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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

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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.

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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.

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T. Phytochemistry 1963b,

2,

71.

Gupta, R. K.; Haslam, E. J .

Chem.

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erkin Trans.

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1978,

Gupta, R.

K.;

aslam, E. “Polyphenols n Cereals and Legumes”;

Hagerman, A.

E.;

Butler, L. G. J .

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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.

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Chem. 1981,58,234.

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947.

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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.