zhang hui 2012213034
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Determination of Hydrolyzable Tannins (Gallotannins and Ellagitannins) after Reaction with Potassium Iodate
Zhang Hui
2012213034
J. Agric. Food Chem.2002,50,1785-1790.
Contents
Introduction
Materials and methods
Results and discussion
Conclusion
Introduction
Tannins
hydrolyzable tannins
condensed tannins high molecular weight polyphenolics found in
higher plants
gallotannins
ellagitannins
HO
HO
HO
COOH HO
HO
OH
OH
O
O
O
O
Structural formulas of representative hydrolyzable tannins from plants. Compound 1 is pentagalloyl glucose, comprised of the gallate (G,2) esters of glucose. Compound 3 is the simple ellagitannin eugeniin, comprised of both the gallate esters and a single HHDP (G−G,4) ester. Compound 5 is EGCG, the gallate ester of the flavanoid epigallocatechin. Compound 6 is oenothein B, a macrocyclic hydrolyzable tannin. All four of thehydrolyzable tannins react in acidic methanol to yield methyl gallate (7)
OOO
OO
O
GG
G
G
G
1
G=OH
OH
OH
O
2
OOO
OO
O
GG
G
G
G
3
O
OH
OH
OHHO
OH
OO
OH
OHHO
5
OH
OH
OH
O
O
7
OHHO
HO
O
HO OH
OH
OG-G=
4
HO
HO
OH
O
O
HO
HO
O
O
O
O
OO
OH
OH
HO
HO
O
OH
OH
OH
OH
OH
O
O
OH
OH
OH
O
G
O
OO
OH
OO
O
G
6
Introduction
Individual hydrolyzable tannins can usually be determined by HPLC, but methods for conveniently screening large numbers of samples with variable hydrolyzable tannin compositions have not been developed.
The KIO3 method has been employed in some studies of oaks and maples.
The purpose of this work was to optimize the KIO3
method for analysis of hydrolyzable tannins.
Materials and methods Reagents tannin acid ， gallic acid, methyl gallate, epigallocatechin
gallate (EGCG) Methanolysis and pH Adjustment anhydrous methanol, 85 , 20h℃ , pH: 5.5±0.1 Analysis 30 ℃ water bath， 50 min after KIO3 was added, recorded absorbance at 525 nm HPLC Analysis of Methanolysis Products C18 (100×4.6 mm),1.0 mL/min , ƛmax 525 nm, gradient elution
Results and Discussion
OH
OH
OH
O
+ KIO3 A B
max 525 nmƛ yellow
21
Why KIO3 ？Hypochlorite ? the lifetime of A was shorterPotassium periodate (KIO4) ?past the red intermediate to the final product
Disadvantages of KIO3 method
different color yields
in rates of reaction
for structurallydifferent gallotannins
Improve
Why not gallic acid ？
Methyl gallate
OH
OH
OH
O
O
H3C
+ KIO3 A B
max 525 nmƛ yellow
21
Why anhydrous methanol?
traces of water caused production of a mixture of gallic acid and methyl gallate
avoid the disappearance of some methyl gallate formation of unidentified phenolic products observed at higher temperatures
Why 85℃?
Methanolysis
Reaction of KIO3 with methyl gallate as a function of pH. Methyl gallate solutions dissolved in methanol were diluted with a series of solutions comprised of ethanolamine and ammonium acetate buffer prepared at various pH values between 4.5 and 7.0.
How to control pH?
too basic, oxidation was acceleratedand only the yellow end product
too acidic, the chromophore Adid not accumulate
KIO3 Reaction
50 min after KIO3 was added, recorded absorbance at 525 nm
How to control time?
product A was temperature-dependent and time-dependent
at room temperature, there were significant day-to-day variations
How to control temperature?
30 ℃ water bath
KIO3 Reaction
a linear relationship between methyl gallate and absorbance at 525 nm:
Abs = 0.0132 μg + 0.0701
where R2 = 0.996, standard error of Y = 0.0303, and standard error of slope = 0.000 19.
Conversion of hydrolyzable tannins to methyl gallate by 20 h of methanolysis at 85°C.
A Methanolysis samples (s) of tannic acid
B . EGCG
C. Acer leaf powder
Theoretical Production of Methyl Gallate from Hydrolyzable Tannins
Yield of methyl gallate is calculated from molecular weights of tannin andmethyl gallate and is expressed in grams of methyl gallate per gram of tannin.
HO
HO
OH
O
O
HO
HO
O
O
O
O
OO
OH
OH
HO
HO
O
OH
OH
OH
OH
OH
O
O
OH
OH
OH
O
G
O
OO
OH
OO
O
G
Analysis was done with the original KIO3 method (A)
Analysis was done with the new method described (B)
Example:red maple leaves
Theoretical Production of Methyl Gallate from Hydrolyzable Tannins
Yield of methyl gallate is calculated from molecular weights of tannin andmethyl gallate and is expressed in grams of methyl gallate per gram of tannin.
Conclusion
The new KIO3 method will be useful for
examining hydrolyzable tannin in ecological,
physiological, and nutritional studies of plants.
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