J Agric Food Chem 30 (1982) 1087-1089
Transcript of J Agric Food Chem 30 (1982) 1087-1089
8/12/2019 J Agric Food Chem 30 (1982) 1087-1089
http://slidepdf.com/reader/full/j-agric-food-chem-30-1982-1087-1089 1/3
J Agric. Food
Chem. 1982 30
1087-1089
1087
Vanillin Assay for Proanthocyanidins (Condensed Tannins): Modification of the
Solvent for Estimation of the Degree of Polymerization
Larry G. Butler,* Martin L. Price,' and Jeffery E. Br oth ert od
When the reaction of f lavanob w ith vanillin is carried out in glacial acetic acid, the absorb ance produced
is much greater tha n in me thanol, the conventional solvent. In glacial acetic acid, but not in me thanol,
the time course of the vanillin reaction with catechin is similar
to that
of tannin. Th e absorba nce produced
in glacial acetic acid, but no t in m ethano l,
is
approximatelyproportional
to
the concentration of flavan-3-01
end groups a nd th us m easures the concentration of oligomeric molecules rathe r tha n t he total con-
cen tratio n of flavan-3-01units . By use of the reac tion in glacial acetic acid, th e degree of polym erization
of purified tann in sam ples can be determined. T he results agree well with literature values obtained
indep enden tly by more laborious techniques. However, me thanol is th e solvent of choice for determ ination
of tan nin content because in methanol th e reaction is much less sensitive to monom er units such as
catechin than it is to th e polymeric tannins.
T he vanillin assay is widely employed as a me thod for
quantitative determination of condensed tannin (pro-
anthocyanidins) n plan t materials such
as fruits
(Goldstein
and Swain, 1963b), sorghum grain (Burns, 1971), and
forage legumes (Broadhurst and Jones, 1978).
It
is a
sensitive, relatively sim ple assay specific for flavan-3-ols,
dihydrochalcones, and proanthocyanidins (Sarkar and
How arth, 1976; Gu pta and H aslam, 1980). F or conven-
ience, catechin, a monom eric flavan-3-01unit of condensed
tannins, is often used to standardize the assay rather th an
purified condensed tannin, although this leads to a con-
siderable overestimation of ta nnin content (Price et al.,
1978; Gu pta an d Haslam, 1980). In m ethanol, the usual
solvent for th e assay of extra cts of sorghum , catechin and
tannin react with vanillin with quite different kinetic
patterns (Price et al., 1978; Gupta and Haslam, 1980).
We now re port t ha t in glacial acetic acid, the reactions
of catechin and tan nin with vanillin are kinetically similar.
In addition, the reaction produces more chromophore han
in methanol, and the concentrations of chromophore
produced is proportional
to
the concentration of flavan-3-01
end groups present. Thu s, the assay can be used to es-
timate th e degree of polymerization of a proantho cyan idin.
EXPERIMENTAL PROCEDURES
Materials. Catec hin, phloroglucinol, an d vanillin were
obtained fro m Sigm a, an d epicatechin was from Aldrich.
All were used without further purification. Catechin and
epicatechin oligomers were generously provided by Dr. E.
Haslam , Depart me nt of Chem istry, Sheffield University,
U.K. Purified tann in from sorghum grain (NK 300) was
provided by Dr. Haslam. Tan nin from a high-tannin
sorghum grain (BR 54) was purified by our standard
technique (H agerman an d Butler , 1980) by A. Hagerman.
Samples
to
be used for determ ining extinction coefficients
were dried overnight over
Pz05
t room temperature.
Assays.
Vanillin assays in methanol were carried out
as recomm ended by P rice e t al. (1978), in a 30 OC water
bath with a reaction tim e of 20 min. Th e vanillin reagent
contained 4 concentrated HCl and 0.5 vanillin in
methanol. Absorbance was read in a Zeiss PMQ -I1 spec-
trophotometer, at 500 nm, t he wavelength of m aximum
Dep artm ent of Biochemistry, Pu rdu e University, West
lPresent address: ECHO, N. For t Meyers, FL 33903.
?Present address: Depa rtme nt of Agronomy, University
Lafaye tte, Indiana 47907.
of Illinois, Ur ban a, IL 61801.
abso rbanc e, by using a 1-c m cell. Sam ples were dissolved
in methanol.
For assays in glacial acetic acid, the vanillin reagent
contained 4 concentrated HC1 and 0.5 vanillin in
glacial acetic acid. Th e absorban ce was read at 510 nm
as described above. Sam ples were dissolved in glacial
acetic ac id, except for tannin s, which were dissolved in a
minimu m volume of me thanol an d diluted in glacial acetic
acid. Th e reaction tim e was shorten ed to
5
min, and the
methanol conten t was kept as ow as possible to minimize
th e effect of m ethanol o n th e reaction (see below).
RESULTS
We examined several parameters of the vanillin assay
for tann in in addition to those we previously investigated
(Price et al., 1978).
A
brief survey of vanillin
(4-
hydroxy-3-methoxybenaldehyde) nalogues dem onstrated
tha t
2,4-dimethoxybenzaldehyde
ave absorbanc e values
2-5 times greater tha n those obtained with vanillin and
may therefore warrant further investigation if greater
sensitivity is needed. All data presented in this paper were
obtai ned by using vanillin.
Th e rate and e xtent of color developm ent in the reaction
between vanillin and catechin or tan nin were found to be
strongly solvent dependent. Conventionalsolvents for this
reaction are sulfuric acid (Swain and Hillis, 1959) and
methan ol (Burn s, 1971). In both glacial acetic acid and
acetonitrile the reaction produced much more intense
absorption near 500 nm than
was
produced in methanol.
Glacial acetic acid was chosen for further investigation
because th e kinetics of th e reaction are less complex in th is
solvent and the colored product is m ore stable.
Th e change in absorbance at 510 nm vs. time for the
reaction between vanillin a nd catechin in a solution of 4
concen trated HC1 in glacial acetic acid is show n in curve
A of Figure 1. When the same reaction was carried out
in methano l (Price et al., 1978), h e ab sorbance after 5min
was appro xima tely 3-fold lower.
Addition of methanol to the reaction in glacial acetic
acid caused a rapid dec rease in absorbance (curves
B
and
C in Figure 1). Th e change in ASI0 s not d ue to a single
second-order reaction for either the initial color form ation
or
the decrease following the addition of methanol, as
shown by the nonlinearity of the ap pro pria te semilog plots
[log Amm A , ) vs. t or log ( A , Amin)vs. t not shown].
In m ethanol, vanillin reacts mo re slowly and in a more
complex fashion with tannin than with catechin (P rice et
al., 1978; Gu pta an d H aslam , 1980). H owever, in glacial
acetic acid purified tannin behaved kinetically in a ma nner
1982 American Chemical Society
8/12/2019 J Agric Food Chem 30 (1982) 1087-1089
http://slidepdf.com/reader/full/j-agric-food-chem-30-1982-1087-1089 2/3
8/12/2019 J Agric Food Chem 30 (1982) 1087-1089
http://slidepdf.com/reader/full/j-agric-food-chem-30-1982-1087-1089 3/3
Vanillin Assay for
Proanthocyanidins
reaction w ith flavan-3-01 monom ers an d their oligomers
and polymers quite differently, producing complex kinetic
pattern s tha t make standardization of tannin analysis with
monomers such as catechin tenuous a t best.
Replacing metha nol with glacial acetic acid as th e sol-
vent results in
similar
kinetics for monomers and polym ers,
produces severalfold more intense absorption, an d most
impo rtantly gives extinction coefficients th a t are approx-
imately equivalent on a molar basis.
T he reaction in bo th solvents involves condensa tion of
vanillin with th e proanthocy anidin, without depolymeri-
zation of th e proanthocyanidin (Watterson and Butler,
1983),so different chromop horicproducts ar e formed from
different proanthocyanidins. Our data obtained with
monomers, dimers, and trimers suggest that in glacial
acetic acid th e absorbance of the pro duct of th e reaction
with catechin and epicatechin
is
essentially equivalent
to
tha t of oligomers and the polymeric proanthocyanidin . We
conclude tha t only terminal un its of the polymer react with
vanillin in acetic acid. Goldstein an d Swain (1963a) have
previously shown tha t a series of com pounds containing
only one phloroglucinol ring
ll
have about th e same molar
extinction coefficient in the vanillin assay using H2 S0 4as
the solvent. These workers
also
demonstrated th at po-
lymerization of catechin reduces its ability to react with
vanillin more tha n ita reaction with a non specific reagent
for phenolic groups, an observation co nsistent with pref-
erential reaction a t terminal units.
Th is being the case, th e degree of polymerization of a
sample of purified tann in can be readily obtained as the
ratio of absorbance of monomer t o polymer, both deter-
minations being made on equal weights of material. In
Table I, for example, the average ESIO1 f catechin and
epicatechin is 257, which when divided by 48, the ESlo1
of
sorghum tannin (NK 300) purified by E.Haslam , gives
a degree of polyme rization of 5.4. Th is is in reasonable
agreem ent with the value of
6-7
flavan-3-01 unita /poly me r
contained by Gu pta and Haslam (1978) by
an
independent
method on a sample from the sam e source.
A
comparable
valu e of 4.6 unita /poly mer can be calculated from the da ta
in Table
I
for sorghum tannin (BR 54) from a different
line prepare d in our laboratory. Th is technique is much
simpler than the
13C
NM R technique recently developed
(Czochanska et al., 1980).
For estim ation of th e tannin co ntent of a sample, th e
vanillin assay run in me thanol a s previously described
(Price et al., 1978) is the most useful of several method s
tested (E arp et al., 1981). T he relative insensitivity of th e
assay in methano l toward monom ers (Figure 3; Table
I)
is an advantage because absorbance due
to
the polymeric
tannin s, which are of greate st interest, is maximized and
absorbance due to m onomers is minimized. Therefore, it
J Agric. Food
Chem.,Voi.
30,No. 6,
1982
1089
is not expected t ha t glacial acetic acid will replace me th-
anol as the solvent for the vanillin assay of ta nnin content.
Measurement of the degree of polymerization as de-
scribed above requires purified condensed tann in, which
can be weighed to de termine th e tota l number of flavan-
3-01 units. Estim ation of th e average degree of polyme r-
ization can be carried out in cru de extracts without pu-
rification of the tannin if the total content of flavan-3-01
units can be determined by an indepen dent method. We
estimate to tal flavan-3-01 units by spe ctrophotometric
assay of th e anthocyanidin pigments produced from the
proanthocyanidins by heating in H Cl/butan ol mixtures
(Gup ta and H aslam, 1980; Lewak, 1968). Because the
vanillin assay for termi nal flavan-3-01units detects both
polymer units an d free monomers such as catechin, but
the above assay for total flavan-3-01units does not detect
free monomers, the average degree of polym erization tends
to be underestimated in crude extracts th at contain ca-
techin and/o r epicatechin.
Th e following pape r (Butler, 1982) reports relative values
of the average degree of polymerization of proantho-
cyanidins from sorghum grain as a function of extraction
conditions and degree of ma turation .
LITERATURE CITED
Broadhurst, R. B.; Jones, W.
T.J . Sci. Food Agric. 1978,29,788.
Burns,
E.
.
Agron. J . 1971,
3,
511.
Butler, L. G. J .
Agric. Food Chem. 1982,
ollowing paper in th is
Czochanska, Z.; Foo, L. Y.; Newman, R. H.; Porter, L. J.
J
Chem.
Earp, C .
F.;
Akingbala,J. 0 ;Ring,
S.
H.; Rooney, L. W.
Cereal
Goldstein,
J.
L.; Swain,
T. Nature (London) 1963a, 198, 587.
Goldstein, J. L.; Swain,
T. Phytochemistry 1963b,
2,
71.
Gupta, R. K.; Haslam, E. J .
Chem.
SOC.,
erkin Trans.
1
1978,
Gupta, R.
K.;
aslam, E. “Polyphenols n Cereals and Legumes”;
Hagerman, A.
E.;
Butler, L. G. J .
Agric. Food Chem. 1980,28,
Jacques,
D.;
aslam,
E.J. Chem.SOC. erkin Trans.
1
1974,2663.
Lewak,
S.
Phytochemistry
1968,
,
665.
Price, M. .; Van Scoyoc,S.;Butler, L. G. J . Agric. Food Chem.
Sarkar, S.K.; Howarth,R. E.
J . Agric. Food Chem . 1976,24,317.
Swain, T.; Hillis, W. E.
J . Sci. Food Agric. 1959,
O
63.
Thompson, R. S.; acques, D.; Haslam, E.; Tanner, R. N. N. J
Watterson,
J.
J.;Butler, L. G.
J . A g r i c Food Chem., in
press,
1983.
issue.
SOC.
erkin Trans. 1 1980, 278.
Chem. 1981,58,234.
892.
Hulse, J H., Ed.; ID RC Ottawa, Canada,
1980; 15.
947.
1978,26, 213.
Chem. SOC., erkin Tra ns.
1
1972, 387.
Received for review February
22,1982.
Accepted June
18,1982.
Supported in part by USAID Project No.
XI1
PRF
4.
Paper no.
8898
rom the Agriculture Experiment Station, Purdue University.