, the plasma -tocotrienol concentration of the rats treated by Triton injection was measured in experiment 2. Triton treatment elevated the plasma triacylglycerol concentrations of all groups after oral administration of the emulsion containing triolein (Fig. 3), suggesting that the triacylglycerol from the emulsion accumulated in the blood due to inhibition of triacylglycerol-rich lipoprotein catabolism. The plasma -tocotrienol concentration of the Triton-treated complex group was higher than that of the Triton-treated T3 and T3 þ CD groups, in contrast to the equal triacylglycerol concentrations of the three groups. In experiment 3, we determined whether dietary intake of tocotrienol/-cyclodextrin complex for 6 weeks would elevate the -tocotrienol concentration in the rat tissues. Dietary intake of neither tocotrienol/ -cyclodextrin complex nor -cyclodextrin affected the body or tissue weights of the rats (Table 3). Although the major vitamin E isoform in all the diets was tocotrienol, the -tocotrienol concentration in the serum, liver, jejunum, heart, muscle, and testis of all the groups was much lower than the various -tocopherol concentrations (Table 4). The serum -tocotrienol concentra-

Bioavailability of Tocotrienol/-Cyclodextrin Complex

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A γ -Tocotrienol (nmol/ml)

γ -Tocotrienol (nmol/g)

Plasma

800 600 400 200

Liver γ -Tocotrienol (nmol/g)

Jejunum

1,000

30 20

10

0

40 30 20 10

0

1

0

3

1

Time (h)

3

1

Time (h)

3 Time (h)

b

400 a

0

a a

200

Kidney

b b

100

4 2

T3+CD Complex

T3

a

0

T3+CD Complex

Spleen

b ab

a

50 0

0

b ab

10

150

a

Liver

20

5

T3

γ -Tocotrienol (nmol/g)

γ -Tocotrienol (nmol/g)

30

10

0

8 6

50 40

15

T3+CD Complex

T3

10 1

b

γ -Tocotrienol (nmol/g)

200

ab

Plasma

γ -Tocotrienol (nmol/g)

20

Jejunum 600

γ -Tocotrienol (nmol/ml)

γ -Tocotrienol (nmol/g)

B

T3

60

Heart

40

a

T3+CD Complex

b

a

20

0

T3

T3+CD Complex

T3

T3+CD Complex

Fig. 2. -Tocotrienol Concentrations in the Jejunum, Plasma, and Liver at 1 and 3 h (A), and in the Plasma and Tissues at 3 h after Oral Administration of Tocotrienol (B) in Experiment 1. Rats were administered tocotrienol mixture ( , T3), tocotrienol mixture and -cyclodextrin ( , T3 þ CD), or tocotrienol/-cyclodextrin complex (, Complex). Values are means  SEM, n ¼ 8. Means not sharing a letter differ, p < 0:05.

Triacylglycerol (mg/100 ml)

Plasma triacylglycerol b

4,000

b

b

T3 T3+CD Complex

3,000

Table 3. Body and Relative Tissue Weights of Rats Fed Diets Containing Tocotrienol Mixture (T3), Tocotrienol Mixture and Cyclodextrin (T3 þ CD), or Tocotrienol/-Cyclodextrin Complex (Complex) for 6 Weeks in Experiment 3 T3

2,000

1,000

a

a

Initial body weight Final body weight

a

0

Control

Triton

Relative liver weight Relative spleen weight

γ -Tocotrienol (nmol/ml)

Plasma γ-tocotrienol 40

b

T3 þ CD

Complex

g 144  3 144  3 144  4 269  6 276  6 270  6 g/kg body weight 24:7  0:3 24:2  0:2 24:8  0:3 2:2  0:1 2:3  0:1 2:1  0:05

Values are mean  SEM, n ¼ 8.

30 20

a 10

a

a

a

a

0

Control

Triton

Fig. 3. Effects of Triton on Plasma Triacylglycerol and -Tocotrienol Concentrations after Oral Administration of Emulsion Containing Triolein and Tocotrienol in Experiment 2. Rats were administered the tocotrienol mixture (T3), the tocotrienol mixture and -cyclodextrin (T3 þ CD), or the tocotrienol/-cyclodextrin complex (Complex). Values are means þ SEM, n ¼ 5 (Control) or 7 (Triton). There were Triton effects (p < 0:0001) on the triacylglycerol concentration and effects of Triton (p < 0:0001), tocotrienol (p < 0:0001), and their interaction (p ¼ 0:0006) on the -tocotrienol concentration. Means not sharing a letter differ, p < 0:05.

tion of the complex group tended to be higher (p < 0:1) than those of the T3 and T3 þ CD groups, but the -tocotrienol concentrations in the various tissues of the complex group were not different from those of the other groups.

Discussion Cyclodextrin forms inclusion complexes with hydrophobic molecules due to the formation of supramolecular complexes with their polymer chains. Cyclodextrin forms pseudorotaxane-like supramolecular complexes with polyethylene glycol25) and coenzyme Q10,26) suggesting that -cyclodextrin also forms pseudorotaxane-like complexes with tocotrienol, including an unsaturated phytyl chain, which is similar in structure to coenzyme Q10. The tocotrienol/-cyclodextrin complex

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Table 4. Vitamin E Concentrations in Sera and Tissues of Rats Fed Diets Containing Tocotrienol Mixture (T3), Tocotrienol Mixture and Cyclodextrin (T3 þ CD), or Tocotrienol/-Cyclodextrin Complex (Complex) for 6 Weeks in Experiment 3 T3 Serum

-Tocotrienol -Tocopherol

0:05  0:01 3:40  0:31

Liver

-Tocotrienol -Tocopherol -Tocotrienol -Tocopherol -Tocotrienol -Tocopherol -Tocotrienol -Tocopherol -Tocotrienol -Tocopherol -Tocotrienol -Tocopherol -Tocotrienol -Tocopherol

1:0  0:5 16:5  0:9 9:9  0:9 46:2  2:3 6:3  0:8 63:9  5:1 4:1  0:4 18:7  1:5 1:9  0:2 56:8  6:7 50:2  7:3 23:9  3:4 35:2  3:2 14:2  2:0

Jejunum Heart Muscle Testis Epididymal fat Perirenal adipose tissue

T3 þ CD nmol/ml serum 0:04  0:01 3:25  0:19 nmol/g tissue 0:6  0:3 15:3  1:0 10:7  1:2 44:1  3:0 6:4  0:5 62:1  2:0 3:7  0:2 17:8  2:2 1:7  0:2 53:1  1:9 56:2  7:9 25:3  3:4 35:9  5:0 12:2  1:6

Complex 0:09  0:02 3:16  0:20 0:6  0:2 14:1  0:5 10:2  0:8 45:2  3:6 6:7  0:5 61:2  2:5 3:7  0:5 14:4  1:5 2:2  0:3 52:6  2:1 42:0  3:4 18:1  1:8 41:0  5:0 11:9  1:5

Values are mean  SEM, n ¼ 8.

was poorly soluble in water, but an emulsion containing the complex, distilled water, and triolein was stable and homogenous for more than 1 h (Fig. 1). In order to determine the bioavailability of the tocotrienol complex with -cyclodextrin, the effects of the tocotrienol/-cyclodextrin complex on the tocotrienol concentrations in rat plasma and tissues were studied. Complexation of tocotrienol with -cyclodextrin elevated the -tocotrienol concentration in the plasma and tissues at 3 h after oral administration of tocotrienol, although simultaneous administration of tocotrienol and -cyclodextrin did not affect the tocotrienol concentration (Fig. 2). Complexation of tocotrienol with -cyclodextrin also elevated the plasma -tocotrienol concentration of the Triton-treated rats (Fig. 3), suggesting that complexation with -cyclodextrin elevated the -tocotrienol concentration in the plasma and tissues by enhancing intestinal absorption of -tocotrienol. In contrast to - and -cyclodextrin, which are resistant to amylase, -cyclodextrin is completely digested by salivary and pancreatic amylase.21) Therefore the fact that the plasma -tocotrienol concentration of the Triton-treated complex group was higher than that of the other Triton-treated groups although the dose of tocotrienol in experiment 2 was the same suggests improvement of the solubility and stability of fat-soluble tocotrienol in the gastrointestinal tract due to complexation. The solution containing the tocotrienol/-cyclodextrin complex, distilled water, and triolein was homogeneous and stable for 1 h, in contrast to solution containing -cyclodextrin, distilled water, and triolein (Fig. 1). This suggests that complexation of tocotrienol with -cyclodextrin enhances intestinal absorption of tocotrienol by improving its solubility and stability in the gastrointestinal tract. In experiment 3, rats were fed a diet containing the tocotrienol/-cyclodextrin complex for 6 weeks to determine the effects of dietary intake of the tocotrienol/-cyclodextrin complex on -tocotrienol accumulation in the various tissues. Although high levels of -tocotrienol accumulated in the adipose tissues, as

reported previously,9) the -tocotrienol concentration in the serum and tissues, except for the adipose tissues, was much lower than the -tocopherol concentration (Table 4). Dietary intake of the tocotrienol/ -cyclodextrin complex for 6 weeks did not affect the -tocotrienol concentration in the various tissues, including the adipose tissues. The dietary tocotrienol/ -cyclodextrin complex tended to elevate the serum -tocotrienol concentration (p < 0:1), but the elevation was less effective, because the serum concentration was lower than 0.1 nmol/ml. This suggests that complexation of tocotrienol with -cyclodextrin enhances intestinal absorption of tocotrienol and elevates the tocotrienol concentration in the plasma and tissues for a few hours after administration, and that tissue accumulation of tocotrienol is less regulated by its intestinal absorption. The dietary vitamin E isoform is absorbed in the small intestine and transported to the liver. -TTP in the liver cytosol catalyzes vitamin E transport to cell membrane, and vitamin E is subsequently transported to the various tissues by VLDL.27) Because of the high affinity of -tocopherol for -TTP,12) -tocopherol, among the vitamin E isoforms, is preferentially transported to the various tissues. The other isoforms and excess amounts of -tocopherol are catabolized mainly to CEHC in the liver and are excreted in the urine. The pathway of catabolism involves !-hydroxylation of the phytyl chain and subsequent -oxidation,28) and the rate-limiting step is !-hydroxylation by CYP4F2.17) In comparison with -tocopherol, tocotrienol is preferentially catabolized due to its high affinity for CYP4F2.15) We reported recently that CYP-dependent catabolism of vitamin E was a critical determinant of the vitamin E concentrations in the various tissues.29) Ketoconazole, a CYP inhibitor, clearly increased the tocotrienol concentrations in the various tissues of rats. These results suggest that dietary intake of the tocotrienol/-cyclodextrin complex was less effective at increasing the tissue tocotrienol concentration in experiment 3 because tocotrienol catabolism to CEHC is more important for accumulation of it in the tissues.

Bioavailability of Tocotrienol/-Cyclodextrin Complex

In conclusion, we found that complexation of tocotrienol with -cyclodextrin elevated the -tocotrienol concentration in rat plasma and tissues after oral administration by enhancing intestinal absorption of it, and that dietary intake of tocotrienol/-cyclodextrin complex did not affect the tissue accumulation of -tocotrienol. These results suggest that complexation of tocotrienol with -cyclodextrin enhances tocotrienol bioavailability for several hours after oral administration.

10)

Acknowledgments

14)

This study was supported in part by a Grant-in-Aid for Research KHC1023 on Publicly Essential Drugs and Medical Devices from the Japan Health Sciences Foundation.

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