ANALYTICAL EDITION
May 15, 1942
use of certain solvents and concerning t h e study on the effect of radiation during the measurements.
Literature Cited Baxter, J. G., and Robeson, C. D., Science, 92, 203 (1940). (2) Baxter, J. G . , and Robeson, C. D., personal communication, (1)
1941. (3) Embree, N. D., IND. ENG.CHEM.,ANAL.ED., 13, 144 (1941). (4) Ewing, D. T., Vandenbelt, J. M., Emmett, A. D., and Bird, 0.D.,Ibid., 12, 639 (1940).
(5) Hogness, T. R., Zscheile, F. P., and Sidwell, A. E., Jr., J . Phys. Chem., 41,379 (1937).
(6)
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Holmes, H. N., and Corbet, R. E., J. Am. Chem. Soc., 59, 2042 (1937).
(7) McFarlan, R. L., Bates, P. K., and Merrill, E. C., IND.EXG. CHEM.,ANAL.ED., 12, 645 (1940). (8) Mead, T. H., Biochem. J., 33, 589 (1939) (9) Mitchell, J. H., Jr., and Kraybill, H. R., IND. ENG.CHEM..
ANAL.ED., 13, 765 (1941). (10) Munch, R. H., J. Am. Chem. SOC.,57, 1863 (1935). (11) Wilkie, J. B., IKD.ENG.CHEW,ANAL.ED., 13,209 (1941). (12) Zscheile, F. P., and Comar, C. L., Bot. Gaz., 102,463 (1941). (13) Zscheile, F. P., White, J. W., Jr., Beadle, B. W., and Roach, R., Plant Physiol. (in press).
Determination of Certain Quercetinlike Substances Using a Klett-Summerson Photoelectric Colorimeter CLARENCE W. WILSON, California Fruit Growers Exchange, Ontario, Calif., AND LEROY S. WEATHERBY A N D WILLIAM Z. BOCK, University of Southern California, Los Angeles, Calif.
I
T WAS observed b y one of the authors t h a t boric acid dried with lemon juice gave a brilliant yellow coloration
(3). Further work led to the conclusion t h a t this coloration was produced b y a reaction between the boric acid and a flavone or group of flavones similar in structure t o quercetin:
i t to effloresce completely in air at 30" to 40' C., followed by heating in a thin layer to 100" for 2 hours.) However, on long standing with boric acid, citric acid produces a yellow coloration, so that i t was necessary to mix the two materials immediately before use. This was done b y mixing equal parts of two filtered solutions prepared as follows: A. Acetone 100 ml., anhydrous citric acid 10 grams B. Acetone 100 ml., boric acid to saturate
More specifically i t has been postulated that the reaction is due to the grouping within the dotted line (2, 4). Included in the materials thus reacting is citrin, believed b y Szent-Gyorgyi t o have vitamin activity and tentatively called b y him vitamin P (1). T h e necessity of a flavone-free diet in physiological work with citrin makes practical t h e test to be used, as its sensitivity will detect amounts as low as 2 or 3 micrograms. T h e Wilson boric acid test has been discussed and used considerably in a qualitative manner. However, it was the object of this work t o develop some method of accurate quantitative measurement of flavones, using this test. T h e color-forming substance, insoluble in toluene and chloroform, will dissolve in acetone saturated with boric acid t o give a yellow coloration, the intensity of which is a quantitative measure of the amount of flavone present. For color measurement i t was convenient to use a KlettSummerson photoelectric colorimeter with adaptor for the use of a test tube graduated in 5- and 10-ml. divisions. This eliminates the necessity of matching colors and is a n accurate measure of the intensity of color produced. T h e instrument has a logarithmic scale, thus making possible the production of linear curves on ordinary graph paper. T h e intensity of color is deepened considerably b y a n acid medium, b u t the strong acids react to give yellow colors with flavones. Acetic acid is too weak to develop the color fully. Anhydrous citric acid has been found suitable, considering strength, acetone solubility, and availability. (Citric acid hydrate may be conveniently rendered anhydrous b y allowing
This mixture is referred to hereafter as borocitric reagent. These separate solutions are apparently stable indefinitely. It was found that metaboric acid gives a more intense coloration, but the sensitivity of t h e solution t o traces of moisture, as shown b y copious precipitates of boric acid, was too great, and the use of metaboric acid was discontinued. The authors recognized the possibility of advantageous use of metaboric acid if its concentration was restricted to the equivalent of boric acid saturation. As the depth of color is affected greatly b y traces of moisture, attempts were made t o dehydrate the solvent acetone. As these methods of dehydration gave values which varied slightly, it was considered best to standardize with quercetin and to use one sample of acetone for all reagents. Merck's "Blue Label" acetone was found adequate as t o dryness.
Method of Standardization The standard used for the work with quercetin was pre ared by C. E. Sando (Food Research Division, Bureau of Agricurtural Chemistry and Engineering, Washington, D. C.) and re urified by Wilson. Weighed amounts of quercetin were dissofved in acetone and diluted to 100 ml. in volumetric flasks. The first sample of 56 mg. was found to give a color far too intense for the scale of the colorimeter. Therefore, further work was done with samples between 10 and 15 mg. Using the industrial test-tube model of the Klett-Summerson colorimeter, the test tube was filled t o the 10-ml. mark with a mixture of equal volumes of solutions A and B, and the zero point set for this reagent, using the blue filter KO.42 accompanying the instrument (value 400 to 465 millimicrons). The acetonequercetin solution was then dropped into the test tube, using a serological pipet, graduated in 0.01-ml. divisions. After the addition of each 0.1-ml. the tube was removed from the instrument, shaken well t o ensure proper mixing, and re-inserted and a reading xyas taken. The addition of 1 ml., in 0.1-ml. intervals, was found sufficient to determine response curves. As the quercetin solution is itself yellow, it was necessary also t o obtain a curve of colorimeter response to the acetone-quercetin
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The tissue was dried in a 60' oven under vacuum, then ground, and a sample was taken, generally from 1 to 5 grams. This was extracted with methyl alcohol in a Soxhlet extraction apparatus, and the extracted liquid was evaporated to dryness on a water bath. Chlorophyll, fats, resins, etc., were next removed from the dry extract by digestion with chloroform. Sometimes, because of the quantity of substances extracted by the methyl alcohol, this chloroform extraction is incomplete, chlorophyll often remaining. In such cases the residue may be redissolved in a small amount of methyl alcohol, a few milliliters of toluene added, the methyl alcohol removed by evaporation, and the chloroform digestion repeated. The resultin residue is dissolved in acetone, filtered, and diluted to 100 in a volumetric flask. The readings with borocitric reagent shown in Table I1 were made by taking sufficient of the acetone solution of the extract to give a reading in the desired range (suitably 100 to 250) and adding sufficient reagent to fillthe tube to the 10-ml. mark. Blank determinations were then made, using the same quantities of the extracts and a mixture of equal parts of solution A and acetone. The net reading is the difference between these two skts of readings, and the quercetin equivalent value was the result of calculating the quantity found on the net curve back to the original material;
A.
Results on some typical plant and animal materials are shown in Table 11. MICROGRAMS QUERCETIN/lO mL.SOLUTION
FIGUF~E 1
TABLE I. CALIBRATION OF COLORIMETER WITH QUERCETIN (Quercetin strength: 10.2 mg. per 100 ml.) Micrograms of Scale Reading with: Quercetin per 10 MI. Borocitric Citric acidNet of Solutions reagent acetone Reading 0.1 10.1 28 6 22 0.2 20.0 55 10 45 0.3 29.7 81 14 67 0.4 39.2 105 18 87 0.6 57.7 155 22 133 0.8 75.6 202 26 176 1.0 92.7 248 31 217 a Quercetin solution was added to 10 ml. of reagent, but concentration of quercetin is calculated as micrograms per 10 ml. of total solution. M I . of Solution Added to 10 MI. of Reagent
I n order to check the accuracy of the method of extraction, 0.82 gram of dried bean leaves (representing 8.2 grams of fresh material) was added to an acetone solution of quercetin containing 1.70 mg. of quercetin. The acetone was evaporated and the fortified leaves were extracted as outlined above. The final residue was taken up in acetone and diluted to 50 ml. Using 1 ml. of this solution, the net reading was 115, equivalent to 50 micrograms of quercetin or 2.50 mg. of quercetin for the 8.2 grams of fresh leaves. According to Table I1 this quantity of bean leaf contained 0.86 mg. The recovery of added quercetin was 2.50 - 0.86 or 1.64 mg.
Readings with smaller amounts of bean leaf and lemon peel extracts showed a possibility of detection of amounts as small as 2 or 3 micrograms of quercetin with the equipment used. Amounts below this would be TABLE 11. FLAVONE DETERMINATION IN BIOLOGICAL MATERIALS difficult to detect. Volume of Reading with. hiiorograms Quercetin EquivaWeight Taken Solution Borocitric Citric'acidNet of Quercetin lent Mg. per Gram Rabbit liver, a n animal Material Fresh Dry Taken, MI. reagent acetone Reading Equivalent of 'Fresh Weight tissue, showed no detectable 0.105 Bean leaf 17.53 1.753 1.0 151 113 38 18.4 quercetin, whereas the plant 1.71 44.0 Lemonpeel 25.75 6.799 0.1 113 13 114 1.65 loo 48.8 Lemon peel 5.92 1.563 0.5 142 tissues seem to be excellent 1.74 60.0 Lemon eel 6.89 1.820 0.5 170 30 140 Rabbitfivera ... ... .. .. ..28 .. .. sources. This may be explained a Undetectable. in several ways: the unlikely Dossibilitv of a flavone-free 'diet; t h e possibility t h a t the solution, free of the boroflavone reaction. This was done by flavones are changed in the animal body into materials mixing it with 5 ml. of solution A and 5 ml. of pure acetone and which do not give the boric acid color reaction; and the posusing the measuring technique described above. Thus a curve sibility t h a t the flavones are conjugated to some form not could be plotted for the boroflavone reaction and then for the solubilized b y the procedure employed. acetone-quercetin solution alone, The net reading of these two curves gave the curve due to the boroflavone reaction with quercetin. From this curve were taken the data needed in Summary determining quercetin equivalents of natural materials. A method for quantitative determination of quercetinlike The reaction is so sensitive to moisture that all precautions must be taken to exclude even traces of moisture from the acetone substances wTith the use of a Klett-Summerson photoelectric and from the materials used. As little as 1 per cent of moisture colorimeter has been described. Quercetin equivalent condecreases the color response by half. It was necessary to dry tent of bean leaves was found to be 0.105 mg. per gram and the quercetin in a vacuum desiccator for 2 days before use and of lemon peel, 1.70 mg. per gram. No flavone could be to use care in mixing the borocitric with the quercetin solution. It was advisable in shaking the test tube to cover its lip with thin detected in a sample of rabbit liver. Pure quercetin added tin foil (previously washed in acetone) rather than to run the to dried bean leaves was quantitatively recovered. risk of picking up moisture from the fingers.
...
The net curves based on these data are shown in Figure 1. As these are straight lines, it is both simple and advisable to replot them for each lot of reagents.
Extraction Procedure For the determination of the quercetin equivalents of tissues, the following procedure was used:
Literature Cited (1) Russnyak, I., and Szent-Gyorgyi, A., Nature, 138, 27 (1936). (2) Wilson, C. W., J . Am. Chem. SOC.,61, 2303-6 (1939). (3) Wilson, C. W., U. S. Patent 2,255,341 (Sept. 9, 1941). (4) Wolfram, M ,L., hfahan, J. E., Morgan, P. W., a n d Johnson, G . F., J. Am. Chem. Soc., 63, 1248-53 (1941). PRESENTED before the Division of Agricultural and Food Chemistry at the 102nd Meeting of the AMERICAN CHEXICAL SOCIETY, Atlantio City, N. J.
