No. 40 Studies on the selectivity for complex formation of saturated and unsaturated fatty acid triglycerides with α-Cyclodextrin
Summary
α-Cyclodextrin (α-CD) is a beneficial dietary fiber to reduce blood glucose level,neutral fat level and also body weight.
It was reported that α-CD 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 α-CD.
Tristearin was selectively complexed with α-CD from the mixture of tristearin and triolein.
![Fig. 1. Structures of tristearin and triolein](img/research_e_040_01.png)
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%
- α-CD - AIN-93G + α-CD 5%
- Complexed α-CD - AIN-93G + α-CD 5% (lipid and 5%α-CD 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](img/research_e_040_02.png)
Experimental design following Szejtlis' method2)
![](img/research_e_040_03.png)
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 :
Evaporative Light Scattering Detector (ELSD) analytical condition
system : ELSD-LT II (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 α-CD solution
Experiment 1 selection of organic solvent
After adding various organic solvent to 10% aqueous α-CD 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](img/research_e_040_06.jpg)
A : selection of organic solvent / B : TG solubility test by organic solvent
Pre-test (selection of washing solvent for TS adhered to TS-α-CD)
Purpose
selection of washing solvent for TS adhered to TS-α-CD
Experiment
- TS-α-CD(TS 30w/w% contained) was prepared by conventional method.
- After washing TS-α-CD with various organic solvent, the TS-α-CD was analyzed using DSC.
Results
- DSC analysis of TS-α-CD 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-α-CD by various solvent](img/research_e_040_07.png)
The result showed that THF has washing capability for TS adhered to TS-α-CD.
Pre-test (THF Washing test for TS adhered to TS-α-CD)
Purpose
Washing effect with THF on TS adhered to TS-α-CD
Experiment
After washing TS-α-CD with THF one time or more, the TS-α-CD was analyzed using DSC and HPLC-ELSD.
Results
- DSC analysis was performed for washing evaluation for TS adhered to TS-α-CD. HPLC-ELSD analysis was performed for the evaluation of TS content in TS-α-CD. 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-α-CD disappears by washing 2 times.
- HPLC-ELSD analysis showed that there was no difference in TS content in TS-α-CD by 3 times washing compared with 2 times washing (Fig. 5-B).
- This result means TS content in TS-α-CD comes not from adhered one, but from encapsulated one, that is, no adhered TS remains.
![Fig. 5. THF washing test for TS-α-CD](img/research_e_040_08.png)
The result showed that THF washing remove adhered TS, but does not remove TS encapsulated in α-CD.
Complex formation selectivity test for TG with α-CD
Purpose
investigating the complex formation selectivity of saturated and unsaturated fatty acid triglycerides with α-CD
Experiment
- Experimental method was shown in the slide of "Experimental design".
- Formulation was as follows (table 1).
![Table 1. Formulation of TS, TO and α-CD](img/research_e_040_09.png)
α-CD concentration of 3 samples was different.
Appearance of TG and α-CD 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 α-CD.
- 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 α-CD addition.
![Fig. 6. Appearance of TG and α-CD mixture](img/research_e_040_10.jpg)
TG analysis of TG-α-CD (A3 sample)
- The endothermic peak derived from the melting point of TS disappeared by washing TS-α-CD 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-α-CD was higher than that of original mixture (Fig. 7).
The result showed that tristearin was selectively complexed with α-CD.
![Fig. 7. TG analysis of extract of A3 sample](img/research_e_040_11.png)
1 : TO / 2 : TS
Pre-test
(selection of washing solvent for TS adhered to TS-β-CD or TS-γ-CD)
Purpose
selection of washing solvent for TS adhered to TS-β-CD or TS-γ-CD
Experiment
- TS-β-CD and TS-γ-CD (TS 30w/w% contained respectively) was prepared by conventional method.
- After washing each TS-CD with various organic solvent, the TS-CD was analyzed using DSC.
Results
- DSC analysis of TS-β-CD and TS-γ-CD before washing showed the endothermic peak derived from the melting point of TS (Fig.8-A, B red line).
- The endothermic peak of TS-β-CD and TS-γ-CD disappeared by THF washing.
The result showed that THF have washing capability of TS adhered to TS-β-CD and TS-γ-CD.
![Fig. 8. DSC analysis of washed TS-β-CD and TS-γ-CD by various solvent](img/research_e_040_12.png)
A : TS-β-CD / B : TS-γ-CD
Pre-test
(THF washing test for TS adhered to TS-β-CD or TS-γ-CD)
- DSC analysis showed that the endothermic peak derived from the melting point of TS disappeared by THF washing TS-β-CD (for 3 times, Fig. 9-A1) and TS-γ-CD (for 2 times, Fig. 9-B1).
- HPLC-ELSD analysis showed that TS content in TS-β-CD was reduced by THF washing 3 times or more (Fig. 9-A2). But TS content in TS-γ-CD was not reduced by THF washing 3 times (Fig. 9-B2).
![Fig. 9. THF washing test for TS-β-CD and TS-γ-CD](img/research_e_040_13.png)
A-1 : DSC analysis of TS-β-CD / A-2 : ELSD analysis of TS-β-CD
B-1 : DSC analysis of TS-γ-CD / B-2 : ELSD analysis of TS-γ-CD
Complex formation selectivity test for TG with CDs
Purpose
investigating the complex formation selectivity of saturated and unsaturated fatty acid triglycerides with α-CD, β-CD and γ-CD
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).
![Table 2. Formulation of TS, TO and CDs](img/research_e_040_14.png)
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](img/research_e_040_15.jpg)
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: α-CD was 7 times, β-CD was 4 times γ-CD 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-α-CD or TG-γ-CD was higher than that of original mixture (Fig. 11).
The result showed that tristearin was selectively complexed with α-CD and γ-CD.
![Fig. 11. TG analysis of extract of TG-CD](img/research_e_040_16.png)
1 : TO / 2 : TS
Conclusion
Tristearin was selectively complexed with α-CD from the mixture of tristearin and triolein.