THl3 FATTY ACIDS OF CHRYSALIS OIL

BY WERNER BERGMANN*

(From the Department of Chemistry, Yale University, New Haven)

(Received for publication, February 6, 1936)

The systematic researches of animal depot fats carried out by Hilditch, Tsujimoto, Klenk, and others have demonstrated that specific differences characterize the fats from marine, fresh water, and land sources. As far as land animals are concerned the in- vestigations have dealt mainly with vertebrates. Our knowledge of the fats of invertebrates, such as insects, is rather limited. What is known has been largely derived from the study of the fat of a herbivorous insect, the chrysalis oil of the silk moth, Bombyx mori, which has been the subject of several qualitative analyses (1). In order to make an intelligent comparison between fats from insects and from other animals, a more quantitative study of the chrysalis oil seemed desirable, and it was the object of the present investigation to supply the necessary data.

These data, which are shown in Table III, demonstrate that the fatty acids of chrysalis oil contain four major components: pahnitic, oleic, linoleic, and linolenic acids. As minor components stearic, palmitoleic, and unsaturated GO to Cn acids were found. The main difference which distinguishes this insect fat from the fat of higher organized land animals is the relatively high content of linolenic acid.

Commercial Chrysalis Oil

The chrysalis oil used in the present investigation was of the same stock as the oil which had been employed for the preparation of bombicysterol (2). Its properties are shown in Table IV. The oil was found to contain less than 1 per cent of acetone-in-

* Holder of a Textile Foundation, Inc., Research Fellowship, 1935-36. This paper is published with the approval of the Directors of the Textile Foundation, Inc., Washington.

27

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28 Fatty Acids of Chrysalis Oil

soluble material. On the bottom of the flask containing the oil a white precipitate was observed, which disappeared on warming. On long standing the amount of precipitate increased considerably, reaching over 10 per cent of the oil.

White Preci$ate-Piutti (3) claims that a white precipitate which he observed in chrysalis oil was stearic acid. In his opinion this acid owes its origin to a partial hydrolysis of the oil caused by the killing of the chrysalis with toxic gases. Stearic acid, how- ever, as will be shown later, occurs in chrysalis oil only in small amounts, the bulk of the solid acids being palmitic acid. It seems, therefore, Ohat Piutti’s statement is erroneous.

When carefully purified, the white precipitate from commercial oil melted at 67” (stearic acid, m.p. 70.5’). Its crystals were quite different from those of stearic acid. The substance was found to be neutral and to give palmitic acid and glycerol on saponification. From the yield of palmitic acid obtained it was concluded that the glyceride was a dipalmitate. The presence of a free hydroxyl group in the glyceride could be demonstrated by the formation of an acetate and a bensoate.

Of the two possible isomers of glyceryldipalmitate, which have been synthesized by Griin (4), only the symmetrical glyceride exhibits properties identical with those of the substance from chrysalis oil. One can, therefore, conclude that the white precipi- tate is glyceryl-1 ,3-dipalmitate.

This peculiar glyceride is absent in oils obtained from living chrysalises. It is apparently a product of decomposition caused either by the stifling of the cocoon or by aging. It could, how- ever, not have been formed by a partial hydrolysis of any one of the eight glycerides which Suzuki and Yokoyama (5) have found in chrysalis oil, because none of them contains 2 molecules of pahnitic acid to 1 molecule of glycerol.

Isolation of Glyceryl-i ,3-Dipalmitate-The white precipitate contained in the oil was collected by centrifuging and washed with petroleum ether. The substance was then recrystallized four times from boiling petroleum ether and twice from alcohol. The glyceride which was obtained in a yield of 5 per cent crystal- lizes in small needles and melts at 67”.

Saponijication Number-92961 gm. of substance required 10.37 cc. of 0.1 N KOH. Calculated for (C1~H&0&C3H60H, 197.34; found, 196.6.

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W. Bergmann 29

7.4 gm. were refluxed with 5 per cent alcoholic potassium hy- droxide for 1 hour. The acids were then isolated in the usual way, giving 6.61 gm. of palmitic acid or 89.2 per cent. Calcu- lated for a dipalmitate 90.13 per cent, for a tripalmitate 95.26 per cent, and a monopalmitate 77.57 per cent.

The palmitic acid after one recrystallization from alcohol melted at 62”.

0.4310 gm. of substance required 16.93 cc. of 0.1 N NaOH. Calculated for CleH3202, 256.25; found, 254.6.

When the substance was mixed with pure pahnitic acid, no depression of the melting point could be observed.

From the neutral part of the saponification mixture 0.7 gm. of glycerol was obtained.

fi’-Acetoglyceryl-1,3-Dipalmitate-The glyceride was refluxed for 3 hours with 10 times its weight of acetic anhydride. After cool- ing the acetate was precipitated by the addition of water and re- crystallized four times from alcohol. It crystallizes in the form of small needles, and melts at 47”. On cooling, the liquid sub- stance solidifies at 29” and then melts constantly at 33”. The same behav$or is shown by the synthetic substance.

Anulysis-0.3036 gm. of substance required 14.74 cc. of 0.1 N

KOH. Saponification number calculated for Cs,H,OO~, 275.7; found, 272.5.

bBenzoglyceryl-1, S-Dipalmitate-Benzoyl chloride was added to a solution of the glyceride in pyridine. After 3 hours standing the mixture was poured into sufficient dilute H&J04 to neutralize the pyridine. The benzoate was precipitated as an oil which gradually solidified in the cold. It was filtered off, washed with water, and recrystallized four times from alcohol. The benzoate crystallizes in the form of long felty needles and melts at 44’.

Analysis-O.4298 gm. of substance after 3 hours refluxing with alcoholic potassium hydroxide had required 19.16 cc. of 0.1 N

KOH. Saponification number calculated for C41H,202, 250.3; found, 250.1.

The Fatty Acids-The oil in which the white precipitate was redissolved by warming was saponified with alcoholic potassium hydroxide in an atmosphere of nitrogen. The non-saponifiable material was then removed by extraction with petroleum ether and the acids isolated in the usual way. They represented 91 to

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30 Fatty Acids of Chrysalis Oil

92 per cent of the oil, were of a dark color, and showed an equiva- lent weight of 290 to 300 and an iodine number of 90 to 100.

The determination of solid acids by the Twitchell method met with difhculties. On addition of lead acetate to the alcoholic solution of the acids considerable amounts of a brown precipitate were formed, which proved to be almost insoluble in boiling al- cohol and which interfered considerably with the determination. Since this behavior indicated the presence of oxidation products, the latter were removed with petroleum ether, in which they are rather insoluble.

For that purpose the acids were stirred for some time. with 50 times their weight of petroleum ether. After standing for several hours the solution was then poured off the smeary brown oxida- tion products, and the solvents driven off under nitrogen. The remaining acids were redissolved in petroleum ether, and the solution to which some norit was added was kept standing over- night. After filtration and evaporation of the solvents a slightly yellow oil remained. The acids thus obtained represented 83.7 per cent of the oil and showed an equivalent weight of 284.1 and an iodine number of 122.1.

The acid mixture contained 23.85 per cent solid acids as de- termined by the Twitchell or 24.9 per cent as determined by the modified Bertram (6) method. For the isolation of larger quan- tities of solid and liquid acids the lead soap-ether method was used.

Solid Acids-The solid acids melted at 61-62” and showed an equivalent weight of 264.2 and an iodine number of 2.0.

The acids were esteri6ed by refluxing them with absolute ethyl alcohol containing 5 per cent of concentrated sulfuric acid and the esters extracted with petroleum ether. 50 gm. of the ethyl esters were then subjected to a careful fractionation in the distil- ling apparatus described by Jantzen and Tiedtke (7).

The residue was distilled from a small Claisen flask, m.p. 35”, refractive index, 1.4352, acid m.p. 69-70°, equivalent weight 290.2 (0.3277 gm. required 10.17 cc. of 0.1 N NaOH).

The results of the fractionation (Table I) show that there can only be little if any of a CM acid. The bulk of the acids is repre- sented by a pahnitic acid, but stearic acid also is present in con- siderable quantities. The presence of small amounts of an acid

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W. Bergmann 31

higher than C18 is indicated by the high equivalent weight of the acid obtained from the last fraction. In agreement with the observation of Ueno and Ikuta (8) isopalmitic acid as found by Kawase et al. (9) could not be detected.

Liquid Acids-The liquid acids showed an equivalent weight of 285.5 and an iodine number of 161.0.

The liquid acids were hydrogenated in ether solution with plati- num oxide as a catalyst. The hydrogenated acids melted at 69’. Equivalent weight 284.9, iodine number less than 2.

TABLE I

Fractionation of Ethyl Esters of Solid Acids

Ethyl eatem Acid8 _

Yield Refrm-

tive ndex at M.p.

42.5” --

-2.

1.4310 24-25 1.4311 24-25 1.4310 24.5 1.4310 24.5 1.4310 24-25 1.4310 24.5 1.4310 24.5 1.4310 24-25 1.4310 24.5 1.4312 24.7 1.4318 24-25 1.4338 25-28 1.4346 33 1.4349 34-35

M.p.

-

-

-

T- Sub- 0.1 N stsmce NaOH

-- cc.

OLO 23.91

Theory

M.p.

“C.

Cd 62.5

Id. wt.

llm.

3.84 4.33 4.67 2.33 2.62 2.31 2.65 4.90 4.11 2.96 1.40 5.29 2.25 3.55

“C.

60-61 61.5 61.7

257.2 80, 256.3

61.5 256.5

1 2 3 4 5 6 0.2578 10.05 7 8 9

10 0.5651 22.06 11 0.2355 10.88 12 0.5194 19.10 Es 13 0.4340 15.25 14 0.3835 13.37 I - The hydrogenated acids were ethylated and distilled as above. Fraction 1 (Table II) indicates the presence of palmitic acid

probably derived from the hydrogenation of pahnitoleic acid. Fractions 2 to 10 contain ethyl stearate almost exclusively and Fraction 11 indicates the presence of acids higher than C18.

In order to obtain more evidence for the presence of a palmi- toleic acid, 250 cc. of the methyl esters of the mixed saturated and unsaturated acids were divided into four fractions by a frac- tional distillation in a Claisen flask. The 6rs.t fraction (66 gm.)

62 61-62 65-67 67.8 69

257.5 262.3 272.1 284.0 287.0

-

,

-

C181 70.5

40s 284.3

-

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32 Fatty Acids of Chrysalis Oil

was then subdivided into eleven fractions in the Jantzen and Tiedtke distilling apparatus. Fractions with similar refraction indices were united for further investigation.

Fractions 1 to 6 which contained the Cl6 acids weighed 30.9 gm. The iodine numbers observed ranged between 6 and 7, and the equivalent weights of the corresponding acids between 253.8 and 254.6. It is evident that the amount of palmitoleic acid pres- ent in the CM fraction can only be rather small, certainly not over

TABLE II

Fractionation of Ethyl Esters of Hydrogenated Liquid Acids

1 2 3 4 5 6 7 8 9

10 11

I Ethyl e&m

Yield

om.

2.08 11.24 8.72 5.80 5.00

10.01 5.68 4.48 4.00 1.32 0.95

6 per cent. ture failed.

Refrac- 1 tin

index at M.P. 42.b”

-- CC.

1.4319 32-33 1.4333 33.5 1.4340 33.7 1.4343 33.7 1.4343 33.7 1.4343 33.7 1.4343 33.7 1.4345 33.7 1.4345 34 1.4347 34 1.4364 35-37

-

-

M.p.

“C.

63-64 67-68 68-69 68.7

268 282.2 283.0 284.5

Sub- 0.1 N stance NaOH

-- pm. cc.

0.1785 6.81 0.4305 15.14 0.4856 17.16 0.6806 23.93

M.p. MoLwt. --

c.

CJL~O~ 62.5 1256.3

CmHmOz 70.5 234.3

69 284.4 0.2387 8.39

69-70 286.1 0.2950 10.32 C&boO~ 70-72 301 .o 0.1545 5.13 75.1 1312.4

Acids

Attempts to isolate palmitoleic acid from the mix-

The next two fractions (9.0 gm.) were mixtures of Cl6 and Cl8 acids and were not further investigated.

Fractions 9 to 11 (13.7 gm.) contained the Cl8 acids. The iodine numbers of these fractions ranged from 131 to 132. 4.3245 gm. of the ester mixture were saponified and the solid acids de- termined by Twitchell’s method. 0.325 gm. or 7.94 per cent of stearic acid with a melting point of 69” and an equivalent weight of 284.7 was obtained.

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W. Bergmann 33

Fatty Acids of Freshly Prepared Chrysalis Oil

Since the commercial chrysalis oil had shown signs of decompo- sition, fats extracted from living chrysalis of Bombyx mori were studied for comparison. Investigations were carried out with samples imported from Japan, China, and Italy and it was found that there are only insignificant differences in the composition of oils of different origins. As an example, the investigation carried out with the chrysalis of an Italian, yellow race of Bombyx mori will be described.

Isolation of 031-739 chrysalises, weighing 1160 gm., were ground in a meat grinder and the pulp mixed with acetone. After a few hours the mixture was centrifuged, the supernatant liquid poured off, and the residue stirred with more acetone. After the procedure was repeated twice the acetone was replaced by ether and finally the residue was extracted with ether in a Soxhlet apparatus. The acetone was distilled off in a vacuum and the remaining aqueous liquid extracted with ether. All ether ex- tracts were combined, dried, and evaporated to dryness. The entire procedure was carried out in an atmosphere of nitrogen.

The oil which weighed 58 gm. was of a clear yellow color. It did not deposit a precipitate on standing and was easily soluble in petroleum ether. Some of its properties are shown in Table IV.

By repeated treatment of a solution of the oil in ether with acetone, the oil was separated into an acetone-soluble and an in- soluble fraction. The insoluble phosphatide fraction represented 8 per cent of the oil. It was a slightly yellow amorphous powder, the composition of which will be the subject of a separate inves- tigation.

The acetone-soluble oil was saponified in the usual way. The soap solution was then repeatedly extracted with petroleum ether to remove the non-saponifiable fraction. This fraction, 2.5 per cent of the oil, possessed a deep yellow color quite different from the almost colorless non-saponifiable part obtained from com- mercial chrysalis oil. Otherwise their compositions are quite similar (2).

After acidification the soap solution yielded 94.08 per cent of a slightly yellow acid mixture which was completely soluble in petroleum ether. With Twitchell’s method 4.6148 gm. gave

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34 Fatty Acids of Chrysalis Oil

1.1394 gm. or 24.7 per cent of solid acids. Equivalent weight 264.1, iodine number less than 2, m.p. 61.5-62”.

The liquid acids isolated by the lead soap-ether method showed an iodine number of 163.4 and a thiocyanogen (Kaufmann) num- ber of 123.3. From these figures it can be calculated that the mixture of unsaturated acids contains 50.49 per cent of oleic, 13.48 per cent of linoleic, and 35.93 per cent of linolenic acid.

4 gm. of unsaturated acids were placed in a small centrifuge bottle and dissolved in petroleum ether. To the solution, which had been cooled to -lo”, bromine was added until a slight yellow color remained. After 1 hour’s standing the solution was centri- fuged, the precipitate washed with ice-cold petroleum ether, and then extracted with ice-cold ether. 3.65 gm. of a white bromide remained, which were completely soluble in boiling benzene and which after one recrystallization from benzene melted at 17% 180” (hexabromide of linolenic acid, m.p. 179”). From the hexa- bromide value and the iodine number of the unsaturated acids it can be calculated that the mixture contains 47.91 per cent of oleic, 18.56 per cent of linoleic, and 33.50 per cent of linolenic acid. These values agree reasonably well with those calculated from the iodine and thiocyanogen numbers. They differ, however, from the values reported by Kimura, who found 29.8,29.2 per cent of oleic, 48.9, 35.9 per cent of linoleic, and 21.3, 34.9 per cent of linolenic acid. The fhst values were determined by the bromide method (10) and the second by the combination of the iodine and thiocyan- ogen numbers (11).

Mixtures of saturated and unsaturated acids obtained from dif- ferent oils but having similar properties were united and methyl- ated. 90 gm. were then divided into twenty-three fractions in the Jantzen and Tiedtke apparatus. The iodine numbers and melting points of the different fractions were determined, as were the amount of solid acids in the acid mixture after saponification, and the melting points and equivalent weights of the hydrogenated acids. The results of these observations are essentially the same ss those obtained from commercial chrysalis oil. No de6nite evidence for a Cl4 acid could be found. The Cle acids show a slight degree of unsaturation, the iodine number being 6 to 7. The Cl* acids showed an average iodine number of 137 and contained 8.06 per cent of stearic acid. The last two small fractions (1.55

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W. Bergmann 35

gm.), having an iodine number of 138.6, gave after hydrogenation and saponification an acid which after one recrystallization from acetone melted at 72-73’. Its equivalent weight was 201.1 (0.3248 gm. required 10.79 cc. of 0.1 N NaOH). Here again the presence of CB acids was indicated.

From all the results obtained it can be deduced that the average composition of the mixture of fatty acids from the chrysalis oil of Bombyx mori is as represented in Table III.

TABLE III

Per Cent Composition of Acid Mixture

Saturated acids Umaatumted acids

Cl4 Cl6 Cl8 cwcn CM CM CSQ-CX -- ~-

-He -H, -Hc ? 20 4 <l 2 I I 35 12 28 l-2

Chrysalis Oil of Tent Moth, Malacosmu americana

For reasons of comparison the oil of a native American silk- spinning moth was investigated. Living tent moth chrysalises, collected during June and July, 1935, were extracted in the same manner as the chrysalis of Bombyx mori. From 870 chrysalises, representing 480 males and 390 females, and weighing 361 gm., 19 gm. of a clear yellow oil were obtained. For properties of this oil see Table IV.

18.2 gm. of oil gave 1.3 gm. or 7.1 per cent of an acetone-in- soluble phosphatide. The acetone-soluble oil was saponified, giving 1.2 to 1.3 per cent of a yellow crystalline non-saponifiable fraction and 94.7 per cent of an acid mixture completely soluble in petroleum ether.

6.6324 gm. of mixed acids gave 2.9818 gm. of solid acids or 31.0 per cent. Equivalent weight 261.8, iodine number below 2.

The methyl esters of 4.8 gm. of solid acids were subjected to a fractional distillation. Of the four fractions obtained the first three contained methyl pahnitate exclusively, while the fourth yielded an acid with a melting point of 65-66” and equivalent weight of 272. From the residue an acid was obtained which

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TABL

EI

IV

Com

paris

on

of V

ario

us

Oils

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khpq

ni-

NOW

po

len-

Rdcb

- 3

samp

le De

nsity

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&&~

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‘“N&i?

sa

poni-

Ac

id No

. er

t- He

-hmr

,S$

* aS

able

Sk0

No.

. .

“%’

lII&W

id ----

__-__

__

g.

per

cent

pm

ce

nt

m

Vario

us

oils

(l)*.

. .

. D

1~.

~0.9

280

2WO

.147

57

189.

9 10

5-13

2 1.

6-10

8.

5-62

.8

3.2

94.5

23

.8

0,

Da0

0.91

05

20”0

.147

07

25

Com

mer

cial

oil..

.

. D

26

0.92

30

25O

O.1

4720

18

9.9

109

1.5

7.1

3.0

24.4

Fr

esh

oil..

.

. . .

. D2

6 0.

9210

25

OO

.147

24

191.

7 12

3 2.

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8 0.

7 0.

5 94

.0

24.7

f

Tuss

ah

oil

(10,

11)

. .

. .

D20

0.92

59

20”0

.147

07

192

133

2.08

25

Te

nt

mot

h oi

l..

. .

. .

D 2

6 0.

9231

25

’0.1

4712

19

1.5

106.

2 1.

25

0.7

0.5

94.7

30

.1

E

* Th

e fig

ures

in

pa

rent

hese

s re

pres

ent

bibl

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c re

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. &

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W. Bergmann 37

after two recrystallizations from acetone melted at 69”. Equiva- lent weight 282.1.

The liquid acids showed an iodine number of 150 and a thio- cyanogen number of 111.3. Calculated, 57.45 per cent oleic acid, 19.7 per cent linoleic acid, and 22.85 per cent linolenic acid.

5.3 gm. of liquid acids were dissolved in 15 cc. of absolute alcohol and neutralized with 2 N lithium hydroxide solution. Sufficient water was then added to make the final volume 30 cc. After having been cooled for 12 hours, the salts of the lower unsaturated acids were filtered off and the illtrate evaporated to dryness. The residue was dissolved in a little water, dilute hydrochloric acid was added, and the acids were extracted with ether. 700 mg. of unsaturated acids having an iodine number of 208.8 were obtained. On hydrogenation stearic acid was formed with a melting point of 69-70” and equivalent weight of 285.1. The lower unsaturated acids obtained from the insoluble lithium salts were also hydro- genated. Here too stearic acid was obtained. The combined fractions of hydrogenated acids were methylated and the methyl esters were subjected to a fractional distillation. Four fractions were obtained all of which gave stearic acid on saponification. The material available for investigation was insufficient for a de- tection of minor components such as palmitoleic or CZO to CzZ acids.

A comparison of the oils from the silk moth and tent moth demonstrates a great similarity.

SUMMARY

Samples of commercial chrysalis oil as well as the oil obtained from living chrysalises of several varieties of Bombyx mot-i have been investigated. It was found that commercial oil contained considerable quantities of a solid material which was identified as glyceryl-1,3-dipahnitate.

The mixture of fatty acids obtained from chrysalis oil consisted of 20 per cent palmitic, 4 per cent stearic, 2 per cent pamitoleic, 35 per cent oleic, 12 per cent linoleic, and 28 per cent linolenic acid, besides less than 1 per cent of saturated and 1 to 2 per cent of unsaturated acids containing more than 18 carbon atoms.

The composition of the chrysalis oil of the tent moth, Maluco- somu americana, was very similar to that of Bombyx mori.

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38 Fatty Acids of Chrysalis Oil

BIBLIOGRAPHY

1. Griin, A., and Halden, W., Analyse der Fette und Wachse, Berlin, a, 491 (1929).

2. Bergmann, W., J. Viol. Chem., 107, 527 (1934). 3. Piutti, A., Rend. accad. SC., Nap& 33, 106 (1927). 4. Griin, A., Ber. them. Ges., 38, 2286 (1905). Grtin, A., and Corelli, O.,

2. Chem., 26, 665 (1912). angezu. 5. Suzuki, B., and Yokoyama, Y., Proc. Imp. Acad., Tokyo, 4, 161 (1928). 6. Bertram, S. H., 2. Untersuch. Lebensmittel, 66, 179 (1928). 7. Jantzen, E., and Tiedtke, C., J. prakt. Chem., 137, 227 (1930). 8. Ueno, S., and Ikuta, H., J. Sot. Chem. Znd., Japan, 37, suppl., 124

(1934). 9. Kawase, S., Suda, K., and Fukuzawa, A., J. Chem. Sot. Japan., 24,

181 (1921). 10. Kimura, W., J. Sot. Chem. Znd., Japan, 30, 858 (1927). 11. Kimura, W., J. Sot. Chem. Znd., Japan, 33, suppl., 262 (1930).

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Werner BergmannTHE FATTY ACIDS OF CHRYSALIS OIL

1936, 114:27-38.J. Biol. Chem. 

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