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.
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© Mike Watson 2005
