Aug., 1916
T H E J O C R N A L OF INDUSTRIAL A N D ENGINEERING CHEMISTRY
there is no significant difference; t h a t is. t h e protein in t h e younger plants was more profoundly affected b y this condition t h a n t h a t from the seed stage, which was very little changed. a-The differences between total and pure protein are greater in t h e alfalfa cured in the shade t h a n in t h a t cured in t h e sun or open field; t h a t is, t h e proportion of pure protein decreases during t h e process of drying. This is most noticeable in t h e earlier stages. The hay cured in t h e sun contains a larger per cent of pure protein t h a n t h a t cured in t h e shade. I n comparing t h e figures obtained on the samples for 1914 with those of 1915, important differences are noticed. The per cent of pure protein in the total protein is less in every instance in the samples of 191j 3 and differences between t h e alfalfa cured in t h e shade and t h a t cured in t h e sun are less. The more unfavorable drying conditions of 191j , with the necessity of curing under cock covers, allowed a prolongation of the vital activity of t h e protoplasm, resulting in protein cleavage. The per cent of pure protein in the total protein is from 6 t o 9 per cent less in the alfalfa dried in t h e shade in 1915 t h a n in t h e same lot in 1914, while t h e 191j samples dried in the open field show about 9 per cent less t h a n t h e 1914 samples. T h e larger, heavier plants of 1915 took longer t o dry. T h e stems, which dry the slowest, show t h e greatest differences in comparing t h e samples of the two years, a n d t h e leaves, which d r y t h e fastest, show t h e least difference. These facts point t o a very profitable line of investigation in regard t o t h e chemical changes which t a k e place under different conditions of haymaking. These changes no doubt include not only t h e proteins, b u t also t h e carbohydrates, fats, and phosphorus compounds. T h e feeding value of h a y is affected not only b y mechanical losses due t o handling, and the changes due t o bacterial action, b u t also b y chemical changes which are little known or noticed.
.
SURIMARY
I-The alfalfa cut in t h e bud stage had t h e largest a s h and crude protein and t h e smallest crude fiber and nitrogen-free extract. 11-In each successive stage t h e crude fiber a n d nitrogen-free extract increases, and t h e crude protein and ash decrease. I n pounds per t o n t h e alfalfa cut in t h e earlier stages has more of crude protein a n d less of crude fiber. 111-The total amount of a n y or all nutrients produced per acre depends t o a large extent on t h e yield, as shown b y t h e fact t h a t in 1914 t h e greatest amount of nutrients was obtained in t h e bud stage, while in 191 j t h e full bloom gave t h e greatest amount of total nutrients. IV-The leaves and stems differ in content of ash, e t h e r extract, and nitrogen-free extract, b u t the greatest difference is in t h e per cent of crude protein and crude fiber. The leaves contain over a 1 / 2 times as much protein as the stems, while t h e stems contain over 2 l / p times as much crude fiber as t h e leaves.
729
V-In harvesting and handling there is a large loss of leaves, which loss affects t h e composition of the hay in a n increase of crude fiber and a decrease of crude protein. VI-The alfalfa cured in the sun has a larger pure protein content as determined by Stutzer's method, t h a n t h a t cured in t h e shade. This difference is s o great as t o more t h a n offset t h e influence of the loss of leaves. The differences in respect t o pure protein content were most pronounced in the alfalfa cut in t h e earlier stages. CHEMICAL DEPARTMENT .kGRICULTURAL
EXPERIMENT STATION
MANHATTAN, KANSAS
THE VOLATILE OIL OF CALYCANTHUS OCCIDENTALIS By CHARLES C. SCALIONE
Received April 11, 1916
Calycanthus occidentalis, or Butneria occidentalis, also popularly known as spice-bush, belongs t o t h e family Calycanthaceae, 4 or 5 species of which are found in t h e United States. The species named is limited t o northern California and southern Oregon and is rarely found in large patches, b u t usually scattered along stream banks or river bottoms or on t h e lower slopes of hillsides. It is an unarmed shrub, which sometimes reaches a height of I O ft. It bears a n abundance of ovate t o oblong lanceolate leaves, somewhat scabrous and of a bright green color, and The leaves, dark red flowers from I t o 1'/2 in. long. bark and wood are all strongly aromatic; the flowers less so. Considerable interest has been attached t o some of the other species of this genus on account of their physiological action. An alkaloid was discovered in t h e seeds of C. glaucus C.Jerfilis, b y R . C. Eccles,' in 1888, and named by him Calycanthine. This was confirmed by H. W. Wiley2 in 1889. H. 1 4 . G ~ r d i n ,in ~ a number of papers, has shown t h e presence of two alkaloids, calycanthine and isocalycanthine. The volatile oil of Floridus has been studied b y Miller4 and his co-workers and found t o consist of pinene, cineol, borneol, bornyl acetate, salicylic acid, possibly linalool and some other esters besides the bornyl acetate. The cineol predominated. 1
EXPERINENTAL
Four hundred pounds of leaves and twigs from Oriental, California. t h a t had been kindly supplied by officials of t h e United States Forest Service, a t San Francisco, were submitted t o steam distillation under a pressure of 4 lbs. The yield of oil obtained from t h e united distillates amounted t o about 0 . 2 7 per cent of t h e weight of t h e original material. I n transit considerable decomposition of the leaves had taken place and the water distilling over with 1 2
Eccles, Proc. Am. Phaym. Assoc., 36, 84 and 382. U'iley, A m . Chem. J., 11, 5 5 7 . Gordin, Proc. A m . Phaum. Assoc.. 62, 345; 53, 224. Miller. J . A m . Chem Soc., 26, 2182.
i30
T H E J O 1 7 R J A L O F I S D C S T R I A L Ah-D E S G I X E E X I X G C H E M I S T R I -
the oil ll-as strongly alkaline. After removing the oil the alkaline distillates were treated v i t h a n excess of hydrochloric acid and evaporated t o dryness on the water bath. The residual salts were found t o have an odor X7ei-y suggestive of aliphatic amines; they were crystallized from absolute alcohql and the chlorine determined in both the alcohol-soluble salt and the alcohol-insoluble salt. The former determination gave 3 7 . 4 8 per cent chlorine. which corresponds with t h e theoretical percentage of chlorine in trimethylamine hydrochloride (i, e . , 3 7 , 1 7 per cent). The alcohol-insoluble salt gave 66.I 8 per cent chlorine which corresponds with t h e percentage of chlorine in ammonium chloride ( i . e., 6 6 . 3 7 per cent). A distillation on some fresh leaves failed t o show either of these constituents and whether t h e trimethylamine and ammonium hydroxide were due t o t h e decomposition of the protein matter alone or whether they were derived from t h e protein material plus some easily decomposable alkaloid has not been definitely settled. Qualitative determinations with phosphomolybdic acid demonstrated t h e presence of basic bodies in t h e leaves. These bodies failed t o respond t o tests for tannins: they were probably alkaloidal in character. T h e total nitrogen content was found t o be 3 . 7 j per cent b y weight of t h e dry leaf. This nitrogen content is abnormally high if due t o protein material alone. A second sample of t h e plant, which mas grown in a garden a t Berkeley and weighed some 30 lbs., was separated into twigs and leaves and t h e two portions distilled separately while still fresh. The yield of oil from t h e leaves was about 0 . 1 5 per cent and from the twigs 0 . 3 7 per cent. PHYSIC.AL C O K S T A K T S OF T H E O I L
The oil had a greenish yellow appearance, a bitter taste, and a camphoraceous odor. The constants were as follows: Density a t 25' C . , , . , . , 0,9295, Rotation.. . . . . . . , , . . . . 7 7 ' 2 8
Free acid.. . , , , , , , , . . , , . Saponificntion value.. . . .
54.3
Index of refraction N',"
Acetyl v a l u e .
. . ... . . . , .
33.5
1,4713
0.05
The oil is soluble in all proportions in 90 per cent alcohol, and in I j t o 16 volumes of 7 0 per cent alcohol by volume. CHEMICAL EXAMIKATION O F THE O I L
KETONES-The oil did not respond t o decolorized magenta or sodium bisulfite solution. When tested with phenylhydrazine a distinct cloudiness was produced. This xTould indicate the presence of a ketone t h a t does not respond t o the magenta or bisulfite test. such as camphor. ALDEHYDES AKD
E R C C acID-The free acid value of the oil was low t h a t it was impossible t o identify the acid.
SO
COUBISED ACIDS-One hundred grams of oil were saponified with alcoholic potassium hydroxide b y heating on t h e water b a t h with a reflux condenser for half a n hour. V a t e r was added t o the mixture and the oil layer separated out. After distilling off
T-GI. 8 , XO. 8
the excess of alcohol on the water bath the alkaline solution mas extracted with ether t o rcmovc any adhering oil. The remaining solution was evaporated t o a small volume, acidified with sulfuric acid and tiistilled 7%-ith steam. The distillate was extracted with ether. The ether was exyaporated spontaneously, leaving a reddish brown crystalline residue. T h e n tested with ferric chloride a purple color was f o r m e d , indicating salicylic acid. The aqueous portion remaining after the ether extraction was neutralized with sodium carbonate., An aliquot of one-tenth of t h e 'original solution was concentrated t o a small bulk and precipitated in three fractions with silver nitrate. Fraction I yielded I . 0 j 6 0 g. of Ag salt; Fraction' 2 , 0 . 543 I g. of Ag salt; Fraction 3, 0 . 2114 g. of Ag salt. A perceptible reduction of t h e silver nitrate showed t h e presence of formic acid. On ignition t h e following percentages of silver were obtained from the silver salt: Fraction I , 6j.8 j per cent Ag, Fraction 2 : 5 3 . I per cent 4 g , Fraction 3, 3 9 . 9 per cent Ag. Acetic acid yields 64.6 per cent Ag, butyric acid jj.3 per cent Ag. valerianic 5 1 . 6 per cent .4g. and capric 3 8 . 6 7 per cent Ag. The combined acids consisted mainly of acetic and salicylic with traces of formic, capric and possibly butyric, or valerianic, or a mixture of t h e t v o . FRACTIONATIOh- O F T H E O I L
T h e oil was fractionated with a 3-bulb Le Bei fractionating column, t h e results being given in the table. TABLEI-FRICTIOIIATION SHOWING
OF THE OIL OF CALYCANTHUSOCCIDEIITALIS P H Y S I C A L PROPERTIES OF EACA F R A C T I O N
Specific Specific Frac- Fractionation P e r c e n t Index of Gravity Rotation tion Temperatures Distilled Refraction 15' C. a t 15' C. No. over 15' C. "C. I 124-170 8.51 0.8810 +16.4Z0 1.4590 2 1,0-180 54.83 0.9119 4- 5 . 8 5 1.4585 3 180-190 9.47 0.9215 6.56 1.4650 4 190-200 2.48 0.9290 +- 9 . 5 2 1.4665 5 200-220 8.75 0,9635 +36.42 1.4817 220-ovFr 10.22 0 9365 +18 85 1.4793 i 5.01 0.9171 ... 1 ,4884 Fractions 5 , 6 and 7 were made under reduced pressure a t 150--180°. T h e boiling points of these fractions %!ere redetermined a t atmospheric pressure.
+
e
From the fractions in Table I it will be seen t h a t this oil contains j groups of constituents. the first distilling between I 54-1 7 o o , the second between 170-190°, the third between 1 9 0 - 2 2 0 ~ ; the fourth between 2 2 0 - - 2 3 0 ° , and the fifth over 2 3 0 ' C. CHEMIC4L
EXAMINATIOS O F
THE
FRACTIOSS
PIh-EKE-Fraction I consisted mainly of pinene. The nitroso chloride was prepared according t o the method of TTallachl and was found t o melt a t 1 0 2 . 3 '; the pure nitroso chloride melts a t 1 0 2 - - 1 0 3 ~ .The medium value of the specific rotatory power indicates t h a t although the pinene is mainly of the dextrorotatory form some of the laeyo-rotatory form i s also present. X quantitative estimation of pinene in the original oil mas made b y distilling off a fraction betTTeen I j j and 170' 6.and placing a portion of this fraction in a cassia flask -and adding a j o per cent 1 XTaIlach,
Liebig's A n n . , 245, 251; 253, 251
Aug.. 1916
T H E J O LT R N A L O F I N D USTRI A L A iVD E X G I N E E RI N G C H E M I S T R Y
resorcinol solution, which absorbed t h e cineol. From t h e residual volume i t was estimated t h a t about 8 . 3 per cent of t h e original oil consisted of pinene. cImoL-Fractions 2 , 3 a n d 4 combined a n d refractionated between 175 a n d 180'. After twice refractionating, t h e portion boiling a t 176' was collected. T h e physical constants of this fraction and of t h e melting point of t h e iodol show t h a t this fraction is mainly cineol. Roiling. p o i n t . . . . . . . . . . . . . . . . . . Density a t 20' C . . . . . . . . . . . . . . . I n d e x of refraction a t 20' C . . . . . Melting point of iodol. . . . . . . . . . .
Fraction 176- 17 7 0.930 1,456; 11 1 . 2
P u r e Cineol 176' 0.929
1.45:9 112
A quantitative determination b y t h e resorcinol method gave 60.32 per cent cineol b y weight of t h e original oil. B O R N E O L A N D CAMPHOR-Fractions 4 a n d 5 were combined a n d redistilled under vacuum. T h e y had a strong camphoraceous odor a n d on freezing crystals separated out which were very difficult t o remove from t h e adhering oil. An a t t e m p t t o prepare t h e semicarbazone b y t h e method of Tiemannl gave no results. Phenylhydrazine produced a distinct cloudiness, showing t h e presence of a ketone. When t h e fraction was heated t o vaporization, a n d t h e vapor permitted t o cool on a microscopic slide, .a white sublimate was obtained which was found t o consist of two distinct substances, one crystallizing in plates, t h e other in smaller irregular aggregates. This would point toward a mixture of borneol a n d camphor. A portion of this fraction was oxidized with Beckmann's chromic acid mixture, t h e acid neubalized and. t h e mixture distilled with steam. The semicarbazone was prepared f r o m t h e product and was found t o have t h e melting point 23 5 . 8 ': camphor semicarbazone melts a t 236-238'. This fact, as well as t h e evidence of a ketone in t h e original oil, points t o a mixture of borneol a n d camphor. If t h e acetyl value be considered due t o borneol t h e n there would be 9 . 2 1 per cent borneol present. This value is a little high, as some of t h e sesquiterpene alcohols present combined t o form t h e ester with acetic anhydride, t h u s raising this constant. M E T H Y L SALICYLATE-Fraction 6 h a d a strong pleasa n t odor of esters. Since salicylic acid was found in t h e original oil, methyl salicylate was suspected. This fraction was treated with cold N / 5 potassium hydroxide solution in order t o form t h e soluble potassium methyl salicylate salt. After extracting t h e remaining oil t h e alkali solution was neutralized with dilute acid and again extracted with ether. On spontaneous evaporation of t h e ether a small q u a n t i t y of oil remained, having t h e characteristic odor of methyl salicylate. Such a small quantity was obtainable t h a t t h e index of refraction alone was determined a n d found t o be I . 5298. T h e index of refraction of pure methyl salicylate is I . 536 t o I . 538 a t 20' C. A further amount of t h e oil was saponified with concent r a t e d hot alkali solution. T h e solution was acidified a n d extracted with ether. The ether extract yielded 1
Tiemann's B e y . , 27, 1, 81.5
-,
/51
a few crystals which gave t h e characteristic coior reactions for salicylic acid. From these d a t a and from t h e boiling point of t h e fraction in which this substance appears it can be safely concluded t h a t a small quantity of methyl salicylate was present in t h e oil. L I N A L Y L A C E T A T E - T ~ ~ remainder of Fraction 6 h a d a very pleasant bergamot odor. When saponified with alcoholic potash t h e saponification number was found t o be 277.9. T h e alcoholic solution was diluted and t h e excess alcohol distilled off on t h e water b a t h . T h e solution was acidified a n d extracted with ether. Silver nitrate was added t o t h e solution; t h e per cent of silver in t h e silver salt obtained was found t o be 64.12 per cent Ag: acetic acid combines with 6 4 . 4 6 per cent Ag. T h e empirical formula for t h e ester would t h e n be C l ~ H 1 7 0 0 C . C H 2 .The ether extract from this solution was evaporated spontaneously a n d t h e residual oil refractionated. The greater proportion distilled between 197 t o 198'. T h e specific gravity was found t o be 0 . 8 7 9 3 ; t h e index of refraction a t zoo C. was q.4691. If from this d a t a t h e molecular index of refraction is calculated it is found t o be 48. 7 7 . T h e theoretical molecular index of refraction for a compound of formula C I O H I 7 0 H with two double bonds is 48.86. This would indicate linalool. The only other alcohol having this structure is geraniol which has a higher boiling point and a greater specitic gravity t h a n linalool. The presence of linalool was confirmed b y preparing the phenyl urethane derivative which h a d a melting point a t 6 j . 4 " : linalool phenyl urethane melts a t 65 t o 66". S E S Q U I T E R P E N E ALCOHOLS-These remained in t h e fractions boiling above 2 3 0 ' C. Owing t o t h e small quantity of material and due t o t h e fact t h a t considerable polymerization had taken place, it was impossible t o determine what they were. They constituted less t h a n j per cent of t h e original oil. S U MM A R Y
The volatile oil of Calycanthus occidentalis is made u p of t h e following constituents in t h e proportion given: Per cent
. . . . . . . . . 8.30 . . . . . . . . . 60.32 9 . 21 Borneol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Camphor. . . . . . . . . . . .......
.... M e t h y l salicylate. , . Linalyl acetate. . . . . . . . . . . . . . . . . . . . . . . . . . . 1S : 99 Sesquiterpene alcohols., . . . . . . . . . . . . . . . . . . . . . .
I n t h e above estimation of t h e percentages of constituents present, t h e pinene a n d cineol were determined directly. The borneol was calculated from t h e acetyl value: this is perhaps slightly in error, due t o t h e presence of sesquiterpene alcohols. The linalyl acetate was estimated from t h e saponification value, a n d is slightly in error, due t o a small amount of methyl salicylate. I n conclusion, t h e writer wishes t o t h a n k Professor W. C. Rlasdale, of t h e Chemistry Department of t h e University of California, for valuable suggestions in carrying out this investigation. CHEMICAL LABORATORY EXIVERSITY OF CALIFORMA, BERKELEY
