investigations on loco weeds. v. further studies on ... - ACS Publications

[CONTRIBUTION FROM THE

DEPARTMENT OF CHEMISTRY OF

COLUMBIA UNIVERSITY]

INVESTIGATIONS ON LOCO WEEDS. V. FURTHER STUDIES ON THE CONSTITUENTS OF ASTRAGALUS EARLEI ARTHUR STEMPEL AND ROBERT

C.

ELDERFIELD

Received July 1, 19$8

In preceding communications (1, 2) the results of a preliminary investigation of the constituents of Big Bend loco weed (Astrugahs earEei) have been reported. While the isolation of the toxic principle, or principles, was not accomplished, two nitrogenous substances called at that time “a-and P-earleine” were separated from the weed along with considerable amounts of d-pinite. With the limited quantity of weed available at the time the early work was carried out, and because of the very small yield of the two bases, it was not possible to characterize them further. In the meantime an additional supply of weed has been obtained and a further study of the two bases, as well as an extension of the general study of the constituents of the weed, has been carried out. The general preliminary extraction of the weed was the same as that used in the earlier work. After extraction of the ground whole weed with 70% alcohol, extraneous inactive material was removed by precipitation with basic lead acetate. The filtrate from this precipitate, after removal of lead as lead sulfide, was concentrated to a syrup which was thoroughly extracted with absolute alcohol, a procedure which has been shown to remove the active substance. The concentrate from the absolute alcohol extract (fraction A) has been used for the work here described. At the outset, further investigation with larger quantities of the so-called “aand @-earleines” showed that they were identical with betaine and choline respectively, and a preliminary note to this effect has already appeared (3). A study of the thermal decomposition of the latter provided the clue to its identity. When “@-earleine”was heated, trimethylamine and acetaldehyde were isolated from the decomposition products as the picrate and 2,4-dinitrophenylhydrazone respectively. From this the identity of “a-earleine” with betaine was surmised and the identity of both bases with choline and betaine was further confirmed by preparation of other derivatives. “P-Earleine” produced a typical choline effect on mice (4). As additional experience with the weed has accumulated, it has been found that the precipitation of the toxic constituent with phosphotungstic acid may possibly be explained by adsorption of the poison on the rather bulky precipitate. In common with the earlier work (l),a considerable amount of activity has been found in the filtrate from the phosphotungstic precipitate. In order to show that the toxicity of this solution was not due to unprecipitatedcholine, the total nitrogen content of the fraction was determined by the Kjeldahl method. On the assumption that all the nitrogen thus found was due to choline, an amount of choline chloride corresponding to the nitrogen value found, was fed to cats in the same dosage as obtained with the weed extracts. In no case 432

CONSTITUENTS O F LOCO WEED

433

did this produce any symptoms of locoism in the animals, although one cat died. On autopsy the cause of death appeared to be starvation. Attention was then turned to the filtrate from the phosphotungstic acid precipitation. In the more recent work particular attention has been paid to thorough washing of this precipitate, with the result that a larger portion of the activity of the weed is found in the filtrate, although it has not been conclusively demonstrated that such activity as is found in the precipitate is adsorbed. However, this now appears to be likely. The extreme solubility of the active material in water and alcohol suggests that the molecule is strongly polar and that it probably is highly hydroxylated. If one assumes that the appearance of the active material in the phosphotungstic acid precipitate is due to adsorption, then the further statement can be made that it is not truly precipitable by this reagent. It was felt that acetylation of the substances found in the phosphotungstic acid filtrate would render them soluble in other organic solvents and hence more amenable to separation. Accordingly the filtrate (fraction C) was freed of phosphotungstic acid with barium hydroxide and the resulting solution was concentrated to dryness. The residue was then acetylated in pyridine with acetic anhydride and the product after such treatment was then separated by extraction with chloroform into a chloroformsoluble fraction (D) and a fraction not extracted by chloroform from water (fraction E). The entire fraction D was deacetylated with barium methoxide and yielded a mixture which was not active in cats. However, fractional distillation of the acetylated material yielded three substances, acetyl d-pinite, glycerol triacetate, and a diacetoxyvalerolactone, or an isomer thereof. The presence of three saponifiable groups in the so-called diacetoxyvalerolactone was established by a quantitative determination. Furthermore, two of these groups were Characterized as acetyl groups by the method of Elek and Harte ( 5 ) . This diagnosis was confirmed by a study of the product obtained by deacetylation of the acetyl lactone by means of either barium methoxide or hydrochloric acid. The product thus obtained was neutral, and on saponification, one equivalent of alkali was consumed. The hydroxy lactone was further characterized by the preparation of a phenylhydrazide of the corresponding acid. With the limited amount of material available it has not been possible to establish the exact structure of the lactone. The following evidence is offered in a preliminary sense, pending the accumulation of larger quantities of the substance. The possibilities to be considered are a ,P-dihydroxy-y-valerolactone, @-6-dihydroxy-y-valerolactonel /3 ,y-dihydroxy-6-valerolactone, a,6-dihydroxy-y-valerolactone and 01 ,y-dihydroxy-6-valerolactone,or branched chain isomers thereof. On the assumption that a straight chain is present, /3,6-dihydroxy-7-valerolactone and /3 ,y-dihydroxy-6-valerolactonecan be eliminated because of the failure of the lactone to undergo dehydration under the conditions attending deacetylation of the acetoxy lactone with hydrochloric acid. Under such conditions, these /3-hydroxy lactones would be expected to suffer dehydration and to lead to the corresponding unsaturated lactone. Evidence

434

ARTHUR STEMPEL AND R. C. ELDERFIELD

CONSTITUENTS OF LOCO WEED

435

secured by a study of the action oflead tetraacetate on the lactone points to the presence of vicinal hydroxyl groups in a trans relationship to each other. In Figure 2 is shown the curve representing the course of the oxidation of the lactone by lead tetraacetate according to the method of Hockett and McClenahan (6), together with curves obtained by these authors for other sugar derivatives. If this interpretation is correct, the structure of the lactone in question narrows down to one of the isomeric a ,8-dihydroxy-y-valerolactones. Such an assumption appears to be warranted by the observed failure of the lactone to display mutarotation in aqueous solution, a behavior which would be expected in the cases of a,6-dihydroxy-y-valerolactoneand a,y-dihydroxy-6-valerolactone. Eight possible stereoisomers of a ,P-dihydroxy-y-valerolactonemay occur, namely, d- or Z-arabomethylonic lactone, d- or 1-xylomethylonic lactone, dor Z-lyxomethylonic lactone, and d- or 1-ribomethylonic lactone. E-Arabomethylonic lactone is reported as melting a t 123" and showing a value for [aJDof -44.7" in water (7). The lactone in question melts a t 52-53' and shows a value for [a],of - 64.7" in water. We have prepared the phenylhydrazide of d-xylomethylonic acid, which melts at 132-133" and shows a value for [a],of 33", which compares with constants of 114-115" and 42" for the phenylhydrazide of the acid corresponding to the lactone under consideration. The new dihydroxy lactone, therefore, is not an arabo- or xylo-methylonic lactone. Neither of the ribomethyloses or their lactones have been described in the literature. d-Lyxomethylonic lactone is reported by VotoEek (8) as showing a value of [a],of 44.2". However, Clark (9) reports Z-lyxomethylonic lactone as melting a t 111" and showing a value of [a],of -63.65'. In view of this conflict of data, it does not appear warranted to exclude a lyxose configuration for the unknown lactone. On the assumption that the unknown lactone is one of the straight-chain methyltetronic acids, the configuration of 1-lyxonic lactone would seem to be probable on the basis of Hudson's lactone and phenylhydrazide rules. Definite corroboration for this suggestion awaits accumulation of larger amounts of the lactone and reconciliation of the conflicting data on lyxomethylonic lactone. These points are under investigation. Whether this hydroxy lactone is a primary constituent of the weed or whether it is formed by transformation of some precursor during the isolation process must be left open for the present, although the comparatively mild treatment undergone by the extract up to the acetylation would hardly be expected to cause degradation of other carbohydrate constituents. However, the possibility that the lactone is an artifact cannot be overlooked. The other constituents of the acetylated fraction were identified as glycerol triacetate and acetyl-d-pinite. The latter obviously arises from traces of pinite carried along mechanically in the solutions. The presence of glycerine, taken together with the occurrence of choline, suggests the unlikely possibilitydhat phosphotides might have been originally present in the active extracts. However, phosphorus determinations on both the original absolute alcohol-soluble part, and the material unextracted by absolute alcohol, showed the absence of phosphorus.

436


Fraction E, which was not extracted by bhloroform, was concentrated and yielded a mixture of substances which was highly toxic to cats. While this fraction still gave a positive Molisch test, it no longer reduced Fehling’s solution. It gave a reddish-blue ninhydrin test. It was obvious, therefore, that a considerable concentration of the activity had been achieved. On destructive distillation of the residue from concentration of fraction E, the vapors gave an intense, red, pine-splinter test for pyrrole and a deep purple color with Ehrlich’s reagent. In contrast to this, an aqueous solution of fraction E gives no pine-splinter test and a faint green color with Ehrlich’s reagent. The color tests are, therefore, due to decomposition products of the bases present. The apparent inability of phosphotungstic acid to precipitat,e all of the nitrogenous material, taken together with the above color tests, indicates the presence of nitrogen in a nonbasic compound of the pyrrole series. Since not all of the nitrogen present in the weed is basic, it was of interest to determine the distribution of the nitrogen in each fraction. This was done as given in the experimental part, and a t least 11% of the total nitrogen in the weed was shown to be non-basic nitrogen. Whether the total activity is to be found in this non-basic nitrogen fraction remains to be seen. Since apparently the nitrogen in the active material is non-basic, and since some indication had been obtained that a derivative of pyrrole wm involved, the use of precipitants such as Reinecke salt or ammonium rhodanilate was suggested. When the filtrate from the phosphotungstic acid precipitate, after being freed from phosphotungstic acid (fraction C), was treated with Reinecke salt, a precipitate was obtained which after decomposition with pyridine in the usual manner, gave an almost colorless, highly active solution (fraction G). The solution gave a positive ninhydrin test and on concentration yielded a residue from which crystalline material was obtained. On thermal decomposition the crystalline material gave off an odor of a lower aliphatic amine and the vapor showed a strong pine-splinter t,est for pyrrole. Elementary analysis .showed the presence of nitrogen and sulfur in this substance, along with carbon, hydrogen, and oxygen. The sulfur is neither disulfide nor sulfhydryl sulfur, and the absence of sulfate and thiocyanate groups was shown. No primary amine groups are present and the compound gave a negative ninhydrin test. The substance is also precipitated by ammonium rhodanilate, and may likewise be obtained from fraction E. With the limited amount available, it has not been possible to characterize this material further. Furthermore, it has not been possible to demonstrate conclusively whether this sulfur-containing substance is responsible for the activity of the weed or whether the active material is to be found in the part of fraction G which gives the ninhydrin test. In our attempts to isolate the active constituent of the weed, the presence of large amounts of carbohydrate material, and possibly glycosides, has occasioned much difficulty. It was, therefore, felt that, if some method could be found whereby this inert material could be removed, the separation of nitrogenous components would be facilitated. For this purpose we have applied the procedure of Rabat6 (lo), which consists in gently heating the dry mixture of carbo-

CONSTITUENTS O F LOCO WEED

437

hydrate material with magnesium oxide. It was found that, when this treatment was applied to the mixture extracted by 95% alcohol, 73% of the reducing sugars had been removed along with 9% of the sugar occurring as glycosides and polysaccharides, while the activity of the extract was comparatively unaffected. However, the ease of isolation of nitrogenous constituents was not improved by such treatment. Crawford (11)reports that Astragalus lambertiihas been used by the Mexicans for making beer, and that in some cases symptoms of locoism developed. If this indication that fermentation does not affect the activity of the weed is true, then a simple way for eliminating some of the carbohydrate material is offered. The phosphotungstic acid filtrate (fraction C) was, therefore, fermented with yeast and the resulting solution, after removal of proteins, was fed t o cats. The activity was not decreased and 66% of the sugars present, determined as glucose, had been removed. Likewise Kj eldahl determinations revealed no change in nitrogen content. Despite this removal of the bulk of the sugars, the isolation of nitrogenqus constituents was not facilitated. It is interesting to note that the absolute alcoholic extract before precipitation with phosphotungstic acid did not ferment with yeast, possibly because of the presence of an inhibitor precipitated by phosphotungstic acid. The filtrate from the phosphotungstic acid precipitate (fraction C) contains glycosides, possibly of the active material. Therefore, attempts were made to determine the effect of enzymatic hydrolysis on the activity of this fraction. By following the increase in reducing power of solutions under the action of takadiastase and emulsin respectively, a t least one glycosidic linkage has been found. The activity of the fraction was not decreased by such treatment, but the evidence as to whether the toxic material occurs as a glycoside, was inconclusive. In the above work we have used cats as experimental animals, although there is a great need for a more satisfactory assay method. The present method requires from four to six weeks for definite symptoms to appear. I n order to try to provide a more satisfactory laboratory animal, we have fed an active extract of the weed to guinea pigs, with no effect. Subcutaneous injections of the extract in chicks, starting when they were a day old, did not affect either growth or stability. Finally, although adult cats react well to the weed, kittens did not react when fed active extracts. We wish to acknowledge our appreciation for the kind cooperation of S. B. Penick & Company of New York City, and of Parke, Davis and Company of Detroit, Michigan, in carrying out preliminary extraction of the weed, which was secured with the aid of Dr. Frank P. Mathews of the Loco Weed Laboratory, Alpine, Texas. Our thanks are also due to the American Academy of Arts and Sciences for a grant for technical help in this investigation. EXPERIMENTAL

All melting and boiling points are corrected for stem exposure. The concentrate used in this work was obtained exactly as described by Pease and Elderfield (I). The concentrate of the absolute alcohol extract of the resin was used.

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Identification of choline and betaine. The picrates of the bases formerly called "a- and 8-earleine" were isolated as previously described (1). When free "B-earleine" was thermally decomposed in a stream of nitrogen, trimethylamine and acetaldehyde were isolated from the decomposition products. The picrate of the former melted a t 228-229" and gave no depression of melting point when mixed with a known sample. Anal. Calc'd for CsH9N.CeHsNs0,: C, 37.5;H , 4.2;N, 19.5. Found: C, 37.8;H, 4.2;N , 19.7. Acetaldehyde was identified as the 2,4-dinitrophenylhydrazone,which melted a t 164". Anal. Calc'd for C8H8N404:C, 42.9;H , 3.6;N, 25.0. Found: C, 43.1;H, 3.4;N , 24.8. TABLE I Choline Picrate Anal. Calc'd for C6H14N0.CJ-&Ns07:C, 39.8;H , 4.8;N , 16.9 Found: C, 39.8;H, 4.7;N , 16.9 M.P. Reported: 240" (uncorr.) (12);found: 247" Acetylcholine Picrate Anal. Calc'd for C7Hl6NO2-C&Ng07: C, 41.7;H , 4.8;N , 15.0 Found: C, 42.0;H, 4.9;N, 15.1 M.P. 111.5-112.5° Choline Chloroplatinate Anal. Calc'd for (C6Hl~NO)z.H*PtCls:C, 19.4;H, 4.9; Pt, 31.5 Found: C, 20.0;H , 5.1;Pt, 31.8 M.P. Reported: 234-235" (dec.) (13);found: 234-236" (dec.) Betaine Picrate Anal. Calc'd for C 6 H I I N 0 2 ~ C ~ H ~C, N ~38.2; O ~ :H, 4.1; N, 16.2 Found: C, 38.3;H , 4.1;N , 15.6 M.P. Reported: 183" (14);found: 184" Betaine Styphnate Anal. Calc'd for C6HllN02*CJIsNaO~: C, 36.5;H , 3.9;N , 15.5 Found: C, 36.8;H , 4.1;N, 15.0 M.P. 186-188" (dec.) Betaine Hydrobromide Anal. Calc'd for C6H11NOz.HBr: C, 30.3;H, 6.1;N , 7.0;Br, 40.4 Found: C, 30.8;H, 6.2;N, 7.0;Br, 40.1 M.P. Reported: 233" (15);found: 225' From this i t appeared likely that "8-earleine" in reality is choline, and i t could be surmised that "a-earleine" in reality is betaine. This interpretation was confirmed by reexamination of the data a t hand, both old and new, on the two bases as shown in Table I. I n cases where there was no decomposition, mixed melting points of known samples with the derivatives were taken, and no depressions were noted. AcetyZation of the phosphotungstic acid filtrate (fraction C). An aqueous solution of the phosphotungstic acid filtrate equivalent to 25 lbs. of dry weed was concentrated to dryness i n vacuo and dried by two distillations with absolute alcohol and benzene. The residue was extracted by stirring with 400 cc. of dry c.p. pyridine on the steam-bath. A small amount did not go into solution. This residue was reextracted with 100 cc. of pyridine and the combined pyridine extracts were cooled in ice to 0". Four hundred cubic centimeters of acetic anhydride was added slowly. The solution warmed up slightly during the addition. After all of the acetic anhydride had been added, the solution was allowed to remain a t room temperature for seven days. It was then poured into 1.5 liters of ice and allowed to stand for 2.5 hours with occasional stirring. The aqueous solution was then extracted several times with chloroform. The combined chloroform extracts were washed several times with 2.5 N hydrochloric acid to remove pyridine, the solution being cooled with ice


439

during the washing. Excess acid was then removed by shaking with a saturated sodium bicarbonate solution. The chloroform solution was dried over calcium chloride, filtered, and the chloroform removed by distillation in vacuo. The residue'was then distilled a t a pressure of about 5 X 10-8 mm. and the following fractions were collected: 1, up to loo", 2, 100-130', 3, 130-150'. These fractions were then redistilled. Fraction 1. This was a slightly brownish mobile liquid which was redistilled; i t boiled a t 85-89' a t 0.15 mm. It furnished analytical figures corresponding to glycerol triacetate. A n a l . Calc'd for CoH1406: C, 49.5; H, 6.4. Found: C, 49.8; H, 6.5. The acetate obtained as above was deacetylated by use of barium methoxide. T o a n ice-cold solution of 4.5 g. of the acetate in 125 cc. of absolute methanol was added 5 cc. of 0.5 N barium methoxide solution in absolute methanol. After standing for 2 days in the refrigerator, barium was removed as the sulfate, and the filtrate from barium sulfate was concentrated to yield a viscous syrup. This was benzoylated with benzoyl chloride in pyridine and yielded glycerol tribenzoate, which melted at 72-73' after crystallization from alcohol, and showed no depression in melting point when mixed with a known sample. A n a l . Calc'd for Cs4HzoOs: C, 71.4; H, 5.0. Found: C, 71.4; H, 5.3. Fraction $. This fraction amounted to about 11 cc. It was redistilled at 0.2 mm., and the material boiling from 124-130" was collected. On standing, the heavy oil crystallized. After recrystallization from aqueous alcohol, 2.0 g. of an acetate which melted at 86-87' was obtained. Analyses indicated the presence of two acetyl groups and one lactone. [a]: -7.09" (c = 2.822 in chloroform); 25.2" (c = 2.124 in alcohol). A n a l . Calc'd for CoHt20s;C, 50.0; H, 5.6; 2 CHsCO, 39.8. Found: C, 50.0,50.3; H, 5.5,5.8; CHaCO, 38.8,38.9. Molecular weight (Rast method in camphor) Calc'd: 216. Found: 195. The saponification equivalent from 0.1102 g. of substance was obtained using 0.1 N sodium hydroxide in dilute acetone at 0' and back titrating with 0.1 N sulfuric acid. Calculated for 3 equivalents: 15.30 cc. of 0.1 N NaOH; found: 15.38 cc. The acetyl lactone was deacetylated either with hydrochloric acid or barium methoxide. I n the former case, 0.3 g. of the substance was heated a t 60" with 60 cc. of 0.5 N hydrochloric acid. The substance slowly dissolved and was completely in solution after an hour, After four hours, the solution was concentrated to dryness under reduced pressure, and the residue was thoroughly dried by azeotropic distillation with absolute alcohol and benzene. On crystallization from ether-petroleum ether (Skellysolve B) long white needles were obtained which melted a t 52-53'. [a]: -64.7" (c = 0.580 in water). No change after two days. A n a l . Calc'd for CsH804:C, 45.5; H, 6.1. Found: C, 45.4; H, 6.2. Saponification equivalent, calc'd: 132; found: 131. The deacetylated lactone obtained by the more cumbersome barium methoxide method was identical in all respects with that obtained with hydrochloric acid. The phenylhydrazide of the hydroxy acid corresponding to the lactone was prepared by heating the lactone with a slight excess of phenylhydrazine on the steam-bath for one hour. After cooling, the oilymaterial was rubbed up with ether until i t solidified, and then washed several times more with ether. On recrystallization from benzene-alcohol, i t formed clusters of needles which melted at 114-115'. [a] 42" f 2" (c = 0.558 in methanol); 45" (c = 0.462 in water). A n a l . Calc'd for C11H16N204: C, 55.0; H, 6.7. Found: C, 54.9; H, 6.6. d-Xylomethylonic acid phenylhydrazide was prepared in a similar manner from d-xylomethylonic acid prepared by oxidation of d-xylomethylose with bromine water. The phenylhydrazide crystallized as plates from benzene containing a trace of alcohol and melted at 132-133". [a]E 33" (e = 0.640 in methanol); 21" (c = 0.398 in water). A n a l . Calc'd for CllHlsNz04:C, 55.0; H, 6.7. Found: C, 54.9; H, 6.7.

440


Oxidation of the hydroxy lactone with lead tetraacetate. The procedure was that of Hockett and McClenahan (6). To a solution of 35 mg. of the lactone in about 70 cc. of glacial acetic acid was added 25 cc. of 0.1288 N lead tetraacetate in glacial acetic acid and the resulting solution was made up to exactly 100 cc. At intervals, 5-cc. samples were removed, added to 10 cc. of a solution of sodium acetate and potassium iodide, and the liberated iodine was titrated with 0.02 N thiosulfate. The course of the oxidation is shown in Figure 2, together with a curve obtained similarly by Hockett and McClenahan for 8-methyl-d-xylopyranoside and a- and 8-methyl-d-glucopyranoside. From the similarity of the curves i t is suggested that the hydroxyl groups in the lactone are trans to each other. Fraction 5. This was an extremely viscous oil distilling a t 137-145" a t 10- mm. It was obtained crystalline from ether-ligroin and melted constantly a t 97-98'. It was identified as acetyl pinite by mixed melting point and rotation. [a]: 7.0" (c = 1.920 in alcohol). Isolation of the nitrogen and sulfur compound. To a solution of 75 g. of Reinecke salt in 800 cc. of absolute methanol cooled in an ice-bath, was added with vigorous stirring, 1 liter

/.25 Loo

0.75 050

0.S 0

0

25

7s

/00

/25

150

I75

ZDO

h e in Hours FIGURE 2 of an aqueous solution of the filtrate from the phosphotungstic acid precipitate (fraction C) which had been freed from phosphotungstic acid by treatment with barium hydroxide. This was equivalent to 25 lb. of dry weed. A precipitate formed almost immediately. After refrigerating overnight, the precipitate was filtered off and suspended in 200 cc. of water. After the addition of 6 cc. of pyridine, the mixture was shaken for an hour and filtered. Several drops of acetic acid were added to the filtrate to remove the last of the Reineckate, the solution was filtered again and the filtrate was concentrated to dryness under reduced pressure. The residue was taken up in absolute methanol and the solution deposited crystals on standing. After several crystallizations, both from methyl and ethyl alcohol, the substance appeared homogeneous and formed glistening white plates. The substance does not show a sharp melting point but decomposes about 320". The yield was 50mg. The analytical data, obtained from two different preparations, are difficult to reconcile with a satisfactory formula a t present. A n a l . Found: C , 33.0,33.0; H, 7.1,7.1; N, 6.5; S,17.7, 17.1. The compound is optically inactive, extremely soluble in water, but sparingly soluble in cold alcohol. I t s aqueous solution shows no turbidity with barium chloride, and a negative nitroprusside test for sulfhydryl and disulfide sulfur. The ninhydrin reaction is negative and no amino nitrogen can be detected by the Van Slyke procedure. That the sulfur did not come from the Reinecke salt was shown by a negative ferric chloride test for thiocyanate. On thermal decomposition, a strong odor of a lower aliphatic amine appears, and the vapors give a strong pine-splinter test for pyrrole.

441


While the solution obtained on decomposition of the Reineckate produces typical locoism in cats, due to the extremely small amount of material available, i t has not been possible to demonstrate whether the above compound is responsible for such symptoms or whether a still unisolated substance carried down in the precipitate is the active material. This point is under active investigation. The methanol filtrate, after removal of the Reinecke precipitate, was concentrated under reduced pressure, the residue was taken up in water and freed from Reinecke salt by use of pyridine in the usual manner. Concentration of the final filtrate left a syrup, which, on acetylation, yielded the same three acetates described above. Ammonium rhodanilate can be used for the above precipitation, but it is not as satisfactory and does not give as clean a product as Reinecke salt. TABLE I1 FRACTION

Absolute alcohol extract.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Filtrate from phosphotungstic acid precipitate (fraction C). . . . . . . . . . Above filtrate after a second phosphotungstic precipitation. . . . . . . . . . Unacetylated part (fraction E ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

N P E P LB. OF DPY WEED

YG.

306 98 68 34

Enzymatic hydrolysis of the phosphotungstic acid filtrate (fraction C ) . The substrate used was a solution of the filtrate, freed from phosphotungstic acid, of which 25 cc. corresponded to 0.022 Ib. of dry weed. The following solutions were used: (a) 25 cc. of substrate, 2 cc. of M/5 acetate buffer, 2 cc. of 1% takadiastase (Wallerstein), and 2 cc. of water. pH of the solution: 4.70. (b) Same as (a) except that 2 cc. of 1% emulsin (prepared from almonds) was used instead of the takadiastase. p H of the solution: 4.59. (c) Same as (a) except that 2 cc. of 1% emulsin and 2 cc. of 1% takadiastase were used. p H of thesolution: 4.65. The solutions were kept in a thermostat at 35" i0.05" and 2 cc. samples were removed a t intervals. Reducing sugar was determined by the Hanes modification of the HagedornJensen method (16). The results are shown in Figure 3. Action of magnesium oxide on the phosphotungstic acid filtrate (fraction C ) . The method of Rabat6 (10) was used in an effort to remove some of the troublesome carbohydrates. A portion of fraction C equivalent to 0.08 lb. of dry weed was found to contain 48 mg. of reducing sugar determined as glucose by the Hanes, Hagedorn, Jensen method. This solu-

442


tion was stirred up into a paste with magnesium oxide and dried in an oven a t 38”. The dry powder was pulverized and extracted with alcohol ; the solution thus obtained contained 13 mg. of reducing sugar, indicating that 73% of free reducing sugar had been removed. A larger run was then made, and the product produced typical symptoms of locoism in cats. I n order to ascertain whether appreciable amounts of glycosides and polysaccharides had been removed, the following control experiment was done. A solution of fraction C equivalent to 0.08 Ib. of dry weed was made 0.2 N in sulfuric acid and refluxed for 5 hrs. An exactly similar experiment was run on the material after magnesium oxide treatment. After the acid hydrolysis the untreated phosphotungstic acid filtrate contained 82 mg. of reducing sugar calculated as glucose, an increase of 34 mg., and the filtrate from the magnesium oxide treatment contained 44 mg. of reducing sugar, an increase of 31 mg. From this i t is apparent that about 9% of the glycosides and polysaccharides have been split during the magnesium oxide treatment. While this method for concentrating the activity has shown signs of great usefulness, i t has not been pursued further at present because of the more convenient Reinecke precipitation which apparently requires no preliminary concentration other than precipitation of choline and betaine with phosphotungstic acid. Distribution of nitrogen i n extracts of the weed. Since not all of the nitrogen isolated thus far from the weed is basic, i t became of interest to follow the distribution of the nitrogen in the various fractions. This was done by the Kjeldahl method and the results are shown in Table 11. From this i t is apparent that a t least 11% of the nitrogen originally present in the weed may be accounted for as non-basic nitrogen. Evidence at present available points to the occurrence of the active substance in this fraction.

The microanalyses here reported were performed by Mr. Saul Gottlieb of these laboratories. SUMMARY

1. The substances previously called “a- and P- earleine” have been shown to be identical with betaine and choline respectively. 2. It appears likely that the reported precipitation of the active constituent of Astragalus earlei by phosphotungstic acid is due to adsorption on the precipitate. 3. Reinecke salt precipitates a highly active fraction from which a crystalline substance has been isolated. 4. Bases which give a strong ninhydrin test are also precipitated by Reinecke salt. 5. A dihydroxyvalerolactone, or an isomer thereof, has- been isolated from extracts of the weed along with glycerine. Possible structures for the lactone are discussed. 6. Enzymatic action of yeast, takadiastase, or emulsin affects the carbohydrate constituents of t,he weed, but does not apparently affect the activity. 7. A practical method for removing most of the reducing sugars from an extract of the weed without affecting the activity is suggested. NEW YORK,N. Y. REFERENCES (1) PEASE AND ELDERFIELD, J . Org. Chem., 6, 192 (1940). (2) PEASE, REIDER,AND ELDERFIELD, J . Org. Chem., 6,198 (1940). (3) STEMPELAND ELDERFIELD, J . Am. Chem. Soc., 63, 315 (1941).


(4) Private communication, Dr. Oliver Kamm, Parke, Davis & Co. (5) ELEKAND HARTE,Ind. Eng. Chem., Anal. Ed., 8,267 (1936). (6) HOCKETT A N D MCCLENAHAN, J . Am. Chem. SOC.,61, 1667 (1939). (7) HUDSON A N D CHERNOFF, J . Am. Chem. SOC.,40, 1005 (1918). (8) VOTOEEK, Collection Czechoslov. Chem. Commun., 2, 36 (1930). J . B i d . Chem., 64, 65 (1922). (9) CLARK, (10) RABAT&, J . pharm. chim., [SI, 24,311 (1936). (11) CRAWFORD, Bull. 129,u. s.B. Plant Industry (1909). (12) SUZUKI, SRIMIMURA, ODARE,Biochem. Z., 43,100 (1912). (13) Handbuch der Pflanzenanalyse IV/I, 111,p. 278 (Vienna, Julius Springer, 1933). (14) BEBESCHIN, 2. physiol. Chem., 72, 385 (1911). 2 . physiol. Chem., 92,471 (1914). (15) STOLTZENBERG, (16) HANES,Biochem. J.,23, 99 (1929).

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