We made liposomes with lucifer yellow (see Thunnissen, J Neurosci methods). Now note that your substance added to the liposomes is not only inside the liposome, but often in the membrane and just outside, coupled to the membrane. Several substances taken up in fat/membranes will show colour change in light.

this maybe due to oxidation of curcumin by sulphate radical anion.

see this article: Kinetics and mechanism of oxidation of curcumin by sulphate radical anion in aqueous acetonitrile solution (Bulgarian Chemical Communications, Volume 42, Number 2 (pp. 126–129) 2010)

Dear Marani, I'm gonna see that reference. Exactly that, curcumin is bounded to the membrane because we actually made the thin lipid film with the curcumin already, the hydration medium is only PBS. Do you know another references of changes of colour when association with fat or membranes?

Dear Agiba, we thought about some oxidation, but the UV spectrum of pure curcumin and curcumin after the liposome production is exactly the same. Maybe the oxidation doesn't change the UV spectrum but is it possible? I'm gonna read the reference.

Thanks a lot.

What about the UV spectral change/no change in the non-oxidative states when curcumin is ligand bound!?  

Dear Khan, can you explain more about it? Thanks a lot.

To your question, we did liposome research with acridine orange, never published it, but a colour-shift was present too. I don't have the results anymore and don't trust the memory of a 70 year old prof. Sorry I cannot be of more help. Enrico Marani

Dear David,

Use dry, neutralized (of any acid-base traces which might be there due to Nitrogen purification and drying step) Nitrogen gas flushing during your preparation and try to maintain an inert (especially Oxygen-free, but other components too!) environment. Get the UV spectrum beforehand of the curcumin, Curcumin + a, b, c- you are using, giving time to each sample with the ratio you experimented; may be run the spectra under simulated conditions- to locate the exact change point (if your BLACK coloration observed change was immediate, this may not be the point). Another point, the densification may also be one reason of the color change, though should be small contribution to color change and , most likely, should not turn black, I guess it was not full, pitch black but a blakish one; and this time you should able to catch the color change point which should be able to provide more exact, localised reasoning and mechanistic aspect of your observation. More importantly, what about the color intensity of the 'got-black color' after your process (immediate preceeding process) was over!!???

Dear Doktorovova,

We have already measured the pH of the solution and it's between 6 or 7, so we guess that curcumin is not being subjected to basic pH. When we dissolve the liposomes in pure ethanol, so the liposome is broken, the free curcumin look light yellow again. 

Dear Khan, the black coloration is immediate. And we have recorded the UV spectrum of free curcumin (in ethanol) after the production of the liposomes and it looks the same as the pure curcumin in ethanol. When we hydrate the black thin lipid film with PBS, the color black dissapear but remains a redish one and brown. But after some days, the color turns back a little bit dark yellow. But it's never again light yellow as pure curcumin.

If you got the exact UV spectra, the curcumin is safe! The coloration is, seemingly (though I don't see from so far), from the solvent and other associated molecular take up including any traces of water, medium molecules and perhaps, ions ETC. (if present in the medium of the preparation). The densification of the medium and optical activities are known phenomenon. The later ease up/grade up in color might be medium's polarity/osmolarity adjustments. If you get the (different) color after filtering out (a small part of the product), and resuspending it in the medium of your preparation (minus your product) and IMMEDIATELY LYOPHILIZE, the color change effects can be known/confirmed in this part.  

Yes it is the exact UV spectra. I don't know if degradation products of curcumin absorb in another wavelenghts. We thought the coloration depends on the interaction with the lipids because when we dissolve the liposomes in ethanol, the solution turns back from brown to light yellow. Maybe because the curcumin is out of the mebrane. If you see the video, when the chloroform is evaporated, the color black is proportional to the evaporation of the chloroform and the production of the lipid film. When we rehydrate with PBS, the dark colour remains but it's a little bit reddish, maybe because the PBS relax a little bit the curcumin in the membrane. When we purify the liposomes with centrifugation, the pellet is brown and the supernatant is dark yellow, so we think that the densities and organization and localization of curcumin in the membrane is the thing. The colour remains before and after extrusion. 

Yes indeed I am not good with this yet. I don't know how to assess of there's some degradation products. The quantification went right, giving 100% curcumin but once again it was by UV and it's not that reliable because it only measures the part of the molecule that absorbs. 

The structural changes tend to produce UV absorption change(s) IN A MOLECULE, how small that be, from the main chromophore or add-up or removal of a group which will have SOME cumulative effect on the spectrum, the question is can you record/observe at that level! If you got some degradation, although the question is what's the level and extent of degradational change in terms of the structural part and whether you were able to record/observe that or not. The UV is quite sensitive, ng scales. All you could do to confirm degradation is to do simulated degradation studies, like suggested before and increase the concentrations of the degradants (molecules and IONS of acidic-basic characters) gradually. The degraded curcumin WILL not provide EXACTLY same spectrum at the same concentration, absorbance. So, look more carefully. The back and forth coloration are not indicative of the degradation at least to the levels of the retained coloration and this has to be juxtaposed with the degradation studies/trials if you carry them out. The other point is, all the curcumin degraded? if it degraded! 

Thanks, we want to expose our curcumin liposomes to basic conditions and the record changes in UV spectrum and other assessments. I will let you know. 

Update: We performed preparation of curcumin liposomes in acidic conditions (pH=5.8) and the changes in color still remain. When liposomes are disrupted in ethanol to perform UV spectrophotometry, curcumin looks again yellow as its original color. Any other ideas? I find this color changes highly interesting and I am very sure it's not due to degradation of curcumin. I think it's more related to the interaction between curcumin and phospholipids and specially highly cationic stearylamine (zeta potentia +70 mV). Thanks!!

please find file  attached

Thanks but we think it's not due to basic degradation of curcumin since it is yellow again when liposome are dissolved in ethanol. It's more a effect of concentration and organization between membranes.

Yes, I think that's the case. But literature is kind of weak about solvatochromic shifts. Or chromic shifts when curcuming interacts with biological membranes.

Thanks a lot for your suggestion.