SOME MISCELLANEOUS UNSAPONIFL\BLE LIPIDS 107 



The foregoing discussion illustrates some of the complexity which may be involved 

 in studying anthraquinone derivatives as they occur naturally in plants. Adding to the 

 problem is the fact that more than one type of derivative may be present, and frequently 

 the nature of the constituents varies with the age of the plant. For example, in the com- 

 mon rhubarb (Rlieiti)i Hiicliilatit»i) young leaves contain mostly anthranol glycosides whereas 

 older leaves have glycosides of anthraquinones. 



The physiological activity of several of these anthracene derivatives has made them 

 important cathartics for many hundreds of years. Only recently has it been shown that 

 the free anthraquinones or the glycosides are ineffective, and the pharmacologically im- 

 portant compounds are free anthranols. The fact that pharmacopeias recommend storage 

 of purgative plants for periods up to one year before use is explained by the necessity for 

 slow hydrolysis of the glycosides to free anthranols. If storage is too long, however, the 

 anthranols are oxidized to anthraquinones. Anthraquinones are active as cathartics only 

 because they are reduced to anthranols by intestinal bacteria. Nothing is known regarding 

 any function of these various anthracene derivatives in plants. The case with which the 

 reaction anthraquinone— anthrone may be brought about in the laboratory has raised the 

 possibility that these compounds may somehow participate in hydrogen transfer or oxida- 

 tion-reduction reactions. 



Isolation procedures depend on whether free aglycones or the various glycosidic 

 derivatives are desired. For the first, extraction of the plant with rather non-polar sol- 

 vents such as ether or benzene is effective. The sugar derivatives, however, are ex- 

 tracted using water, ethanol, or water -ethanol mixtures. If anthrones or anthranols are 

 to be isolated, care must be taken to avoid their oxidation by oxygen in the air. This oxi- 

 dation is particularly rapid in alkaline solutions and leads to the formation of dianthrones 

 and polyanthrones as well as to anthraquinones. After extraction a solution of glycosides 

 may be concentrated under reduced pressure. to obtain crude crystals. These crude crys- 

 tals may then be purified by repeated crystallization from acetone-water. The glycosides 

 on heating with acetic acid or dilute (e. g. 5%) alcoholic HCl are readily hydrolyzed within 

 one hour at 70°. After hydrolysis a 1: 1 mixture of ethanol-benzene is added and then di- 

 luted with 0. 5% aqueous HCl. A layer of benzene separates containing the aglycones. 

 The aglycones obtained by hydrolysis or direct extraction of plant materials may be puri- 

 fied by extraction from benzene into dilute alkali and precipitation with acid. (Aglycones 

 with free carboxyl groups can be extracted from benzene using sodium bicarbonate solu- 

 tion and a second extraction with sodium hydroxide used to remove any less acidic sub- 

 stances. ) This crude precipitate is crystallized from benzene or alcohol. Purification of 

 the aglycones by column chromatography is successful if rather weak adsorbents are used 

 for example magnesium oxide, polyamide, or calcium phosphate. Some indication as to 

 the nature of the compounds is given by the way they migrate when chromatographed on 

 magnesium oxide (31). Thus, ortho-dihydroxy phenols are not eluted with even as strong 

 an eluant as acetic acid. 



For identification of anthraquinone derivatives the Borntrager reaction is routinely 

 used. Some of the unknown material is boiled in dilute, aqueous potassium hydroxide for 

 a few minutes. This not only hydrolyzes glycosides but also oxidizes anthrones or anthra- 

 nols to anthraquinones. The alkaline solution is cooled, acidified, and extracted with ben- 

 zene. When the benzene phase is separated and shaken with dilute alkali, the benzene 

 loses its yellow color and the alkaline phase becomes red if quinones are present. The 

 test is not specific for anthraquinones; naphthoquinones also give a positive reaction. If 

 partially reduced anthraquinones are present, the original solution does not turn red im- 

 mediately on making alkaline but turns yellow with green fluorescence and then gradually 

 becomes red as oxidation occurs. If desired, the oxidation may be hastened by adding a 

 little 3% hydrogen peroxide. The Borntrager reaction can also be made the basis of a 

 quantitative colorimetric determination. Direct spectral observations of a benzene solu- 

 tion may also be made for characterization of anthracene derivatives. Anthraquinones 



