the non-volatile organic acids of green

THE NON-VOLATILE ORGANIC ACIDS OF GREEN TOBACCO LEAVES*

BY HUBERT BRADFORD VICKERY AND GEORGE W. PUCHER

(From the Biochemical Laboratory of the Connecticut Agricultural Experiment Station, New Haven)

(Received for publication, December 24, 1930)

Accurate information on the organic acids of leaves is surpris- ingly meager; even the identity of the acidic substances in many of the plant tissues that have been investigated is still in doubt. Franzen and Keyssner (1) pointed out some years ago that the identity of the malic acid had been conclusively proved in only 15 of 235 plants reported in the literature to contain this substance and a series of critical reviews by Franzen and his associates (2-4) showed that the available information for the distribution of citric, tartaric, succinic, and lactic acids is almost equally unsatis- factory. Franzen’s own extensive studies of the acids of fruits and leaves (e.g. (5,6)) demonstrated the nature of the predominating acids as well as the identity of the accompanying acids but permitted statements only of the relative proportions of these substances, not of their absolute amounts, in the tissue.

Malic, citric, tartaric, and lactic acids are probably the most widely distributed of the aliphatic organic acids commonly found in aqueous extracts from green leaves. Oxalic and succinic acids are also frequently found; fumaric, malonic, and glycolic acids have been occasionally observed. The function of these substances in the metabolism of the leaf is unknown. They obviously play some part. in the stabilization of the reaction of the cell as most of them have considerable buffering capacity at hydrogen ion activities within the range usually observed in leaf extracts, but opinions differ as to whether they are metabolites

*The expenses of this investigation were shared by the Connecticut Agricultural Experiment Station and the Carnegie Institution of Washing- ton.

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Organic Acids of Tobacco Leaves

carbohydrates or of protein. Many recent workers, however, arrpear to incline to the latter view (7, 8).

The strongly basic alkaloid nicotine occurs in the leaves of the tobacco plant in relatively high concentration, frequently more than 3 per cent of the dry weight. The reaction of extracts from these leaves is, nevertheless, almost invariably faintly acid (about pH 5.5). A study of the acidic substances present is therefore a prerequisit’e to the understanding of the acid-base relationships in the cells. Garner, Bacon, and Foubert (9) have obt.ained data on the oxalic, malic, and citric acid content of samples of dried green leaves and partially cured leaves of several varieties of Connecticut tobacco. They employed an indirect method proposed by Kissling (10) .l Cured and fermented tobaccos of oriental types have been extensively investigated by the Russian workers at Krasnodar, and a summary of t,heir results was recently given by Schmuck (11). Fumaric, oxalic, malic, and citric acids were isolated and identified positively in the course of theirquali- tative investigations; t,he identification of succinic and caffeic acids was less satisfactory. Malic, citric, and oxalic acids were likewise identified in green leaves (12).

Although malic, citric, and oxalic acids appear to be the predominating acids of tobacco leaves, the other acids being present

1 Kissling mixed 10 gm. of finely ground tobacco with an equal weight of 20 per cent sulfuric acid and a small quantity of pumice, extracted the mixture with ether for 20 hours, and dissolved the ether extract in water. Half of this solution was treated with calcium acetate in the usual way for the determination of oxalic acid. The other half was neutralized with barium hydroxide, and alcohol was added to make 20 per cent by volume. The precipitate was filtered off at once and the alcohol concentration of the filtrate was raised to 70 per cent. The precipitate so produced was filtered after several hours. The two precipitates were ignited and the small amount of barium oxide formed was converted to carbonate by the addition of a little ammonium carbonate solution followed by gentle ignition. The weights of the ashes, assumed to be barium carbonate, were taken as the equivalents of the sum of the oxalic and citric acids for the first precipitate and as the equivalent of the malic acid for the second. This method, although it gave fairly satisfactory results on mixtures of the three pure acids, was not regarded by Kissling himself as very accurate. He recognized the inadequacy of the separation of the acids and also the difficulty of securing a quantitative extraction by ether. Furthermore it may be pointed out that no account is taken of the possibility that other substances may con- taminate the precipitates.


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H. B. Vickery and G. TV. Pucher

only in small amount.s, the value of the quantitative determinations of t.hese three substances that have been published is dubious, owing to the universal use of indirect methods of analysis. Our invest.igations of t,he acids of green tobacco leaves have been conducted with the view of obtaining information not only of the nature but also of the quantities of these substances present. To this end we have subjected the various methods of organic acid analysis that have been proposed to critical study. The only method that promised to give trustworthy qualitative and quan- tit,ative results is that employed by Franzen and his associates (13) in the investigation of a variety of vegetable tissues. We have devised modifications of this method by which results of approximately quantitative significance may be secured. It is the purpose of this paper to describe these modifications and to show their a.pplication to the determination of the organic acids in the tobacco plant at various stages of growth.

Lead salts have been used by pract’ically all investigators since Scheele (14) as reagents to precipitate the organic acids from plant extracts, basic lead acetate being probably most extensively employed although it has many disadvantages. This reagent is by no means highly selective; much carbohydrate material is found in the precipitates produced by it and Vickery and Vinson (15) found that it precipitates considerable proportions of widely diversified nitrogenous substances from extract,s of alfalfa leaves. The lead salt of succinic acid (16) redissolves if an excess of basic lead acetate is added, and the directions in the literature for the use of this reagent contain many empirical prescriptions on the adjustment of the reaction and on other points that suggest that much difficulty has been experienced with it.

Some years ago one of us observed (17) that barium hydroxide, when added in excess to extracts from alfalfa leaves or from yeast, on the furt.her addition of alcohol, yielded precipitates that contained much non-nitrogenous organic material. Subsequent invest,igation (unpublished) revealed the presence of large amounts of organic acids but. of very lit,tle nitrogen in these precipitates. We have therefore studied the effect of this procedure2 on pure

2 It is interesting to recall that Scheele discovered malic acid as a result of adding alcohol to an apple extract that had been neutralized by boiling with calcium carbonate.


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640 Organic Acids of Tobacco Leaves

organic acids as well as upon extracts from green tobacco leaves. It was found that a mixture of equal amounts of 1 per cent solutions of citric, oxalic, malic, succinic, maleic, and tartaric acids, when t,reated with excess of barium hydroxide and 2 volumes of alcohol, yielded a precipitate that could be extensively washed with 60 per cent alcohol wit,hout passing into solution. After decomposition of the precipitate with sulfuric acid, a solution was secured that contained 99.5 to 101.5 per cent of the original acidity due to organic acid as indicated by titration according to Van Slyke and Palmer (18).

That malic and citric acids are practically completely precipitated as their barium salts from tobacco leaf extracts by the addition of alcohol was shown by an investigation of the barium salts precipitate and of the filtrate from it. The precipitate was decomposed by sulfuric acid and the organic acids were extracted from t’he solution in a continuous ether extraction apparatus, The filtrate was likewise acidified and extracted with ether. Only about 77 per cent of the total ether-extractable acidity was found in the barium salts precipitate, but the filtrate contained no malic nor citric acids nor other acids that form silver salts insoluble at pH 7.0. To make certain that the malic acid had indeed been completely precipitated a study was made of the distribution of malic acid in the different precipitates and filtrates obtained in the large scale operations (see below). In the absence of t,artaric acid, malic acid can be determined by measurements of the optical act,ivity of the acids extracted by ether (19). Careful investigation of the fractions in which tartaric acid should occur showed that this acid was not present in tobacco leaves, and, con- sequently the polarimetric method for the estimation of malic acid could be applied. It was found that about 98 per cent of the malic acid of the leaf extract was precipitated by barium hydroxide and alcohol. As will later appear, citric acid is also completely precipitated from tobacco extracts by these reagents. Inasmuch as these two acids make up nearly 90 per cent of the organic acids of green tobacco leaves, the esters of which can be distilled, and also because of the completeness with which a wide variety of pure acids was precipitated in the preliminary tests, it seemed safe to assume that most of the esterifiable acids of familiar types that occur in tobacco leaf extract could be precipitated as their barium


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H. B. Vickery and G. W. Pucher

salts from dilute alcohol. Precipitation in this way therefore appeared to offer an advantageous quantitative method to remove the simpler organic acids from extracts of tobacco leaves.

The significant features of the procedure employed can be most readily presented in diagrammatic form. Minor details, such as the washing of the precipitates, are omitted. The extract was prepared by plunging leaves, immediately after picking, into boiling water and cooking until the midribs were soft. The leaves were then removed, enveloped in canvas, and pressed at the hydraulic press. The press-cakes were ground in a meat grinder and re- extracted with boiling water twice successively. The extracts were collected quantitatively and concentrated in vacua. Much inorganic material was removed by the two successive alcohol precipitations which were conducted at pH 2 so as to avoid the precipitation of calcium salts of organic acids. These precipitates were washed free from nicotine with 60 per cent alcohol.

The precipitated barium salts were centrifuged and washed once with 60 per cent alcohol and were then decomposed with 50 per cent sulfuric acid. The barium sulfate was extensively washed by decant.ation with hot water and was finally centrifuged. The addition of ammonia to the solution obtained by decomposing the first barium salts precipitate was designed to remove phosphoric acid. The filtrate from the precipitate so produced contained only traces of phosphate.

Filtrates A and B were separately investigated for malic acid. None could be detected in Filtrate A but 4.4 gm. were recovered from Filtrate B. The main organic acid solution contained 190 gm. of malic acid; the precipitation of malic acid was therefore carried out with a loss of not more than 2.3 per cent. The organic acids were extracted by ether in a large scale continuous extraction apparatus of the Hagemann type (20) over a period of 320 hours. The ether extract was freed from ether and from water by re- peated concentration in vacua after the addition of absolute alcohol and the acids were esterified as described by Phelps and Phelps (21), the distillation with alcohol vapor being continued until alcohol of a constant specific gravity came over. The esters were dissolved in ether and the dark red solution was treated with the minimal amount of 30 per cent sodium hydroxide solution


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Fresh leaf 57.27 kilos = 6.65 kilos dry weight

Water extract 5 liters; azidifled Press-cake to pH 2.0 + 1 volume alcohol

I

I i Filtrate Precipitate 1

Concentrated t.o 4 liters; acidified Gums, pectins, inorganic salts; to pH 2.0 + 2 volumes alcohol white granular material, weight

637 gm.; N = 0.49 per cent; ash = 42.8 per cent. [cY]~ = + 19.5”

corrected for ash

I I Filtrate + excess hot satu- Precipitate 2

rated Ba(OH)z solution Weight 298 gm.; N = 0.18

/ per cent, ash = 92.8 per cent

I I Filtrate A Barium Salts 1

Ether-soluble acids = 3500 cc. 0.2 N + HSOa to pH 1 HCl. No malic, citric, nor acid that forms Ag salt insoluble at pH 7

Organic acids + NHaOH to pH 8.0-9.0

I

BaSOa

I I Organic acids + 2 volumes Precipitate 3 alcohol + excess Ba(OH)z Chiefly alkaline earth

I phosphates

I Filtrate B

Ether-soluble acids = 2640 cc. 0.2 N

HCl. Contained 4.4 gm. malic acid

I Barium Salts 2

+ HzSOa to pH 1

I

I I Organic acids 3aSOa

Solids = 463 gm., N = 10.0 gm., total acid = 27,550 cc. 0.2 N HCl, ether-soluble acid = 21,250 cc. 0.2 N HCl

Extracted with ether


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M. B. Vickery and G. W. Pucher 643

I I Ether-soluble acids, Fraction A Ether-insoluble, Fraction B

Total acid = 20,400 cc. 0.2 N HCl Total acid = 6300 cc. 0.2 N HCl Esterified

I I I ! /

, I I

Acids insoluble in 90 per cent Filtrate (Fraction J) alcohol (Fraction I) Carbohydrate, etc.

Solids = 119 gm. = 25.6 per cent of total solids; total acid = 1400 cc. 0.2 N HCl; N =

5.0 gm.

I I

Esters insoluble in alkali (Fraction C) Soluble in alkali (Fraction D) Weight 304 gm. = 207 gm. free acid; total Phenolic substances; acidified acid after saponification = 15,000 cc. 0.2 N HCl; subjected to fractional distillation; esters identified = 272 gm. = 89.5

per cent

I I Ether-soluble (Fraction E) Ether-insoluble (Fraction F)

Phenols and phenolic acids; Weight = 53 gm. = 11.4 per weight = 19.3 gm.’ = 4.15 per cent total solids

cent total solids

required to neutralize the acidity. The alkaline sludge that separated was washed repeatedly with ether and removed.

The main ester fraction so obtained was washed with water until neutral and dried over anhydrous sodium sulfate. It was then freed from ether and the esters were transferred to a 500 cc. 3-necked Pyrex flask, to the central neck of which a series of fractionating columns could be fitted. The smaller necks of the flask carried capillary tubes to admit a slow current of air during the vacuum distillation. The distillation was begun with a small column of the type described by Cooper and Fasce (22). After the low boiling esters (Fraction 1) had distilled, air was admitted and a larger bore column wit,hout dephlegmator was substituted. The apparatus was then evacuated and the malic ester was distilled. A small fraction intermediate between malic and citric esters was collected separately and the distillation was continued


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until the boiling point of citric ester had been passed. The fractionating column was then removed from the apparatus and the distillation was completed without a column. The details of the fractionation are given in Table I.

Fraction 1 yielded no malic dihydrazide nor oxalic dihydrazide. The small amount of hydrazide secured was obviously a mixture. The esters remaining after these tests were therefore saponified by boiling with an excess of sodium hydroxide. Sodium was removed by acidifying the solution to Congo red with sulfuric acid and adding 2 to 3 volumes of absolute alcohol. After filtering

TABLE I

Distillation of Esters oj Organic Acids from Green Tobacco Leaves

Total weight of esters 310 pm.; pressure 7 to 8 mm.

1 2 3 3a 4 5 6

7, 8 Resic h

Boiling point

“C.

53-63 88-90 92-96

103-112 138-140 142-157 165-175 176-178 e

Distillate and residue.

Weight of

f i-action

Pm.

12.4 52.2

200.0 6.4 9.4 4.7 9.5 4.4 4.9

-

Composition

No malic nor oxalic acid dihydrazide Pure malic ester

“ ‘C “

60 per cent malic, 40 per cent citric 43.6 “ “ citric, 56.4 “ “ unknown 16 L‘ ‘I “ 84 ‘L I‘ “

3.14 <t “ “ 96.86 “ “ “ 1.38 ‘I ‘I “ 98.62 “ “ “

303.9 = 98 per cent recovery

off the sodium sulfate the alcohol was distilled off and the excess of sulfuric acid was exactly removed with barium hydroxide. Fractional crystallization of the free acids gave the equivalents of 0.65 gm. of fumaric acid and 0.65 gm. of succinic acid. The fumaric acid melted at 283-284’ in a sealed tube and, when mixed with authentic fumaric acid, showed no depression of the melting point. The succinic acid melted at 185-186” and likewise showed no depression of the melting point when mixed with pure acid. Its o-toluidene derivative melted at 254-255” and showed no depression of melting point when mixed with authentic material. The mother liquors of the succinic acid weighed slightly more than


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H. B. Vickery and G. W. Pucher 645

6.5 gm. (corrected) when evaporated to a sirup and were optically inactive in the presence of uranium acetate. Succinic acid was undoubtedly present but none could be brought to crystallization; oxalic acid could not be detected by testing with calcium acetate. This fraction therefore contained about 8 per cent of fumaric acid, and at least 8 per cent of succinic acid; malic and oxalic acids were absent; the remaining acids have not yet been identified.

Fractions 2 and 3 were essentially pure I-malic ester. The dihydrazide from Fraction 2 melted at 179-180” and that from Fraction 3 at 178.5-179.5”; neither showed depression of melting point when mixed with pure material. The specific rotation of Fraction 2 was [LY]~ = -10.34”, and that of Fraction 3 was [cx]2,0 = -10.88”. This value is slightly greater than that found by Frankland and Wharton (23) (- 10.44”) but was substantiated by observations on samples of pure malic ester secured fromother samples of leaves. The free malic acid secured by saponification of a sample of the ester melted at 100-100.7” and had a neutraliza- tion equivalent of 68 (theory 67.1).

Fraction 3a was a mixture of malic and citric esters. The citric acid was determined by the pentabromoacetone method (24), the malic acid by optical rotation. Malic acid dihydrazide of melting point 179” was also isolated.

Fractions 4 to 8 did not yield solid hydrazides. The citric acid content of each fraction was determined by the pentabromoacetone method and is given in Table I. Fractions 4, 5, and 6 were united, saponified, and the free acids were subjected to fractional crystallization. The equivalent of 3.16 gm. of an insoluble acid was obtained. Fractions 7 and 8 yielded the same acid in an amount equivalent to 0.56 gm. This acid melted at 283-284” in a sealed tube and contained 41.37 per cent of carbon and 3.67 per cent of hydrogen; when mixed with pure fumaric acid the melting point was not changed. Inasmuch as fumaric acid contains 41.37 per cent of carbon and 3.45 per cent of hydrogen, there can be no doubt of the identity of this substance.

The presence of fumaric acid in the ester fractions of high boiling point can be accounted for by the assumption of partial esterification. Fumaric acid is known to be difficult to esterify completely and Anschiitz and Drugman (25) found that the monoethyl


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ester is a solid of melting point 66” which distils at 147” at 16 mm. pressure. Fumaric monoethyl ester should therefore, if present, distil in the fraction that contains citric ester and it is interesting to note that most of the fumaric acid found in our experiments was obtained from the high boiling fractions. The identity of the other acids in these upper fractions has not yet been ascertained. None of the ester fractions contained acids that reduce alkaline solutions of silver nitrate, and Fenton’s (26) test for tartaric acid could not be obtained on the acids from the esters of high boiling point. Furthermore these esters contained only traces of optically active substances.

To recapitulate, the 57.27 kilos of fresh tobacco leaves yielded 304 gm. of distilled esters of organic acids of which 85 per cent was diethyl I-malate, 2.5 per cent was triethyl citrate, 1.6 per cent was mono- and diet,hyl fumarate, and 0.2 per cent was diethyl succinate. The remaining 10.7 per cent of the esters has not been identified but oxalic, malonic, tartaric, and isocitric acids were absent. Nearly three-quarters of the unidentified portion con- sisted of esters of high boiling point.

No oxalic acid was found in the hot water extract of the leaves. The residues of extracted leaves were therefore thoroughly ex- hausted with warm 2 N sulfuric acid and the acid extract was in turn subjected to ether extraction. Only 1.27 gm. of oxalic acid were found, the quantity being ascertained by titration with per- manganate and the identity established by means of the crystalline form of the calcium salt.” Neither malic nor citric acid could be detected. Oxalic acid was therefore present in our specimens of leaves exclusively as an insoluble salt, probably calcium oxalate; the other acids were present in soluble form.

The results of this analysis, calculated as percentages of the dry weight of the leaves and also as percentages of the total acid obtained from the esters are given in Table II. These data are founded upon a procedure that was conducted with quantitative rigor and the identity of the several acids has been established by

3 A specimen of calcium oxalate secured in the same way from young tobacco leaves (Table IV, third column) contained 27.5 per cent of calcium (theory 27.43 per cent). The free oxalic acid secured from it melted at 101” and this melting point was not changed by admixture with authentic oxalic acid.


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means of characteristic derivatives. The examination of the press-cakes demonstrated that all of the esterifiable acids except oxalic had been quantitatively removed by the hot water extraction of the leaves and the study of the filtrates from the barium salts precipitates showed that all save insignificant amounts of malic acid had been precipitated.

The over-all recovery of the malic and citric acids was of the order of 95 per cent. Analysis of the main solution of the organic acids before ether extraction revealed the presence of 190 gm. of

TABLE II

Organic Acids of Mature Green Tobacco Leaves Which Yielded Esters That Could Be Distilled

Oxalic (anhydrous). Succinic Fumaric*. Z-Malic. . Citric (anhydrous) Unknown acids? low

fumaric.. . Unknown acids high

citric.

........ ....... .......

. . . . . . . . . . . . . boiling esters calculated as

boiling esters calculated as

.....................

.....................

.....................

Dry weight

per cent

0.019 0.006 0.05 2.72 0.082

0.114

0.190 6.0

-

_ per cent

0.6 0.2 1.6

85.5 2.6

3.5

* Anschiitz and Reitler (27) observed the formation of small amounts of fumaric acid during the esterification of malic acid. It is therefore possible that the fumaric acid we have found may have been an artifact.

t Contains some succinic acid that failed to crystallize.

malic acid (polarimetric method) and 4.0 gm. of citric acid (pentabromoacetone method). The malic acid in the distilled esters was equivalent to 181 gm. and the citric acid to 5.24 gm. The procedure is therefore esssentially quantitative with respect to the predominating acids of this leaf. Nothing is yet known of the efficiency of the recovery with respect to the acids in the high boiling ester fraclion, but the large scale ether extraction apparatus we employed recovered 96 per cent of the total ether-extractable acidity as compared with results obtained on a small aliquot part of the main solution in a small extractor. It is evident, therefore, that the losses even of these acids could not have been serious.


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Organic Acids of Tobacco Leaves

The barium salts precipitate secured from the water extract of green tobacco leaves contained 465 gm. of organic solids. Of this only 207 gm. or 44.5 per cent could be accounted for as non-volatile aliphatic organic acids. The remaining 55.5 per cent con- sisted of substances that formed barium salts insoluble in dilute alcohol. These, on decomposition, yielded solutions that contained material quantities of organic acids according to the results of titration by the Van Slyke and Palmer method. Of the original acidity of the barium salts fraction (27,550 cc. of 0.2 N acid) the equivalent of only 54.4 per cent (15,000 cc.) was present in the fraction that contained the ether-soluble, alkali-insoluble esters. It is clear, therefore, that only about half the organic acids in the leaves of tobacco are substances of the type usually referred to as plant acids. The chemical nature of the other half of the organic acids is obscure.

The results of attempts to fractionate the various types of acidic substances in the barium salts precipitate are shown in the diagram. About 23 per cent of the total acidity of this fraction was not extracted by ether and remained in Fraction B. Less than one-quarter of this acidity could be precipitated by 90 per cent alcohol and the soluble part contained substances that reduced copper and yielded a mixture of osazones. Substances of carbohydrat’e nature were undoubtedly present but no definite crystalline derivative could be isolated. The alcohol precipitate did not reduce Benedict’s solution and was optically inactive. Its aqueous solution was acid to Congo red and gave an intense green color with ferric chloride.

The alkali-soluble part of the ether-soluble esters (Fraction D) contained 29 per cent of the total esters. This material, after acidification and extraction with ether, yielded 19.3 gm. of an ether-soluble dark red oil (Fraction E) that was acid to Congo red and gave an intense green color with ferric chloride which turned to violet on the addition of alkali. This color test is given by caffeic acid (3 ,4-dihydroxycinnamic acid). The aqueous solution of the oil reduced silver nitrate in the cold and yielded a flocculent yellow precipitate on the addition of bromine. There seems little doubt that phenolic acids were present. Fraction F, when freed from reagents, yielded 53 gm. of a pale yellow, strongly acid oil which was optically inactive and gave no color reaction with


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H. B. Vickery and G. W. Pucher

ferric chloride. This fraction represents ether-soluble material which either failed to esterify or was non-esterifiable. A summary of the data on these different fractions of the acids of green tobacco leaves is shown in Table III.

Little has yet been learned of the solubility of the barium salts of these unknown substances but it is probable that much of the titratable acidity that remained in the different filtrates shown in the diagram was due to their incomplete precipitation. The classification of these acids must await further investigation. It is obvious that they play an important part in the acid-base

TABLE III

Organic Acids of Green Tobacco Leaves That Are Precipitated as Barium Salts by Alcohol

Solids of barium salts precipitate from extract of 57.27 kilos of leaves = 465 gm.

Acids extracted by ether Organic acids from esters, identified.. . .

‘I “ “ “ not identified. Phenolic acids, Fraction E.. . . . Non-phenolic acids, Fraction F.. . .

Acids not extracted by ether Insoluble in 90 per cent alcohol, Frac-

tion I.................................. Soluble in 90 per cent alcohol, Frac-

tionJ*................................

gm.

186.0 21.0 19.3 53.0

119.0

66.7

25.6

I

40.0 14.4

* This fraction contained other substances in addition to organic acids. Some of these are related to the carbohydrates; nitrogenous substances were also present.

equilibria of the cells but until their chemical nature has been established their relationships can only be a matter of conjecture. The presence of these substances shows, however, that indirect precipitation methods for the determination of oxalic, citric, and malic acids, such as that of Kissling, can hardly be expected to yield accurate results.

The figures we have obtained for the proportions of the several acids in tobacco leaves are much lower than those secured by Garner, Bacon, and Foubert on Connecticut Havana seed tobacco by means of the Kissling method and than those of the Russian


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investigators who employed Schmuck’s (11) modification of an indirect method devised many years ago by Fleischer (28). Garner, Bacon, and Foubert’s results indicated the presence in the dried green leaves of IIavana seed tobacco of 1.47 per cent oxalic acid, 5.24 per cent citric acid, and 10.33 per cent of malic acid. Figures of nearly as great an order of magnitude have been given by Schmuck and also by Kissling for certain specimens of cured tobacco. Comparison of these data with ours is meaning- less since both the Kissling and the Fleischer methods are indirect. Although these two methods may give approximately

TABLE IV

Organic Acids of Connecticut Shade-Grown Tobacco

The figures are in per cent of the dry weight.

Acid

Oxalic (anhydrous). Succinic............................ Fumaric............................ I-Malic Citric (anhydrous). Unknown acids? low boiling esters,,

I‘ “ $ high “ “

Total acids. . . . . . .

7

Seed YEFg

per cent per ten,

0.012* 0.078

+ + 0.010 2.76 0.102 0.31 0.012 0.103 0.013 0.059

0.149 I 3.31

-

1

T Mature Cured

leaf leaf

per cent

0.019 0.01 0.057. 2.72 0.082 0.103 o.a90

per cent 0.06

+ 2.11 0.47 0.174 0.25

3.18 -

3.06

* This figure is low as the press-cake was not examined. t Calculated as fumaric acid; probably contains some succinic acid. 1 Calculated as citric acid; represents distilled esters only.

accurate results when applied to a mixture of pure acids of known composition, this is no reason for supposing that they do likewise when applied to a leaf extract in which numerous interfering substances occur. In neither method is there any attempt to control the purity of the precipitates.

The modified methods for the determination of the organic acids described above have been applied to aqueous extracts of oil-free tobacco seed, to the leaves of young and of mature plants, and to cured tobacco prepared commercially from mature leaves derived from the same crop as that here investigated. The details of these other analyses will be given elsewhere (29) but the essential data are shown in Tables IV and V. Malic acid is present in


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traces in the seed but reaches a very high concentration in young plants upon which four to six leaves have developed. This high concentration is maintained up to maturity, but decreases slightly during curing. The great increase in malic acid concentration occurs long before storage of nicotine in the tissue reaches a high level. Rapid increase in nicotine content is a phenomenon of the maturing leaf, not of the young leaf (12). It would seem, therefore, that malic acid synthesis is independent of nicotine synthesis.

Citric acid is the predominating organic acid of tobacco seed. Synthesis of this acid occurs in the young plant although the process is far outstripped by the formation of malic acid. At

TABLE V

Organic Acids of Connecticut Shade-Grown Tobacco

The figures are in per cent of the total acids isolated.

Acid Seed

per cent

Oxalic .................................... 8.1* Succinic .................................. Fumaric .................................. + I-Malic .................................... 6.7 Citric ..................................... 68.5 Unknown acids1 low boiling esters ......... 8.0

“ “ j high “ I‘ ......... 8.7

Young Mature leaf leaf

~___ per cent per cent

2.4 0.6 0.3

+ 1.8 83.4 85.5

9.4 2.6 3.1 3.2 1.7 6.0

per cent

1.9

+ 69.0 15.4 5.5 8.2

* This figure is low as the press-cake was not examined. 1 Calculated as fumaric acid; probably contains some succinic acid. $ Calculated as citric acid; represents distilled esters only.

maturity the leaves contain a much decreased proportion of citric acid, this substance being present, in fact, in smaller proportion than the unknown acids that are found in the high boiling ester fraction. The situation is reversed, however, after curing. The citric acid concentration increases more than 5-fold, while the unknown acids increase only to a small extent. Small amounts of fumaric acid were detected in all the extracts analyzed but only in the mature leaf were results of quantitative value secured.

SUMMARY

Modifications of existing methods for the determination of the organic acids in plant tissues have been devised by which approxi-


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mately quantitative isolation and positive identification of the predominating acids can be secured. These methods have been applied to the determination of the acids present in extracts of the seeds of the tobacco plant, of the leaves of young and of mature plants, and of leaves that had undergone the commercial curing process.

The most important modification consists in the initial precipitation of the organic acids as barium salts in the presence of dilute alcohol. This method is quantitative with respect to the better known organic acids and is superior in many respects, particularly in convenience, to the customary precipitation of the lead salts by basic lead acetate.

It is shown that only about half the total titratable acidity of tobacco leaf ext’racts consists of acids of familiar types such as malic, citric, oxalic, succinic, and fumaric acids. The rest belongs to acidic substances, part of which appear to be phenolic acids, the nature of which has not yet been determined. It is further evident that, in view of the presence of these substances, indirect methods for the determination of oxalic, citric, and malic acids, such as t’hosc of Kissling and of Fleischer do not yield trustworthy results.

BIBLIOGRAPHY

1. Franzen, H., and Keyssner, E., Biochem. Z., 136, 183 (1923). 2. Franzen, H., and Helwert, F., Biochem. Z., 136, 384 (1923); 136, 291

(1923). 3. Franzen, H., and Ostertag, R., Biochem. Z., 136, 327 (1923). 4. Franzen, H., and Stern, E., 2. physiol. Chem., 116, 270 (1921). 5. Franzen, H., and Helwert, F., 2. physiol. Chem., 127, 14 (1923). 6. Franzen, H., and Stern, E., 2. physiol. Chem., 121, 195 (1922). 7. Bernhauer, K., Biochem. Z., 172, 296 (1926); 172, 313 (1926); 1’72, 324

(1926). 8. Wetzel, K., Planta, Abt. E. 2. wissensch. Biol., 4, 476 (1927). 9. Garner, W. W., Bacon, C. W., and Foubert, C. L., U. S. Dept. Agric.

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16. Bachmann, J. A., Einiges iiber Sauren und Aminosauren aus Silofutter, Dissertation, Zurich, 83 (1927).

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PucherHubert Bradford Vickery and George W.

OF GREEN TOBACCO LEAVESTHE NON-VOLATILE ORGANIC ACIDS

1931, 90:637-653.J. Biol. Chem.

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