Recent research finding
No.40 Studies on the selectivity for complex formation of saturated and unsaturated fatty acid triglycerides with α-Cyclodextrin

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Summary

α-Cyclodextrin (αCD) is a beneficial dietary fiber to reduce blood glucose level,neutral fat level and also body weight.

It was reported that αCD administration is effective to increase saturated fats selectively in fecal excretion compared with unsaturated fats1).

Therefore, we investigated the complex formation selectivity of saturated and unsaturated fatty acid triglycerides with αCD.

Tristearin was selectively complexed with αCD from the mixture of tristearin and triolein.

Fig.1 Structures of tristearin and triolein
A; Tristearin (TS)   B; Triolein (TO)

1) D. Gallaher, C. Gallaher, D. Plank, Alpha-cyclodextrin selectively increases fecal excretion of saturated fats, FASEB J;21:A730 (2007)

Background

Alpha-Cyclodextrin Selectively Increases Fecal Excretion Of Saturated Fats
Daniel D. Gallaher, Cynthia M. Gallaher, and David W. Plank

Experimental diet

Male Wistar rats, 125-150 g in initial weight, 10 per group, were fed with one of four diets upon arrival. All diets contained 15% fat by weight.
The diets were as follows:

Control - AIN-93G + cellulose 5%
Chitosan - AIN-93G + chitosan 5%
αCD - AIN-93G + αCD 5%
Complexed αCD - AIN-93G + αCD 5%
(lipid and 5%αCD complexaton)

*AIN:American Institute of Nutrition

Experimental design

Rats were fed with their respective diets for 7 days. Then the animals were fasted overnight, and presented with a 5 g meal of their respective diets containing 14C-triolein and 3H-tripalmitin incorporated into the lipid.
A fecal collection was started four hours later, and was continued 12 hours.

Result

Cyclodextrin appears to selectively increase saturated fat excretion.

Fig.2 Ratio of excretion of radiolabeled tripalmitin to triolein

Experimental design following Szejtlis' method2)

2) J. Szejtli, E. Bánky-Előd, Á. Stadler, Enrichment of the unsaturated components in fatty acid ester mixtures by cyclodextrin complex formation, Acta Chimica Academiae Scientiarum Hungaricae, Tomus, 99 (4), 447-452 (1979)

Analytical method

Differential scanning calorimetry (DSC) analysis

DSC analytical condition
equipment: DSC-60 (SHIMADZU)
temperature: 50℃-100℃ (10℃/min)

Triglyceride (TG) analysis

sample preparation

HPLC analytical condition
system: LC-2010C (SHIMADZU)
column: Luna 5μ C18(2) 100 A, 4.6 mm I.D. x 150 mm (Phenomenex)
temperature: 40℃
flow rate: 1 mL/min
mobile phase: A; acetonitrile   B; THF
program:

Time (min) B%
0 20
20 60
22 60
23 20
28 20

Evaporative Light Scattering Detector (ELSD) analytical condition
system: ELSD-LTⅡ(SHIMADZU)
nebulizer gus: N2
N2 pressure: 360 kPa
temperature: 50℃
gain: 11

Pre-test (selection of dispersion solvent for TG)

Purpose
selection of dispersion solvent for TG in aqueous αCD solution
Experiment 1 selection of organic solvent
After adding various organic solvent to 10% aqueous αCD solution, the sample was observed visually.
Experiment 2 TG solubility test by organic solvent
After adding various organic solvent to TS and TO mixture at 70℃, the samples were observed visually.
Results
Only ethyl acetate sample formed clear liquid (Fig.3-A).
Ethyl acetate could dissolve TS and TO mixture at 70℃ (Fig.3-B).

These results showed that ethyl acetate was proved useful.

Fig.3 Organic solvent selection for dispersion of TG
A: selection of organic solvent   B: TG solubility test by organic solvent

Pre-test (selection of washing solvent for TS adhered to TS-αCD)

Purpose
selection of washing solvent for TS adhered to TS-αCD

Experiment
1. TS-αCD(TS 30w/w% contained) was prepared by conventional method.
2. After washing TS-αCD with various organic solvent, the TS-αCD was analyzed using DSC.

Results
DSC analysis of TS-αCD before washing showed endothermic peak derived from the melting point of TS (Fig.4 red line).
The endothermic peak of TS disappeared by THF washing.
And TO was dissolved easily in THF (data not shown).

Fig.4 DSC analysis of washed TS-αCD by various solvent

The result showed that THF has washing capability for TS adhered to TS-αCD.

Pre-test (THF Washing test for TS adhered to TS-αCD)

Purpose
Washing effect with THF on TS adhered to TS-αCD

Experiment
After washing TS-αCD with THF one time or more, the TS-αCD was analyzed using DSC and HPLC-ELSD.

Results
DSC analysis was performed for washing evaluation for TS adhered to TS-αCD. HPLC-ELSD analysis was performed for the evaluation of TS content in TS-αCD. DSC analysis showed that the endothermic peak derived from the melting point of adhered TS disappeared washing with THF 2 times (Fig.5-A). This result means that TS adhered to TS-αCD disappears by washing 2 times.
HPLC-ELSD analysis showed that there was no difference in TS content in TS-αCD by 3 times washing compared with 2 times washing (Fig.5-B).
This result means TS content in TS-αCD comes not from adhered one, but from encapsulated one, that is, no adhered TS remains.

Fig.5 THF washing test for TS-αCD
A: DSC analysis   B: ELSD analysis

The result showed that THF washing remove adhered TS, but does not remove TS encapsulated in αCD.

Complex formation selectivity test for TG with αCD

Purpose
investigating the complex formation selectivity of saturated and unsaturated fatty acid triglycerides with αCD

Experiment
Experimental method was shown in the slide of "Experimental design".
Formulation was as follows (table 1).
sample reaget weight (g) MW mole ratio
A1 TS 0.1 891.48 1.0
TO 0.1 885.43 1.0
αCD 1.0 (dry) 972.84 10.9
A2 TS 0.1 891.48 1.0
TO 0.1 885.43 1.0
αCD 2.0 (dry) 972.84 21.8
A3 TS 0.1 891.48 1.0
TO 0.1 885.43 1.0
αCD 3.0 (dry) 972.84 32.7

Table 1 Formulation of TS, TO and αCD
αCD concentration of 3 samples was different.

Complex formation selectivity test for TG with αCD

Appearance of TG and αCD mixture
(after 18 hours standing at 4℃ during complexation process)
Aggregating and floating material with cream color was thought that it was not yet complexed oil. Precipitated or suspended material was thought to be the complex of oil with αCD.
That floating material was observed for all samples, but that amount of A3 sample was less than other samples(Fig.6). This picture showed complex proceeds with αCD addition.

Fig.6 Appearance of TG and αCD mixture

TG analysis of TG-αCD (A3 sample)
The endothermic peak derived from the melting point of TS disappeared by washing TS-αCD with THF 3 times (data not shown).
After washing, the extract of A3 sample was analyzed using HPLC-ELSD. The content ratio of TS / TO in TG-αCD was higher than that of original mixture (Fig.7).

The result showed that tristearin was selectively complexed with αCD.

Fig.7 TG analysis of extract of A3 sample
1: TO   2: TS

Pre-test
(selection of washing solvent for TS adhered to TS-βCD or TS-γCD)

Purpose
selection of washing solvent for TS adhered to TS-βCD or TS-γCD

Experiment
1. TS-βCD and TS-γCD (TS 30w/w% contained respectively) was prepared by conventional method.
2. After washing each TS-CD with various organic solvent, the TS-CD was analyzed using DSC.

Results
DSC analysis of TS-βCD and TS-γCD before washing showed the endothermic peak derived from the melting point of TS (Fig.8-A, B red line).
The endothermic peak of TS-βCD and TS-γCD disappeared by THF washing.

The result showed that THF have washing capability of TS adhered to TS-βCD and TS-γCD.

Fig.8 DSC analysis of washed TS-βCD and TS-γCD by various solvent
A: TS-βCD   B: TS-γCD

Pre-test
(THF washing test for TS adhered to TS-βCD or TS-γCD)

DSC analysis showed that the endothermic peak derived from the melting point of TS disappeared by THF washing TS-βCD (for 3 times, Fig. 9-A1) and TS-γCD (for 2 times, Fig. 9-B1).
HPLC-ELSD analysis showed that TS content in TS-βCD was reduced by THF washing 3 times or more (Fig.9-A2). But TS content in TS-γCD was not reduced by THF washing 3 times (Fig.9-B2).

Fig.9 THF washing test for TS-βCD and TS-γCD
A-1: DSC analysis of TS-βCD A-2: ELSD analysis of TS-βCD
B-1: DSC analysis of TS-γCD B-2: ELSD analysis of TS-γCD

Complex formation selectivity test for TG with CDs

Purpose
investigating the complex formation selectivity of saturated and unsaturated fatty acid triglycerides with αCD, βCD and γCD

Experiment
Experimental method was shown in the slide of “Experimental design” (except the amount of both water and ethyl acetate used 3 times more).
Formulation was as follows (table 2).
sample reagent weight (g) MW mole ratio
αCD TS 0.15 891.48 1.0
TO 0.15 885.43 1.0
αCD 3 972.84 20.2
βCD TS 0.15 891.48 1.0
TO 0.15 885.43 1.0
βCD 3 972.84 17.3
γCD TS 0.15 891.48 1.0
TO 0.15 885.43 1.0
γCD 3 972.84 15.1

Table 2 Formulation of TS, TO and CDs

Complex formation selectivity test for TG with CDs

Appearance of TG-CD complex
(after 18 hours standing at 4℃ during complexation process)

Floating materials with cream color were observed for all samples, but that amount of CD added samples was less than that of non-CD sample (Fig.10).

Fig.10 Appearance of TG-CD complex preparation

TG analysis of TG-CD
The endothermic peak derived from the melting point of TS disappeared by THF washing each TS-CD (number of washing: αCD was 7 times, βCD was 4 times γCD was 4 times, data not shown).
After washing, the extract of each samples were analyzed using HPLC-ELSD. The content ratio of TS / TO in TG-αCD or TG-γCD was higher than that of original mixture (Fig. 11).

The result showed that tristearin was selectively complexed with αCD and γCD.

Fig.11 TG analysis of extract of TG-CD
1: TO   2: TS

Conclusion

Tristearin was selectively complexed with αCD from the mixture of tristearin and triolein.