| dc.description.abstract | ABSTRACT
The curcuminoids belong to the group diarylheptanoids and are the colouring principles found in the rhizomes of the plant Curcuma longa L. (Zingiberaceae). The curcuminoids have shown unique properties and many pharmacological effects. There is a great interest in their antioxidant effect and also in their anticancer effect. They are also non toxic in high doses. Because of this, they have a great potential as future drugs or as model substances for treatment of various diseases.
Before this can be reality one has to make a useful pharmaceutical formulation of the curcuminoids. Unfortunately there have been some difficulties doing this. The curcuminoids have very low water solubility, especially at acidic pH. They are also quite unstable, susceptible to both hydrolysis at neutral to alkaline pH and to photochemical degradation. This might also explain their low bioavailability.
Cyclodextrins (CDs) are cyclic oligosaccharides, containing a relative hydrophobic central cavity and a hydrophilic outer surface. They can increase the aqueous solubility of compounds by forming inclusion complexes. It has also been shown that they can increase solubility by forming non-inclusion complexes.
HydroxypropylãCD (HPãCD) and hydroxypropylâCD (HPâCD) are reported to be fairly non-toxic. They have also been shown to improve hydrolytic stability and solubility of the curcuminoids. However, complexation to CDs has shown a photochemically destabilizing effect on some of the curucminoids. Further, it is not known whether the cucuminoids form a 1:1 or a 1:2 complex stoichiometry (compound:CD) with the CDs.
In the present work, curcumin, bisdemethoxycurcumin, diketone (a half curcumin), ethylferulate and acetogalactosidebromide were synthesized, purified and characterized (by melting point in open capillaries, DSC, UV-spectrum, IR spectrum, NMR spectrum).
The acetogalactosidebromide should be used to make the digalactoside of curcumin. It was expected that this compound would have a high aqueous solubility. However, the synthesis failed. It was assumed that the solvent used in the synthesis, pyridine, degraded acetogalactosidebromide and curcumin before they got a chance to form the digalactoside.
Then the DPPH∙ radical scavenging capacity was investigated for the four remaining compounds. Curcumin, diketone and ethylferulate had nearly quantitative reactions with the free radical, showing the importance of the phenolic group, the methoxy group next to the phenolic group and the conjugated double bond for the radical scavenging capacity. The investigation also showed that the two parts of curcumin scavenge radicals independently. The diketone moiety did not however, prove to be very important for the radical scavenging capacity.
The unidentified peak in the HPLC chromatograms of curcumin, bisdemethoxycurcumin and diketone was also investigated by UV-Vis spectroscopy and HPLC chromatography. The peak could not be identified, however, the presence of the peak seems to be related to the presence of the keto-enol structure of the curcuminoids.
The solubility was investigated at neutral to alkaline pH in HPãCD and HPâCD-solutions (pH 6, 7.4 and 8.5 all 0,1 M phosphate buffer, ionic strength 0,3). However, ethylferulate was the only compound which was stable under these conditions. Further, the solubility of this compound was higher at pH 5 (0,05 M citric acid buffer, ionic strength 0,15) than in the neutral to alkaline formulations. Therefore, it was decided that pH 5 was the optimal pH for the formulation.
The third approach to increase solubility, was by adding ethanol to the curcuminoid-CD formulations. However, this decreased the solubility of the compounds. Bisdemethoxycurcumin did however, show an increase at low ethanol concentration, and it was assumed to be caused by ternary complex formation.
Phase solubility investigations and UV-Vis spectrometry were also performed. The data from these studies were evaluated by a curve-fitting program (Kaleidagraph software) to determine stoichiometry and association constants for the curcuminoid-CD complexes.
The curve fitting of Ap-type curves in the phase solubility investigation with the quadratic equation, seemed to be an oversimplification. This method assumes that the components in the solvent show ideal behaviour, and only consider inclusion complexation. The reality seems to be more complex, involving non-inclusion complexation between the compound and CDs, and aggregation of CDs.
It seemed as if curcumin and bisdemethoxycurcumin formed 1:2 complexes with the CDs investigated. Addition of ethanol inhibited complex formation to some degree, resulting in 1:1 complexes. It also seemed as if these 1:1 complexes would aggregate and form micelles, which could further solubilize the compounds.
Ethylferulate did only show 1:1 complexation. But addition of ethanol decreased the complexation efficiency (CE).
The diketone showed 1:2 stoichiometry in HPâCD and 1:1 in HPãCD in formulations without ethanol. Thus, it seemed as if both the enolic part and the phenolic part of the molecule could be accommodated in the CD cavity. Adding ethanol, the 1:2 complex formed in the presence of HPâCD was reduced to a 1:1. Complexation with HPãCD seemed to be more complex, and it was assumed that it also could form 1:2 complexes and that the diketone molecules could be further solubilized by aggregates of these 1:2 complexes.
The UV-Vis spectroscopy investigation gave proof that the compounds were forming inclusion complexes with the CDs in formulations with 10 % (v/v) ethanol. The investigation also showed that all of the compounds formed 1:1 complex stoichiometry with the CDs in 10 % (v/v) ethanol.
These 1:1 complexes have lower solubility than the 1:2 complexes. Thus, competition for the CD cavity between the compound and ethanol explains the reduced solubility upon addition of the latter.
These results show that formulation additives, concentration of the constiuents and the CD batch influence the stoichiometry of the inclusion complex and affect the mechanisms of solubilization. And this may explain the various results on complex stoichiometry between curcuminoids and CDs that are reported in the literature.
The results also show that the diketone moiety and the symmetry of the molecules are the reason for their low aqueous solubility. Thus, ethylferulate could prove to resolve some of the problems observed with curcumin. However, the biological effects of this compound are not as extensively investigated as the effects of curcumin. Also, a more thorough investigation of ethylferulate s hydrolytic stability and photochemical stability is needed before it can replace curcumin as a potential drug. | nor |