Colorimetric assay of shikimic acid against quinic acid

ANALYTICAL

BIOCHEMISTRY

Calorimetric

(1972)

Assay of Shikimic Acid against

TERESA Department

47, 39-45

MOSSOR

AND

RYSZARD

Quinic

W. SCHRAMM

of Biochemistry, Institute of Biology, A. Mickiewicz Fredry 10, Poznali, Poland Received

June

Acid’

University,

23, 1971

The alicyclic hydroxy acids, shikimic and quinic, have been known for a long time, and may sometimes occur in quite considerable amounts in microorganisms and in higher plants. The discovery of the role of shikimic acid in the biosynthesis of the aromatic ring (in the 50’s) first in microorganisms and later in higher plants, has resulted in a need for its quantitative determination. Separation of the two acids from each other, as well as from other acidic compounds, is relatively easy by paper chromatography (2), or by many types of column chromatography: ion exchange (3,4), silica gel (5), and Sephadex (6). However, most of the reactions applied for the identification of shikimic and quinic acid on chromatograms are hardly specific enough for their use as a means of quantitative estimation. Periodate oxidation of shikimic acid yields 2-pentene-1,5-dialdehyde-3carboxylic acid; prolonged treatment appears to effect a further oxidation of the molecule (7,9). Probably oxidation of quinic acid yields the same compound. Various quantitative methods have been proposed involving the formation of colored compounds by reaction of various reagents with the oxidation product (S-15). Some of these methods deal only with shikimic acid while others attempt to estimate both acids in the presence of one another. Det’ailed investigations in our laboratory have shown that all methods based on the periodate oxidation of the two acids are dependent on time and temperature of oxidation and the concentration of reagents; in some casesthe subsequently produced colored compounds are very light sensitire. Furt,hermore, none of these methods differentiates between the two acids with sufficient precision to provide a satisfactory basis for their quantitative determination. We have, therefore, turned from periodate oxidation to an investigation of the direct condensation of the acids with an aldehyde to give a colored compound suitsablefor quantitative assay. ‘This material was part of a thesis of T. Mossor, A. Mickiewicz University, 1979. The principle of the method and preliminary results of this work were presented at the 5th Meeting of the Polish Biochemical Society, Krakbw, 1967 (1). 39 @ 1972 by

Academic

Press,

Inc.

40

XIOSSOR

AND

SCHRAMZI

From many verified nldehydes p-l~ydroxybenzaldel~yde appc~~d the most suitable. The reaction, like others of this type, o~urs in concentrated sulfuric acid. EXPERIMENTAL Reagents The reagents were: p-hydroxybenzaldehyde (Loba-Chemie, Vienna, Austria) ; shikimic, quinic, and oxalacetic acids (Sigma Chemical Co., St. Louis, MO.) ; glucuronic, galacturonic, and tartronic (hydroxymalonic) acids (Calbiochem, Los Angeles, Cal.) ; malonic acid (BDH Chemicals Ltd., Poole, Dorset, U. K.) ; cinnamic, p-coumaric, caffeic, and ferulic acids (Koch-Light Laboratories Ltd., Colnbrook, Bucks, U. K.) ; sinapic acid (Th. Schuchardt, Munich, Germany) ; chlorogenic acid (C. Roth OHG, Karlsruhe, Germany) ; coniferyl alcohol (Fluka A. G., Buchs, S. G., Switzerland). Other chemicals were Polish reagents of the purest commercial grade obtainable. Usnic acid was extracted from Usnen sp. Acetoacetic acid was prepared by alkali hydrolysis of ethyl acetate. ,411 reagents were used without pretreatment. P~rinciples of the Method Shikimic acid heated with p-hydroxybenzaldehyde and concentrated sulfuric acid gives an intensive purple-violet dye. Under the same conditions quinic acid gives no dye at all. Parameters The following were examined:

parameters, which could influence the color intensity, concentration of sulfuric acid, concentration of p-hy-

I &Of

1. Influence

!

I 1.0

FIG.

of the Reaction

of sulfuric

H,SO4

4034

3.0

2.0

(FOR

acid

5ml

on color

OF SAMPLE1

intensity

of the

dye.

ASSAY

z 52 0.6 ~

0.1 I

OF

SHIKIMIC

41

ACID

0.2 I

0.3

0.4

I 20

I 30

40

0.5 /

7b

5 Z L x LL

0.5

P-

04 ~. 10

50 m g /lO

FIG. 2. Influence the dye.

of p-hydrosybenzaldehyde

ml

concentration

on color intensity

of

droxybenzaldehyde, temperature and time of heating of the samples in the bath, lapse of time from the end of heating until measurement, and stability of the dye with time. (a) Conce,nt&ion of szc~jrc& ncid. The influence of the concentration of sulfuric acid on color intensity is shown in Fig. 1. The best results are given with the proportion of concentratecl (96%) sulfuric acid to water 3: 2 (vol) , which corresponds t.o about 73% sulfuric acid. (6) Concentration of p-hydrozybensaldehyde. The results are shown in Fig. 2. The highest extinction value is measured with the concentration of p-hydroxybensaldehyde at 2 mg/ml solut,ion. (c) Time and temperature of heating. The best results, i.e., the quickest appearance and the deepest intensity of the dye, was obtained by heating in a boiling water bath. The influence of time of heating in the boiling water bath is shown in Fig. 3. The dye reaches its maximum in-

15

30

45

60

75

90

MI N UTEY FIG.

3. Effect of heating in a boiling

water bath on color intensity

of the dye.

42

MOSSOR

AND

SCHRAMM

tensity in about 1 hr. The dye becomes fixed in 10 min after heating and remains stable for many hours. (d) The p-hydroxybenzaldehyde solution was stable for several months both at 0°C and at room temperature. Absorption

Curve

The absorption curve of the colored product of the reaction of shikimic acid with p-hydroxybenzaldehycle under optimal conditions is shown in Fig. 4. Details

of Proposed

Method

Reagents. Concentrated sulfuric acid, sp.gr. 1.835 (96%). p-Hydroxybenzaldehyde, 0.2% water solution (dissolved in hot water, then cooled). Procedure. To 1 ml aqueous solution of purified extract, in which shikimic acid is to be estimated, add 3 ml concentrated (96%) sulfuric acid, stirring all the time, then cool. Add 1 ml 0.2% aqueous solution of p-hydroxybenzaldehyde, with stirring. Transfer into a boiling water bath

,

1

1

/

I

I

I

400

450

500

550

600

650

700

WAVELENGTH

4. Absorption pmole/ml) with

FIG.

(0.3

curve of the dye p-hydroxybenzaldehyde

product in

(nml

of the reaction of concentrated sulfuric

shikimic acid.

acid

and heat for 60 min, then cool. After 10 min measure the extinction at 590 nm. Specificity of the reaction. For the specificity of the reaction approximately 80 compounds were examined: organic acids, sugars, phenols and phenolic acids, amino acids (especially aromatic ones), alcohols, polyol~, etc. Only a few of these compounds give, under the described conditions, a positive reaction with p-hydroxybenzaldehyde. They arc: Monosaccharides: an atypical brownish dye. Lower alcohols (met,hanol, ethanol, propanol) : purple-red dye with maximum of absorption 52&570 nm. Uranic acids: red dye (weaker) with large maximum of absorption about 520 nm. Some organic acids wit,h three or four carbon atoms: i.e., malonate, oxalacetate, acetoacetate, and tartronate react weakly to give a purpleviolet dye with maximum absorption at 560 nm. Usnic acid: purple-violet dye with maximum absorption at 580 nm; the intensity is similar to that of shikimic acid. p-Hydroxy and p-methoxy derivatives of cinnamic acid (e.g., p-coumaric, caffeic, ferulic, synapic), chlorogenic acid, coniferyl alcohol: all yield a very intensive purple-violet dye; the acidic compounds give about 3 times the intensity of the color produced by shikimic acid. Cinnamic acid alone gives very low intensity. The color produced by all the compounds has an absorption maximum at 565-570 nm. Purification of sa,mple for determi?lation of shikimic acid. For the determination of shikimic acid the sample must be purified to remove interfering compounds. The simplest method of achieving this was by the use of ion exchangers. The nonacidic compounds (sugars, alcohols) are removed by passing the solution through an anion-exchange resin, on which only the acidic compounds are retained. Shikimic acid can be separated by elution from other acids giving a positive reaction with p-hydroxybenzaldehyde. The best eluent is acetic acid, which separates relatively well the weak acids by elution from the anion exchanger (4,16). Shikimic acid is eluted from the column first, as will be seen from Fig. 5. Malonic acid as well as the phenolic derivatives of cinnamic acid are eluted with much stronger acetic acid. So for the separation of shikimic acid from the other acids 0.3 N acetic acid as eluent’ has been chosen. Many t,rials proved that wit,h 30 ml 0.3 N acetic acid only shikimic acid is eluted. The recovery of this acid from the Dowex l-S10 column was 84.7 + 0.470. Standard cuwes. The standard curves for shikimic acid are presented in Fig. 6: one for water solutions of pure shikimic acid, and the second regarding elution of shikimic acid from anion exchanger (Dowex l-X10). Both curves follow the Lambert-Beer law and shikimic acid can be de-

44

MOSSOR

0.1

AND

10

SCHRAMM

20

30

FRACTION

FIG. Column vessel;

5. Gradient of Dowex conveying

elution of shikimic l-X10, 9 cm length, 2 N acetic acid.

NUMbER

acid and galacturonic and glucuronic acids. 0.6 cm diameter; 150 ml water in the mixing

termined in the range from about 15 to over 120 ng per milliliter, that is, about 0.1 to 0.7 ymole. The method is then about twice as sensitive as the method of Yoshida and Hasegawa (10). pg/ml 5 t

0.8 --

Y ;

0.7-

20 I

40 I

60 I

eo

100 I

012

0.24

0.36

0.46

120 /

140 I

:: 060.5 -

0.60

0.72 ,uM/ml

FIG.

acid; Dowex

6. Standard curves for assay of shikimic (B) under the experimental conditions l-Xl0 column = percentage of recovery).

acid: (after

(A) pure solution of shikimic passing the solution through

With the method described, shikimic acid wa:: tletermined tively in our laboratory in many I)lant cstracts.

cpmtita-

Shikimic acid heated with p-hy(lrosyber~zaldehydc and concentrated sulfuric acid products an intense and stable purple-violet dye with al>sorption martimum at 590 nm, and can be quantitatively determined in the range from 15 to over 120 ng (0.1 to 0.7 pnole/dj. Under the sanlc conditions quinic acid produces no color reaction. Shikimic acid can be eel)aratetl from other acids giving a color with p-hydroxy\wnzaldehycle of varying intensity (uranic acids, phenolic derivatives of cinnamic acid, malonic acid) by clution from a Dowes l-Xl0 column with 0.3 .Y acetic acid. ACKNOWLEDGMENTS We are indebted for the samples of acids to Sigma Chemical CO., St. Louis, MO., for shikimic, quinic, and oxalacetic, to Calbiochem, Los Angeles, Cal., for galacturonic, glucuronic, and tartronic, and to Dr. H. Gertip for usnic. REFERENCES R. W., A~TDMOSSOR,T.. T’. Siclj. Synkp. P. !I’. Bid:. Kmkciw, Abstr. B-4, 16 (1967).Seealsofootnote 1 to the presentpaper. 2. ANET, E. F. I., BND REYNOLDS, T. M., Bust. J. Chem.8, 267 (1955).CARLES, J., SCHNEIDER, A., AND LACOSTE, 8. M.. Bull. Sot. C&m. Biol. 40, 221 (1958). SCHRAMM, R. W., Chem. Anul. 5, 1055 (1960). STEWARD, F. C., HULME, A. C., FREIBERG, S. R., HEGARTT, M. P.. POLLARD. J. K., R.4sso~,R., AND BARR, R. A., Ann. Bot. 24, 53 (1960). 3. HTLME, A. C., J. Erp. Bot. 2, 298 (1951).PALMER, J. Ii., Science126,504 (1957). WEINSTEIN, L.H.. PORTER, C. A., AND LAURENCOT, H.J.. Natwe 194,205(1962). 4. SCHRAMM, R. IV., UAM, Prrtce Il’ydz. BiNoZ, Biol. 2 (1961). 5. WHITING, G. C., n'atztre 179, 531 (1957). 6. SCHEFFER, F., KICKUTH. R.. AND I,~REx, H.. X:rctvrwiss.52, 517 (1965). 7. FISCHER, H. 0. I,., AND DASGSCHXI~. G.. HelLI. Chim. Actu 18, 1204(1935). 8. CARTWRIGHT. R. A.. AND ROBERTS. E. A. H.. (‘hem. Id. (.hndon) 9, 230 (1955). 9. MILLICAN, R. C.. Anal. Biochem. 6, 181(1963). 10. I'O~HIDA, S.. AND HASEGAw4, M., AITJL. Bioclre)n.Biophys. 70, 377 (1957). 11. MANSKAJ.4, S. M.. AND &IDIS.~, L. A., Z/z. f'tikl. R//im. (Leningrd) 32, 2711 (1959). 12. NAG.~S.~WA, M., BdI. Agr. C’hem. SW. Jnpnn 22, 205 (1958). 13. G~IToXDE, M. K.. AKD GORDOS. M. JV.. J. Viol. (‘hem. 230, 1043 (1958). 14. VOIGT, J., AND RAUSCHER. K.. l\'