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The Chemistry of Garlic

 A Review of Analytical Methods

The antibacterial properties of fresh garlic extract were first recognised by Pasteur in 1858 and even as early as 1892 Semmler had managed to obtain a 0.1% - 0.2% yield of volatile oil from garlic cloves by steam distillation. In 1935 Platenius developed a method of flavour estimation whereby the sulphides in oil distillates could be oxidised to sulphate in bromine water, excess bromine driven off by heating and the sulphate precipitated with barium chloride and determined by weight. Ten years later this method was improved upon by Currier who used lower reaction temperatures and converted the sulphur ultimately to methylene blue, thereby effecting a colorimetric determination. Although the use of total sulphur as a measure of garlic pungency was developed further by Kohman and Farber, it was the early work of Cavallito et al in determining the chemical structure of allicin and its reaction with cysteine (1),

(1)  2HSCH2– CHNH2– COOH  +  R–S(O)S–R'  →   R–S–S–CH2CHNH2COOH  +  R'–S–S–CH2CHNH2COOH

and the discovery and characterisation by Stoll and Seebeck of alliinase (2) that pioneered the chemistry of the AIIium species of the last fifty years.

(2)  RS(O)CH2CHNH2COOH  +  H2O  →   [RSOH] + NH3 + CH3COCOOH (pyruvic acid)

Pyruvic acid measurement

From the equation shown in (2), the determination of ammonia or pyruvate suggests a method for the indirect quantitation of thiosulphinates. The large amounts of endogenous ammonia in garlic tissue renders its determination unattractive but the measurement of pyruvate has been applied successfully by a number of workers. The presence of large amounts of pyruvic acid in Allium tissue was first detected qualitatively by Bennet while Morgan proved its presence by isolation of the 2,4-dinitrophenylhydrazine (DNPH) derivative. In 1955, Jager published a method whereby pyruvic acid is reacted with acidified 2,4-dinitrophenylhydrazine and the resultant yellow-coloured derivative measured colorimetrically. This technique was used in the late 1950's by Alfonso et al in a study of Mexican garlic varieties and later in a modified form by Schwimmer et al in the study of alliinase. Further development of the technique, principally by Schwimmer, led to it being employed widely as a measure of AIIium pungency and it is still used today as a simple method for the rapid estimation of onion strength.

Colour reactions for thiosulphinates

Early investigations in Japan into the formation of thiosulphinates in garlic led to the development of a (paper) chromatographic technique that relied on the development of an orange-red colour formed by a reaction with alkaline sodium nitroprusside. This early colour reaction was limited by its inability to distinguish between thiosulphonates and thiosulphinates and that the colour development was not permanent. At almost the same time, during a course of experiments in the USA with homocysteine peptides it was discovered that, when the products of a reaction between N-ethylmaleimide and thiols are made alkaline, a red colour develops. Under the correct conditions, the reaction was found to be extremely sensitive and the stability of the colour made it suitable for the visualisation of thiols or thiol esters separated by paper chromatography and for colorimetric determination. Although this colour reaction was extremely sensitive, it was found to suffer from interference from disulphides and it was subsequently modified by using N-ethylmaleimide in isopropyl alcohol followed by potassium hydroxide in the same solvent. This combination yields pink to red colours with thiosulphinates while disulphides remain colourless. Further improvements in extraction procedures (with diethylether) and colour stability (with ascorbic acid) have produced a reliable method for the colorimetric determination of thiosulphinates.

Chromatographic techniques

Although paper and thin layer chromatography techniques have been developed further, these and the methods involving pyruvate determination lack the resolution, sensitivity and quantitation achievable by modem analytical GC and HPLC methodology.

Gas Chromatography

The first reference to gas chromatography (GC) being used in the field of garlic or allium research was in 1961 when Carson used the technique to separate alkyl di- and tri- sulphides for further identification. GC appears to have been used sparingly throughout the 1960's, presumably because it was a new technique and equipment availability was limited, but as early as 1964 Saghir et al had warned that many of the compounds from allium species detected by GC may be 'artefacts of analysis'.
Because of their excellent resolution and mass identification capabilities, GC and GC-MS have continued to feature prominently in the efforts to characterise allium volatiles but although these tools are of great value in the study of compounds of moderate thermal stability such as those found in distilled oils, thiosulphinates from allium species are known to decompose on heating or attempted GC analysis.
Allicin is quite reactive and unstable, hydrolysing on heating in water to give diallyl disulphide, diallyl trisulphide and the corresponding polysulphides and it was an attempt by Brodnitz et al in 1971 to determine allicin by GC-MS that also provided evidence for an unusual mode of decomposition; they indicated that GC caused diallyl thiosulphinate (allicin) to dehydrate (Fig.1), affording a 2.4:1 mixture of two compounds, 3-vinyl-4H-1,2-dithiin and 3-vinyl-6H-1 ,2-dithiin.


© Mike Watson 2005


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