Sciencemadness Discussion Board

Isolation of flavonoids (anthocyanidins ) from red cabbage juice

neutrino - 22-8-2004 at 14:26

I am trying to isolate flavin from red cabbage juice into its pure form. Here’s what I’ve come up with so far:

1. Take about 1 Kg of red cabbage and extract the juice with boiling water to get 400-600mL of solution.
2. Boil the solution down to 50-100mL.
3. Add enough dehydrated MgSO<sub>4</sub> to the solution to form a solid mass.
4. Crush the mass to a powder, if possible. If you find that you can't crush it due to not adding enough sulfate, grind it under ethanol until the mass breaks up and you're left with a yellow solution and a granular mass.
5. Extract the indicator from the crushed mass with many portions of ethanol. The extraction is finished when the solution extracted is no longer a strong yellow color.
6. Boil the extract down to ~50mL. Make sure to do this with good ventilation and without a flame source! While boiling, you will notice a layer of clear MgSO<sub>4</sub> crystals growing on the bottom of the vessel. These crystals seem to inhibit regular boiling and promote severe bumping and even flash boiling, which is why you don't want a flame source around! You have been warned.
7. Transfer the liquid to Petri dish in a desiccator loaded with CaCl<sub>2</sub>. When most of the ethanol is gone, you should be left with 15-20mL of solution and more MgSO<sub>4</sub> crystals on the bottom.
8. Transfer the solution to a vial and cool it in a freezer. This will form a blackish blob at the bottom.
9. Separate the blob and extract the indicator from it with small portions of ethanol. When the blob starts turning white, discard it.


At this point, you should be left with about 25mL of dark red solution. This can be stored indefinitely at room temperature, but decomposes easily if heated, the decomposition point being slightly above 100*C. If you take a drop of the solution and let it dry, it will turn bright pink. Add some water to this and you will see the original purple color or, if you add more water, a blue color (although this might just be due to my water being basic, so don't be surprised it you don't see it).

Now, this is where I need help. Paper chromatography tells me that there are 3 substances in this: 1)a purple material with a constant, high Rf value; 2)a blue material with a slightly lower, more erratic Rf value that always mixes with the rear portion of the purple material (my paper strips are only 11cm long at best, made from 11.0 cm filter paper); and 3)a brown material that never moves. The solution also contains a nonvolatile solvent keeping me from crystallizing the materials, which I suspect is glycerin. If I can remove this, then I think that it should be easy to remove the brown stuff by fractional crystallization. Does anyone have any suggestions?


[Edited on 12-11-2005 by chemoleo]

JohnWW - 22-8-2004 at 16:32

You are becoming an expert in red-cabbage-ology!

What solvent did you use in the paper chromatography?

The purple and blue substances are either flavin isomers, or other closely related chemical species, e.g. one being an oxidation product of the other. The brown substance that does not move on chromatography is probably degraded chlorophyll.

Flavin, C10H8O2N4, is a complex heterocyclic ketone with 3 fused 6-membered rings , which is also a secondary and tertiary amine with 4 N atoms (2 of each), owing its color to four double bonds conjugated to an aromatic benzene ring. The middle ring has two Ns next to the benzene ring, and the third ring has a N and a keto group next to the middle ring, with another N separated from the other N by another keto group. Flavoproteins, in which it is mostly found in nature, are usually yellow in color.

Flavin, in the form of the coenzyme Flavin adenine dinucleotide, in which the flavin is bonded via a N atom in the central on its 3-ringed system, is biochemically important in the catabolism of fats (fatty acid triglycerides) in the stomach and intestine, after their hydrolysis. It takes part in the reaction step resulting in formation of a double bond at the alpha-carbon of fatty acids, which is subsequently oxidized to a OH and then keto group.

The term "flavin" is also popularly (but wrongly) given to quercetin, a yellow crystalline slightly water soluble substance, C15H10O7, which I think is an anthraquinone derivative, from the bark of quercitron and other vegetable substances. It is used as a yellow dye.

It is unfortunate that there are two different substances called "flavin". I am not sure which one, or a derivative thereof, is sold in pharmacies as the yellow disinfectant "acraflavine", but I suspect the latter.

John W.

Geomancer - 22-8-2004 at 21:51

I think that the red cabbage pigment is a mixture of anthocyanins, mostly di-glycosides of cyanidin. If you're looking to isolate a pure compound, extract with hot acid to cleave the glycosidic bonds, and wash with an non-soluable solvent. You could try an acid-base extraction, but it may be that chromatography is your only bet. I don't yet know how the indicator properties of cyanidin compare to the parent anthocyanins; most likely they show similar color effects.

neutrino - 23-8-2004 at 06:07

I used ethanol for the chromatography. Seeing as it is only paper chromatography, I doubt it could be used to separate large amounts of flavin. I'd mainly like to separate the brown stuff and glycerin (?) from the blue and purple pigment.

Geomancer: could you elaborate on your extractions?

Geomancer - 23-8-2004 at 16:54

I tried to extract some cyanidin today. Short report, requisite scientific posturing included:

A half pound of red cabbage was cut into slices. Samples were removed at intervals for digestion. The cabbage was then steamed about 10-20 minutes, with samples being removed for digestion throughout the process. The resulting cooked cabbage was mixed with just enough water to allow it to be processed in a blender with a few drops of detergent. The mush from the blender was heated to boiling, removed from the heat, and about 10-20% muriatic acid was added. The resulting mixture had the general appearance of raspberry puree. After this point, no further samples were removed for digestion. After sitting for 30 minutes, the puree was filtered, partly by gravity through a coffe filter, the rest manually through two layers of flannel. The fluid was saturated with table salt. 40ml of the fluid were taken and washed with 60ml of naptha, in 5 portions, using a separatory Ziploc bag. The layers showed a great tendency to form a recalcitrant foamy emulsion, even after all the washing. The resulting liquid was still slightly cloudy. NaOH and vinegar were added to adjust the color to blue. The blue solution had a much more opaque appearance. Attempts were made to extract the cyanidin with naptha, engine starting fluid (mostly ether), and isopropyl alcohol. None were successfull.

HaHA! I did it. All actions in passive voice. Damn, I'm scientific.


As to why the procedure was not successfull, there are several possibilities.

  1. Cyanidin just annoyingly hydrophilic.
  2. Cyanidin is not that hydrophilic (perhaps the increase in opacity when basified is precipitating pigment), but instead is just (everything else)-phobic.
  3. The HCl treatment didn't break the glycosidic bonds.
  4. Blue is not the color that indicates the uncharged state.
  5. The cyanidin is complexed with something that is annoyingly hydrophilic, for example, metal ions.

Possible solutions:

  1. Magic solvent. Probably not OTC. Suggest alternatives!
  2. Ditto.
  3. Longer, hotter acid treatment (this might kill the pigment, though).
  4. More research.
  5. Chelation?

Other improvements:

Other lessons learned:

Anthocyanidin is the purple compound in cabbage

chemoleo - 23-8-2004 at 18:22

Anthocyanidin derivatives cause the purple colour of red cabbage, for a chemical structure see this: http://home.howstuffworks.com/question439.htm

Looking at this structure, I am not sure how the colour change upon pH works, however. There are no amino groups. All I can think of is that the hydroxyls are in acid-base equilibria, changing the electron distribution in the heterocyclic rings once they are protonated (or not).

As to the extraction of anthocyanidines:
There is no way you will be able to crystallise that, as long as the glycosidic linkage to sugar (and sugar chains) exists. The pigment is bound to link to various sugar chains, all of which will be heterogeneous. But of course you can only cyrstallise homogenous compounds.

So - the only way I can see to crystallise this is to remove the sugar (chains) from the anthocyanidine. There are enzymes that do this, of course - but not particularly useful here. But this is an ether linkage of sorts, so it should be hydrolysable by refluxing this in strong acid. The question is unfortunately what happens to the pigment itself - I couldnt guarantee that it wouldn't be attacked. But it's worth a shot I guess.

Then, in proper science you'd run it down a column that separates molecules by size (a gel filtration column). Coloured fractions are collected, concentrated and analysed further (mass spec).
I have been often thinking about how to make gel filtration columns at home - but it's difficult, one needs designed resins for that.
Maybe this warrants a post one day.

Another option is to incubate your crude Anthocyanidin extract with biological washing powders ( I am serious). It contains among others, cellulases and other glycosidases. This might remove the sugar entities.

Also - to remove any further impurities:
Essentially one wants to get rid of fats, cellulose derivatives, proteins, and nucleic acids (DNA/RNA). Fats can be extracted with acetone (it shouldn react with the anthocyanidin). Cellulose can be precipitated simply because it's insoluble, after thorough mashing/ grinding (mixer). Proteins - acidify massively. Most will precipitate then. AFter that, basify (add lots of NaOH) to remove acid soluble proteins. Then you will have gotten rid of most proteins. Alternatively, add to your clear proteinaceous extract 4M ammonium sulphate, which will also precipitate 99% of all proteins (it's called salting it out).

So do these things first, i.e. incubate with washing powders to degrade sugars, then remove lipids, protiens and undegraded polysaccharides, and play around with a few solvents (ether for starters) to see whether u can extract the pigment.

[Edited on 24-8-2004 by chemoleo]

neutrino - 24-8-2004 at 05:09

I always thought that the flavin was in the form of a simple salt. Now I know better Thanks, chemeleo, I'll try playing around with that for a while. Now to find some strong acids lying around... Would NaCl work equally well for salting out or does the sulfate ion react with the proteins in some way (I remember hearing about some sulfur-containing ion doing this)?

[Edited on 24-8-2004 by neutrino]

Geomancer - 24-8-2004 at 05:53

Just to clear up the nomenclature here: anthocyanidins are a group of compounds. The figure in the link chemeleo provided is somewhat misleading, as it only depicts the structure for cyanidin (I think) mono-glycosides. In addition to cyanidin, there are six common and at least as many uncommon anthocyanidins. They can also have various patterns of glcosylation (and other forms of modification).

I don't know where referring to red cabbage dye as "flavin" comes from. It appears to have been plagairised around the internet somewhat extensively. CRC shows no reference for "flavin", although anthocyanidins (and other things) are substituted forms of flavan.

I've decided I didn't hydrolyse my extract. It seems to want 80C in rather strong acid for at least an hour. In fact, it is a rather popular lab experiment to observe anthocyanin hydrolysis with TLC. Should be fun for anyone who has the required equipment (I don't). You'll need formic acid, HCl, cellulose TLC plates, and various sundries.

chemoleo - 24-8-2004 at 06:56

Just to point out, in the 'howstuffworks' link I posted above, it does say that derivatives relate to different sugars linked as indicated, and the modification of the hydroxyls, i.e. methoxys.

neutrino - 24-8-2004 at 08:19

I just had a thought about the hydrolysis: could the stuff hydrolysize by itself in water, thus explaining the stuff's short shelf life (~1 week)? Also, could I use a weak acid like CH<sub>3</sub>COOH or NaHSO<sub>4</sub> for the hydrolysis?

[Edited on 24-8-2004 by neutrino]

chemoleo - 6-9-2004 at 18:22

By the way, I recently noticed that the blackcurrant juice from 'Ribena' solely contains anthocyanidins as a colouring agent!
Presumably this may be a purer source of the dye :)

JohnWW - 6-9-2004 at 21:58

It, or derivatives of it, probably is the natural colorant in all other purplish fruits, like blueberries, blackberries, cranberries, boysenberries, tamarillos, plums, blackcurrants, etc. Some reddish fruits, like raspberries, cranberries, redcurrants and peaches, probably have it in other forms; but other reddish fruits like tomatos and chilis owe their color to lycopene and other vitamin A precursors.

John W.

kazaa81 - 10-11-2005 at 13:10

I like the use of anthocyanins for finding the pH of a solution!

Attachment traduced from a foreign source....

Attachment: Anthocyans [or anthocyanines].txt (833B)
This file has been downloaded 1144 times

kazaa81 - 11-11-2005 at 12:33

Attachment traduced from a foreign source...about pirilium salts

Attachment: Pyranes.txt (1kB)
This file has been downloaded 1112 times

neutrino - 11-11-2005 at 17:30

So, the extract should be boiled with a strong acid to break the glycoside linkage. I will try this one day with sulfuric acid, but I'm far too busy at the moment.

chemoleo - 11-11-2005 at 18:14

Correction, anthocyanidins belong to the class called flavonoid, of the following structure:



There are about 250 of them that occur in nature or have been described, whereby the anthocyaniDIN are sugar free, and the anthocyanINE are glycosids (sugar-linked).
In food they come under the code 'E163'.

Black berries contain 1.15 g anthocyane per kg. Red cabbage is also rich in it.

Mg/100 g fruit/vegetable
Aubergine 750
grapefruit ~200
blackberry ~115
blueberry 80-420
rasberry 10-60
cherry 350-400
black currant 80-420
red grape 30-750
red wine 24-35

The fact that the colour (absorption) is affected by the pH is of course explained by the O+ - which is titratable.

All the equilibria are shown here



Some is in german, but it should be clear nonetheless.

What I found intersting is that the anthocyanidins form complexes with Al, Mg, Fe3+, which induces colour changes.





Cool isn't it!?


Oh, and what I didnt know - they are quite healthy! Strong antioxidant, even better than Vitamin C, at least in vitro! MIght be something to use for copper salt reduction to copper! Also they are good for the eyes, antiinflammatory and good against veneral disease!

[Edited on 31-5-2006 by chemoleo]

kazaa81 - 12-11-2005 at 12:27

chemoleo, it's good to correct himself!

I've found some pages on the web that may have some small interest, even if I haven't yet found some writing about specific anthocyanins....

- some about Flavonoids:
http://www.friedli.com/herbs/phytochem/flavonoids.html

-Flavonoid- from wikipedia-
http://en.wikipedia.org/wiki/Flavonoids

kazaa81 - 13-11-2005 at 11:49

To anyone new in this thread and in anthocyans:

anthocyans or anthocyanins - are anthocyanidins bonded with a sugar and are founded in colored plants (purple & blue)

anthocyanidins - aren't found free in vegetables but linked with a sugar (forming anthocyanins).

Do all anthocyanins react with bases or acids changing color? I'll try to free anthocyanidin from an anthocyanin (extracted from red salad) with aq. 30% HCl with boiling....I want to make some Al complexes with it! ;)

Red Cabbage Juice as Indicator

ktw_100 - 31-5-2006 at 05:01

Hi ..

Anybody know a way to make crystals from cabbage juice extract, versus just creating an indicator liquid with the juice? As a liquid, it only lasts a few days, and then goes rancid.

Thanks
Keith

Nerro - 31-5-2006 at 06:51

Are there perhaps preservatives that will make it last longer? Beside that you can boil it down and keep the concentrate in the fridge for a little longer.

unionised - 31-5-2006 at 10:20

Don't bother trying bisulphite as a preservative in this instance. On the other hand, bisulphite does a good job of bleaching the stains from red wine, blackcurrant etc.

Red Cabbage Juice

ktw_100 - 2-6-2006 at 06:38

If the liquid fraction of Red Cabbage juice was boiled off, you'd have a solid with whatever in it. Would this solid deteriorate or decompose by itself in it's solid form?

What about just suspending a concentrated cabbage juice in non-acidic paper or a cloth, for example. Let it dry, and then when needed as an indicator, you just drop the cloth into some IPA, and voila, there's your indicator?

Keith

neutrino - 2-6-2006 at 14:43

You can't boil it to dryness. You get a semi-liquid sludge. Although it is a pain in the ass to handle (it really doesn't flow), it is stable.

That second idea should work.

Ozone - 6-11-2006 at 20:40

Hello,

Get a small pump. Wash some activated carbon in boiling water. Slurry the carbon into a column. Pump the liquid extract though the carbon (better if you can heat this *jacketed column* at about 80°C) to yield clear, colorless liquid. Drain the carbon and dry it (a vacuum oven at 50°C, ~24mmHg works well). Elute the carbon with (DMSO works better, but is very hard to remove) DMF (which is still hard to remove). Remove the DMF with a rotary evaporator or vacuum oven (it does not take much solvent to do this). The residue will contain all of the stuff you are interested in (mostly anthocyanines, polyphenolics, cinnamic acid derivatives, and flavanoid derivatives (quercetin is a flavone).

These can be fractionated from the carbon (or better, c18) using a polarity gradient, viz. pH 4 AcOH against AcCN (or MeOH:CCl3, but the selectivity (a) varies). Look for absorbance of light at 254 and 337nm.

Best of luck, have fun, and be safe,

O3