No. 38 Stabilization of Tocotrienols by γ-Cyclodextrin

Abstract

Tocotrienols (T3) are known powerful antioxidants having various beneficial biological effects like antitumor, cholesterol-lowering, and hyaluronic acid production. But their low bioavailability and low stability are critical problems to be solved for oral intake. Consequently we have already reported the bioavailability enhancement of tocotrienols by the complexation with γ-cyclodextrin1). Here, we report on the stabilization of tocotrienols by γ-cyclodextrin complexation.

1) S. Ikeda, T. Uchida, T. Ichikawa, T. Watanabe, Y. Uekaji, D. Nakata, K. Terao, T. Yano, Complexation of Tocotrienol with γ-cyclodextrin Enhances Intestinal Absorption of Tocotrienol in Rats, Biosci. Biotechnol. Biochem., 74 (7), 1452-1457 (2010).

Intestinal Absorption of Tocotrienol in Rats

Samples

○ : T3 mixture
● : T3 mixture +γ-CD physical mixture
× : T3 mixture / γ-CD complex
(Each sample was distilled water containing 200mg of triolein, 200mg of sodium taurocholate, 50mg of albumin , and 13.9mg of T3 mixture.)

Animals

Male Wistar rats (6 weeks old) were maintained at 24℃ with a 12 h light dark cycle (lights on from 8:00 to 20:00).
Before the start of experiments, the rats were fed a vitamin E-free diet for 4 weeks.
Each 1 mL of above samples was administered to rats by an oral gavage.

γT3 concentration after oral administration

A : In the Jejunum, Plasma, and Liver at 1 and 3 hours.
B : In the Plasma and Tissues at 3 hours.

1) S. Ikeda, T. Uchida, T. Ichikawa, T. Watanabe, Y. Uekaji, D. Nakata, K. Terao, T. Yano, Complexation of Tocotrienol with γ-cyclodextrin Enhances, Biosci. Biotechnol. Biochem., 74 (7), 1452-1457 (2010).

In this work

We reported that the complexation of tocotrienol with γ-CD enhanced γT3 bioavailability in rat after oral adminixtration. This is supposed by improving its stability in the gastrointestinal tract.
In this work, the following three points were studied.

• Stability comparison of αT0 and γT3 after complexation by CDs
• Method for complexation of high oil content
• Evaluate of oil contents to be complexed and Stability

Samples

• T3 oil : Oryzatcotrienol from Oryza Oil & Fat Chemical Co., Ltd (Total T0 and T3 : 82.6%、Total T3 : 41.4%、αT3 : 14.1%、γT3 : 24.7%)
• γ-CD : CAVAMAXR W8 FOOD from Wacker Chemie AG
• MCC : VIVAPUR101 from Riverson & Co., Ltd

HPLC condition of T3 oil

Column : SSCPAK PEGASIL Silica SP100 (4.6 mm I.D. x 150 mm)
Mobile Phase : Hexane : Dioxane : IPA = 496 : 7.9 : 3
Flow Rate : 1.0 mL/min
Temperature : 40℃
Detection : UV 295 nm
Diluent : Hexane

Preparation of complex

After heating, the mixture was homogeneous suspension.
Obtained powder by Freeze drying was white and highly fluid.
Mixture of oil and MCC was brown and sticky.

Stability of αT0 and γT3

Each sample in test tubes was heated at 140℃ for 30, 60, 90, 120 min.
After heating, each sample was extracted by hexane and the content of αT0 and γT3 was analyzed by HPLC.

αT0 and γT3 were stabilized by complexation with γ-CD.
γT3 was more stabilized than αT0

Compare the effect of α, β, and γ-CD

Each complex sample in test tubes was heated at 140℃ for 120 min.
After heating, each sample was extracted by hexane and the content of αT0 and γT3 was analyzed by HPLC.

Stabilization effect of α-CD complex was little.
β-CD complex and γ-CD complex stabilized γT3 and αT0.

Compare the effect of oil content

When mixed with high concentrations of oil, the suspension could not be homogenized by above method.
But more than 20 minutes at 80℃, homogeneous suspension and powder was obtained.

Stability of various oil content with γ-CD or MCC was evaluated.
Each sample was heated at 140℃ for 120 min, and extracted by hexane.
And then the content of αT0 and γT3 was analyzed by HPLC.

The stability of the higher oil content was reduced.
γ-CD complexes more stabilized γT3 and αT0 than MCC mixture under 35% oil content.

Stability of γ-CD complex in various conditions

• 1g of γ-CD complex powder was put into either polyethylene or aluminum bag, and stored in 40℃・75%RH
• 1g suspension of γ-CD complexes was put into vial, and stored at 4℃

Each sample was extracted by hexane and the content of αT0 and γT3 was analyzed by HPLC.

αT0 and γT3 was stable by complexation with γ-CD after 2 months.

Conclusion

• Stability of αT0 and γT3 was enhanced with CDs in the order: γ-CD > β-CD > α-CD.
• Especially, the stability of γT3 was more improved than αT0 by complexation with γ-CD.