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A PRECISE METHOD FOR THE DETERMINATION OF COUMARIN, MELILOTIC ACID, AND COUMARIC ACID IN PLANT TISSUE* BY WILLARD L. ROBERTS AND KARL PAUL LINK (From the Biochemistry Research Laboratory, Department of Agricultural Chemistry, University of Wisconsin, Madison) (Received for publication, March 24, 1937) The relatively unpalatable nature of the common sweet clovers, Melilotus alba and Melilotus oficinalis, and their tendency to be- come toxic to live stock if improperly cured has stimulated bio- chemical studies in this genus. Recently a non-bitter sweet clover was found by Brink (1) which proved to be an annual form of Melilotus dentata. This led to cooperative studies1 which have as their ultimate objective the improvement of the agricultural usefulness of sweet clover by developing more palatable strains which are also free of the tendency to become toxic. A thorough qualitative study of the two species of common sweet clover shows a marked difference in chemical composition as compared with the non-bitter Melilotus dentata. We have found that the seeds and green tissue of the bitter species contain large quantities of coumarin, usually smaller amounts of melilotic acid, and only traces of coumaric acid. The seeds of Melilotus dentata, on the other hand, contain only small amounts of cou- marin. Melilotic acid and coumaric acid, if present, exist only in traces. The green tissue of Melilotus dentata appears to contain, at the most, only minute traces of the three compounds. In order to determine the relationship of these substances to the palatability of the fresh tissue and the toxicity of the spoiled hay, a quantitative procedure for measuring them had to be developed. * Published with the permission of the Director of the Wisconsin Agri- cultural Experiment Station. 1 Cooperative studies with the Department of Genetics, University of Wisconsin, and the Division of Forage Crops and Diseases, United States Department of Agriculture. 269 by guest on June 6, 2018 http://www.jbc.org/ Downloaded from

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Page 1: A PRECISE METHOD FOR THE DETERMINATION OF … · A PRECISE METHOD FOR THE DETERMINATION OF COUMARIN, MELILOTIC ACID, ... gravimetric procedure developed by us2 ... Determination of

A PRECISE METHOD FOR THE DETERMINATION OF COUMARIN, MELILOTIC ACID, AND COUMARIC

ACID IN PLANT TISSUE*

BY WILLARD L. ROBERTS AND KARL PAUL LINK

(From the Biochemistry Research Laboratory, Department of Agricultural Chemistry, University of Wisconsin, Madison)

(Received for publication, March 24, 1937)

The relatively unpalatable nature of the common sweet clovers, Melilotus alba and Melilotus oficinalis, and their tendency to be- come toxic to live stock if improperly cured has stimulated bio-

chemical studies in this genus. Recently a non-bitter sweet clover was found by Brink (1) which proved to be an annual form of Melilotus dentata. This led to cooperative studies1 which have as their ultimate objective the improvement of the agricultural usefulness of sweet clover by developing more palatable strains which are also free of the tendency to become toxic.

A thorough qualitative study of the two species of common sweet clover shows a marked difference in chemical composition

as compared with the non-bitter Melilotus dentata. We have found that the seeds and green tissue of the bitter species contain large quantities of coumarin, usually smaller amounts of melilotic acid, and only traces of coumaric acid. The seeds of Melilotus dentata, on the other hand, contain only small amounts of cou- marin. Melilotic acid and coumaric acid, if present, exist only in traces. The green tissue of Melilotus dentata appears to contain, at the most, only minute traces of the three compounds.

In order to determine the relationship of these substances to the palatability of the fresh tissue and the toxicity of the spoiled hay, a quantitative procedure for measuring them had to be developed.

* Published with the permission of the Director of the Wisconsin Agri- cultural Experiment Station.

1 Cooperative studies with the Department of Genetics, University of Wisconsin, and the Division of Forage Crops and Diseases, United States Department of Agriculture.

269

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270 Coumarin, Melilotic and Coumaric Acids

It is clear from the accompanying formulae that the determination of three compounds so closely related in chemical structure pre- sents a formidable problem.

Coumarin Coumarinic acid (cis form)

Coumaric acid Melilotic acid Melilotic acid (tram form) lactone

Methods for the determination of coumarin or coumarin and melilotic acid have been described by others. However, it was soon learned in the course of this study that none of the existing procedures was capable of the degree of precision and accuracy demanded in this work.

The permanganate oxidation method originally developed by Obermayer (2) for the determination of coumarin has been shown to be faulty. Stevenson and Clayton (3) showed there is consider- able loss of coumarin in the preparation of a sample dry enough for ether extraction, and Duncan and Dustman (4) proved that de- structionof coumarin takes place in the prolonged ether extraction.

The gravimetric procedure developed by Kanewskaja and Fedorowa (5) for the determination of coumarin and melilotic acid is not only subject to the inherent weaknesses of Obermayer’s method, but is also rendered unreliable by the fact that the melilotic acid fraction is grossly contaminated with other organic acids and plant pigments.

Duncan and Dustman (4) improved Obermayer’s method to some extent, but found it desirable to retain the tedious step of

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W. L. Roberts and K. P. Link 271

removing the coumarin from the tissue by steam distillation, which, in their .procedure is repeated six times.

The method developed by Clayton (6), which was subsequently slightly revised by Clayton and Larmour (7) and Stevenson and Clayton (3), is based on the formation of a red dye when diazo- tized p-nitraniline is coupled with coumarin and related com- pounds in an alkaline solution. This method is rapid and valu- able for certain types of work but has two pertinent handicaps. It does not present a procedure which enables the complete sep- arate estimation of coumarin and related compounds. Secondly, these substances are measured under conditions that do not elimi- nate the errors introduced by the presence of interfering plant pigments and certain phenolic bodies.

The calorimetric method described below is a modification of a gravimetric procedure developed by us2 (unpublished) which gave satisfactory results but was too slow and involved too large a sample. In the calorimetric procedure the finely ground green tissue or seed is first extracted with acidulated 10 per cent aqueous acetone. The extracted coumarin, melilotic acid, and coumaric acid are separated from the original acetone solution by contin- uous extractions with selective solvents. The amount of the indi- vidual components (coumarin, melilotic acid, and coumaric acid) are finally measured calorimetrically after coupling with the dia- zonium solution introduced for the purpose by Clayton et al. (6).

EXPERIMENTAL

Description of Apparatus Employed-Pyrex test-tubes. 25 X 200 mm. marked for 50 ml. and 30 X 200 mm. unmarked.

Shaking machine. The motion of the shaking machine should be such as to provide a back and forth movement of the extraction solution-green tissue mixture in the test-tubes.

Liquid-liquid extractors (8). These are conveniently pre- pared from 25 mm. Pyrex tubing. The length over all is 400 mm.; the 12 mm. side arm is exactly centered on the length of the tube. The inner tube, length 410 mm., is prepared from 7 mm. Pyrex tubing. One end carries a bulb perforated with a few small equally spaced holes; the other end a funnel of appropriate size.

2 The gravimetric procedure developed can be used for the isolation and characterization of coumarin and related substances and will be described in a separate paper.

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272 Coumarin, Meliiotic and Coumaric Acids

The holes in the bulb must be small, equal in size, and in the same plane, so that many small bubbles of ether will be evolved rather than single large bubbles from the largest hole. Each extractor is fitted with a West condenser approximately 750 mm. in length. The 125 ml. Erlenmeyer flask containing the solvent is heated in a beaker of water which is in turn heated by a small flame.

Solvents and Xolutions- Petroleum ether. B.p. not over 40”. Ethyl ether. Washed with three consecutive portions of water

for the removal of most of the alcohol. Extraction solution. 1 volume of acetone and 9 volumes of

approximately 0.1 N H&04. Standard coumarin, melilotic acid, and coumaric acid solutions.

Each contains 0.1 mg. per 1 ml. of solution. pNitraniline hydrochloride (Solution A). Dissolve 3.5 gm. of

p-nitraniline in 45 ml. of 37 per cent hydrochloric acid, dilute to 500 ml. with distilled water, and filter. This solution keeps in- definitely if stoppered.

Sodium nitrite (Solution B). Dissolve 5 gm. of sodium nitrite in 100 ml. of distilled water. Keep this solution in a dark bottle away from light and renew it frequently.

Diazonium solution. Thoroughly chill a 100 ml. flask and Solutions A and B in chipped ice. Now pipette 3 ml. of Solution A and 3 ml. of Solution B into the 100 ml. flask, chill for 5 minutes, add 12 ml. of Solution B, swirl, chill for another 5 minutes, fill to the mark with ice-cold distilled water, mix, and place in chipped ice for 15 minutes before using. If kept on ice, this solution will remain stable for 24 hours.

Preparation of Color Standards-Color standards, for comparison purposes, are prepared by pipetting the requisite amounts of the standard solutions in question into test-tubes marked for 50 ml. 5 ml. of 1 per cent Na2C03 solution and sufficient distilled water to make a volume of approximately 40 ml. are then added. The coumarin standards are then heated for 15 minutes at 85”, after which they are cooled. The melilotic acid and coumaric acid standards are not heated. Now add 5 ml. of the diazonium solu- tion, make to volume, mix, let stand for 2 hours, and use for comparison purposes. The standards are good for several days after the color is developed.

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Analytical Procedure

Preparation of Sample-The sample should consist of at least 5 gm. of fresh green tissue. This material is sliced as finely as possible and is thoroughly mixed prior to weighing the portions for analysis. When seeds are to be analyzed, they should be ground in a mortar, and the ground mass well mixed.

Determination of Moisture-Weigh approximately 1 gm. to f. 0.001 gm. into a tared, flat bottom dish. Place in an air oven maintained at 100-105’ for 4 hours. Cool and weigh. From the loss in weight calculate the per cent moisture.

Preparation of “Solution for Analysis”3-Weigh a 3 gm. portion of sliced tissue or 1.5 gm. of ground seeds to fO.OO1 gm. and transfer to a 100 ml. test-tube containing exactly 50 ml. of the extraction solution; close with a rubber stopper and start the shaking.

When the results of the moisture determination are obtained, remove the tube from the shaking machine and, using a burette, add an amount of the extraction solution that will make a total of 25 ml. when added to the volume of water contained in the sample involved. Now add a pinch of dry asbestos, close with the rubber stopper, and shake for a total of 24 hours.

After shaking, filter with suction through a small Buchner funnel fitted with a disk of qualitative filter paper and a mat of asbestos.4 Transfer 50 ml. of the clear filtrate (representing 2 gm. of plant tissue or 1 gm. of seeds) to the extractor and extract for 2 hours with ethyl ether.K Remove the inner tube and transfer the ether layer in the extractor to the extraction flask.‘j Now add 20 ml. of distilled water to the ether extract and carefully evaporate the ether by swirling the flask in a water bath (50-55’). Heat the aqueous solution to about 70” to dissolve water-soluble substances

3 See “Addendum.” 4 The Buchner funnel must be dry. During the filtration it is often

necessary to scratch the surface of the asbestos to increase the rate of filtra- tion. The extract of ground seeds cannot be filtered. In this case centri- fuge and take a 50 ml. aliquot of the turbid supernatant liquid.

6 Since superheating of the ether may cause decomposition, the 125 ml. flask should be immersed in the water bath (kept at SO’) in such a way that one-half of the ether in the flask is above the surface of the water.

6 This is easily done by displacing the ether by means of a carefully in- serted test-tube of the proper size.

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274 Coumarin, Melilotic and Coumaric Acids

and cool. Filter through a small qualitative filter paper into a 125 ml. flask and wash the original flask and the filter with 20 ml. of distilled water. The filtrate constitutes the ‘%olution for analysis.”

Separate Determination of Coumarin, Melilotic Acid, and Coumaric Acid

Coumarin-Add 0.5 ml. of 5 N NaOH to the solution for analysis, heat just to boiling, cool, add 0.75 ml. of 5 N H&SO+ mix thor- oughly, add 0.25 gm. of anhydrous NaHC03, mix, and transfer to the extractor. Rinse the flask with distilled water and transfer to the extractor until the volume is 50 ml. Extract immediately for 3 hours with petroleum ether, remove the inner tube, and transfer the petroleum ether in the extractor to the extraction flask. Add 20 ml. of water to the petroleum ether extract and carefully evaporate the ether by swirling the flask in a water bath (50-55’). Transfer the aqueous solution to a 50 ml. volumetric flask, make to volume, and mix. Pipette a 25 ml. aliquot into a test-tube graduated for 50 ml., add 5 ml. of 1 per cent NaG03 solution, heat in a water bath at 85” for 15 minutes, and cool. Add 5 ml. of the diazonium solution, make to volume, mix, let stand 2 hours, and compare in a calorimeter against a coumarin standard of approximately the same color intensity. If the cou- marin content of the 25 ml. aliquot exceeds 0.8 mg., repeat the color development with a smaller aliquot. Calculate the per- centage of coumarin to the dry basis.

Melilotic Acid-Now add 1 ml. of 5 N H&S04 to the aqueous solution remaining in the extractor, mix, extract for 2 hours with ethyl ether, remove the inner tube, and transfer the ether in the extractor to the extraction flask. Carefully filter the ether extract through a small dry filter paper into a dry 125 ml. flask. Wash the original flask and filter thoroughly with ether and evaporate just to dryness.7 Add 5 ml. of benzene and with occasional swirl- ing boil for a few minutes on a steam cone. Cool for 5 minutes in chipped ice and let stand at room temperature for about 30 minutes. Now carefully decant the benzene extract through a small retentive filter paper (Whatman No. 44 or its equivalent) into a 50 ml. separatory funnel and wash the inside of the flask

7 Water must be absent at this point.

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and the filter three times with a small jet of benzene. The total filtrate, washings included, should not exceed 15 ml. of benzene. This extract contains the melilotic acid. The Erlenmeyer flask and filter paper contain the coumaric acid. Add 15 ml. of 0.2 per cent Na&03 solution to the benzene extract and shake vigor- ously. Let stand a few minutes and draw off the aqueous layers through a qualitative filter paper into a 50 ml. volumetric flask. Repeat the shaking and drawing off process with a fresh 10 ml. portion of 0.2 per cent Na&03 solution and follow with 10 ml. of distilled water. Wash the paper and the funnel with distilled water, make to volume, and mix. This solution contains 5 ml. of 1 per cent Na2C03 solution. Pipette a 25 ml. aliquot into a test-tube marked for 50 ml., add 2.5 ml. of 1 per cent NaG03 solution, and mix. Add 5 ml. of the diazonium solution, make to volume, mix, let stand 2 hours, and compare in a calorimeter against a melilotic acid standard of approximately the same color intensity. If the melilotic acid content of the 25 ml. aliquot exceeds 0.8 mg., repeat the color development with a smaller aliquot and the proper amount of 1 per cent Na&03 solution. (For suitable color development 5 ml. of 1 per cent Na2C03 solu- tion is necessary for the 5 ml. of diazonium solution.) Calculate the percentage of melilotic acid to the dry basis.

Coumaric Acid-Free the coumaric acid-containing Erlenmeyer flask and filter from adhering benzene by drying in a steam oven. Add 5 ml. of 1 per cent NaZC03 solution to the flask, swirl, and pass through the filter into a test-tube marked for 50 ml. Wash the flask and filter with distilled water until the volume in the test- tube is about 40 ml., add 5 ml. of the diazonium solution, make to volume, mix, let stand 2 hours, and compare against coumaric acid standards of approximately the same color intensity. Calcu- late the percentage of coumaric acid to the dry basis.

Coumarin Singly, Melilotic Acid, and Coumaric Acid in Terms of Melilotic Acid

Using the “solution for analysis” determine coumarin as di- rected in the procedure above. After the extraction of coumarin add 1 ml. of 5 N H&304 to the aqueous solution remaining in the

8 Occasional emulsions may be broken by agitation of the affected area with a glass rod.

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276 Coumarin, Melilotic and Coumaric Acids

extractor, mix, extract for 2 hours with ethyl ether, remove the inner tube, and transfer the ether in the extractor to the extraction flask. Add 20 ml. of water to the ether extract and carefully evaporate the ether in the usual manner. Transfer to a 50 ml. volumetric flask, make to volume, and mix. Couple an aliquot with the diazoninm solution as directed under the procedure above for melilotic acid and compare against melilotic acid standards. Calculate to the dry basis and report as melilotic acid and cou- maric acid in terms of melilotic acid.

Coumarin, Melilotic Acid, and Coumaric Acid in Terms of Coumarin

Add 0.5 ml. of 5 N NaOH to the solution for analysis, heat just to boiling, cool, and add 1 ml. of 5 N H&Q; mix thoroughly and transfer to the extractor. Rinse the flask with distilled water and transfer to the extractor until the volume is 50 ml. Extract for 2 hours with ethyl ether, remove the inner tube, and transfer the ethyl ether in the extractor to the extraction flask. Add 20 ml. of distilled water to the ether extract and carefully evaporate the ether in the usual manner. Transfer to a 50 ml.volumetric flask, make to volume, and mix. Couple an aliquot with the diazonium solution as directed under the procedure for co,umarin and com- pare against coumarin standards.,> Calculate to the dry basis and report as coumarin, melilotic acid, and coumaric acid in terms of coumarin.

Results

Table I shows that when the method is applied to pure solu- tions of coumarin, melilotic acid, and coumaric acid in admixture with each other accurate recoveries for the first two substances are realized over the entire range. With coumaric acid the accuracy drops off when the concentration falls below 0.1 mg.

Table II shows good recoveries when the pure compounds are added to a plant extract. Alfalfa tissue was used in this case, because it gave less red color in the various fractions than the particular Melilotus dentata on hand at the time the analyses were made. Here again accuracy and precision are realized except when the coumaric acid content is below a certain level.

Some typical results are shown in Table III. It is interesting

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to note that Me&lotus dentata seeds contain a small amount of coumarin, whereas the foliage appears to be devoid of this com- pound.

Table IV shows similar results obtained by our colleague Mr. Mark Stahmann in additional control determinations. One

TABLE I

Recovery of Varying Quantities of Coumarin, Melilotic Acid, and Coumaric Acid When in Admixture with Each Other in Acidulated 10 Per Cent

Acetone Solution

Run No. i Coumarin

Added Found

ml. ml.

25.12 25.10

10.05 9.98

2.00 2.00 0.10 0.099 0.10 0.099

Melilotic acid I

Coumaric acid

Added Found

WJ. mJ.

2.51 2.51 1.00 1.04 1.00 1.05 1.00 1.04 0.10 0.140

Added Found

ml. mg.

2.02 1.99

1.00 0.98

1.00 0.98

0.50 0.48 0.10 0.036

TABLE II

IZecovery of Varying Quantities of Coumarin, Melilotic Acid, and Coumaric Acid When Added to Acidulated 10 Per Cent Acetone Extract of 2 Gm. of

Alfalfa Tissue

I Coumarin

%

1 2 3 4 5

Added

w7.

20.08 5.02 0.10 0.10

None

ml.

20.10 5.00 0.124 0.134 0.027

ml.

20.07 4.97 0.097 0.107

Melilotic acid I

Coumaric acid

Added Found COP rected’ Added Found

~----

ml. m?. “8. m7. ml.

2.64 2.83 2.65 1.23 1.33 0.88 1.14 0.96 1.24 1.33 5.54 5.71 5.53 0.61 0.64 0.100 0.280 0.100 0.100 0.109 None 0.180 None 0.080

COP reoted*

“8.

1.25

1.25

0.56 0.029

* Corrected for the blank obtained with alfalfa control tissue to which no coumarin, melilotic acid, or coumaric acid had been added.

sample of Melilotus ojicinalis analyzed in quadruplicate shows that the method gives reproducible results. The remaining data in Table IV also show the accuracy of the method. This sample of Melilotus dentata contained many seed pods which are probably responsible for the small percentage of coumarin. The Melilotus dentata in Table III carried no seed and contained no coumarin.

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278 Coumarin, Melilotic and Coumaric Acids

TABLE III

Coumarin, Melilotic Acid, and Coumaric Acid Content of Green Tissue and Seeds of Various Species of Melilotus

Sample / Moisture / Coumarin 1 M$$tiO / “;$riO

Green tissue

per cent per cent on per cent on pet cent on dry basis dry basis dry basis

M. alba (Hubam), 1st cutting July30......................... 72.1 2.08 0.14 0.010

M. dentata (annual). _. . . . . . . . 72.3 0.0000 0.027 0.033 Alfalfa (control). . . . . . . . . . . . . 70.9 0.0047 0.030 0.013

Seed

M. oficinalis (biennial). . . . . . 6.76 “ aZba (biennial). . . . . . 6.81 “ dentata, AC-85 (biennial). _. . 6.87 ,‘ “ AC-89 “ . . 6.70 “ ‘C (annual). . . . . . 8.32

Alfalfa (control). . . . . . . . . . . . . 6.95

TABLE IV

0.63 0.023 0.46 0.020 0.074 0.012 0.040 0.012 0.021 0.0087 0.0058 0.0075

-

-

0.0059 0.0060 0.0088 0.0088 0.0054 0.0053

Analyses by Independent Worker, Unfamiliar with Analytical Procedure, Showing Reproducibility and Accuracy of Method

Sample

M. ojkinalis ............. ; ............ ‘L “ .......................... “ ‘I .......................... “ ‘, .......................... “ ‘< A-9 ...................... ‘I ‘, A-9. ..................... “ alba (Hubam), 2nd cutting Ost. 20. “ t< ,‘ 2nd “ “ 20. “ alba, A-10 ......................... ,‘ “ A-10. ........................ “ ‘I A-43. ......................... “ ‘I A-43. ........................ “ dentata (annual). .................. “ “ “ ...................

Alfalfa (control). ..................... “ ‘< ......................

I

.-

-

Moisture

per cent 70.8 70.8 70.8 70.8 69.5 69.5 77.7 77.7 69.1 69.1 73.5 73.5 75.0 75.0 71.2 71.2

(

-

humarir; on dry basis

per cent

0.69 0.69 0.68 0.69 0.64 0.67 0.086 0.088 0.36 0.37 0.93 0.97 0.018

0.0041 0.0080

per cent

0.043 0.045 0.041 0.043 0.24 0.26 0.40 0.40 0.27 0.27

0.28 0.026 0.025 0.025 0.026

(

i

- _

-

Zoumaric acid on

dry basis

per cent

0.014 0.014 0.020 0.016 0.035 0.038 0.035 0.031 0.048 0.062 0.018 0.019 0.042 0.035 0.025 0.025

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DISCUSSION

The determination of coumarin, melilotic acid, and coumaric acid as outlined in this paper gives satisfactory results with common sweet clover. The red colors in the coumarin and melilotic acid fractions are easily compared against the proper standards. The coumaric acid fraction may offer some difficulty owing to the presence of a yellow color. This difficulty can be largely avoided if the reader concentrates on the intensity of the red color while making the color comparisons.

The results obtained in the analysis of alfalfa and Melilotus dentata appear to be somewhat in error. In the case of Melilotus dentata seeds coumarin is responsible for the red color obtained in the coumarin fraction. We believe that ‘most of the red color produced in the melilotic acid and coumaric acid fractions is due to the presence of other phenolic bodies. The red colors developed are of a different shade than those produced by the pure com- pounds. Some of the naturally occurring compounds which we have been able to couple to form a red dye are p-hydroxybenzoic acid, gallic acid, guaiacol, phenol, phloroglucinol, protocatechuic acid, pyrocatechol, pyrogallol, thymol, and vanillic acid. It is possible that one or more of these compounds or others are re- sponsible for the traces of red color.

In comparative studies on different species of Melilotus, the traces of coumarin and the small amounts of phenolic bodies con- taminating the melilotic acid and coumaric acid fractions can be disregarded. The real interest centers in the use of the method with common sweet clovers which contain appreciable amounts of coumarin, melilotic acid, and coumaric acid which can be measured accurately by the method described.

The plant geneticist and forage crop specialist thereby have at their disposal a chemical method for the absolute and comparative estimation of coumarin and related substances in studies dealing with the selection of species or strains of Melilotus.

SUMMARY

A calorimetric method for the determination of coumarin, melilotic acid, and coumaric acid in small amounts of sweet clover tissue and accurate for the range in which these constituents occur has been described.

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The method is based on the principle of first extracting the com- ponents from the green tissue or seeds with acidulated aqueous acetone (10 per cent). After each component has been separated from the crude extraction mixture, through the use of a selective solvent, it is coupled with a diazonium solution prepared from p-nitraniline. The coupling procedure produces a red dye which lends itself to a calorimetric estimation in an ordinary calorimeter. The color comparisons are made against known standards of coumarin, melilotic acid, and coumaric acid produced by coupling the pure substances with the diazonium reagent.

Summarized tabulations showing the performance of the method with varying amounts of pure coumarin, melilotic acid, and coumaric acid in admixture with each other and when added to alfalfa tissue as control are given. The analyses of various species of sweet clover and alfalfa tissue for coumarin, melilotic acid, and coumaric acid are presented. It is shown that the method can be employed to differentiate between so called bitter and non-bitter species of Melilotus on the basis of their coumarin, melilotic acid, and coumaric acid content.

The writers wish to express their thanks and appreciation to Dr. E. A. Hollowell of the United States Department of Agricul- ture, Washington, and Professor R. A. Brink and Dr. W. K. Smith of the Genetics Department of this institution for financial assist- ance and counsel. The help of Mr. Mark Stahmann, Graduate Training Fellow, on control determinations is also gratefully acknowledged.

Addendum-After the above communication was submitted, it was learned that the estimation of coumarin content of sweet clover tissue and sweet clover seeds (particularly the latter) can be placed on a much firmer basis by including an incubation period for the enzymatic release of bound coumarin. Proceed as follows: incubate the 1.50 gm. of ground tissue or 3.0 gm. of the finely minced green tissue with 10 ml. of water (in the closed test-tube) at 40” for 1 hour. After the incubation period the mixture is made to the desired volume with the extraction solution. Thorough shak- ing and filtering in the manner described yield the “solution for analysis.”

BIBLIOGRAPHY

1. Brink, R. A., Science, 79, 301 (1934). 2. Obermayer, E., 2. anal. Chem., 62, 172 (1913).

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3. Stevenson, T. M., and Clayton, J. S., Canad. J. Research, Sect. C, 14, 153 (1936).

4. Duncan, G. J:, and Dustman, R. B., Ind. and Eng. Chem., Anal. Ed., 6, 210 (1934).

5. Kanewskaja, S. G., and Fedorowa, A. M., 2. anal. Chem., 93,176 (1933). 6. Clayton, J. S., Thesis, University of Saskatchewan (1934). 7. Clayton, J. S., and Larmour, R. K., Canad. J. Research, Sect. C, 13, 89

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Willard L. Roberts and Karl Paul LinkACID IN PLANT TISSUE

MELILOTIC ACID, AND COUMARICDETERMINATION OF COUMARIN,

A PRECISE METHOD FOR THE

1937, 119:269-281.J. Biol. Chem. 

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