GERMICIDAL PROPERTIES OF NITROGEN BASES FROM TRANSFORMER-OIL EXTRACT W. NELSON AXE, DOUGLAS D. HENSON, AND V. T. SCHUHARDT The University of Texas, Austin, Texas
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From time to time a mild interest has been exhibited in the possibility of antibacterial action among the petroleum bases of unknown structure such as those obtained from transformer-oil extract. As a first step toward the possible utilization of these bases in pharmacology, a survey of various roughly differentiated aromatic and nonaromatic fractions revealed F. D. A. phenol coefficients as high as 17. Dilutions of these water-insoluble bases were effected with 0.1 N hydrochloric acid. However, under the conditions of the tests the bases were precipitated by the buffer action of the peptone broth as a fine emulsion. On the other hand, emulsions of the bases in soap solution were without germicidal activity. Phenol coefficients of a selected aromatic and nonaromatic fraction showed a considerable reduction when determined against Staph. aureus, but only a slight drop was noted against E. f y p h i in the presence of horse serum at 37" C. A pronounced inhibitory action against strains of E. typhi and Staph. aureus was also observed.
inaugurated. After a preliminary examination of the bactericidal action of various base fractions, the following determinations were made on a promising aromatic and on a nonaromatic portion having approximate average boiling points of 340" C.: (a) accurate phenol coefficients a t 20' and 37" C . against strains of Eberthella &phi and Staphylococcus aureus, (b) the effect of horse serum on their germicidal action, ( c ) mycostatic action against these bacteria, (d) the effect of a small amount of the bases on agar plates heavily seeded with bacteria, (e) the action of the total acidity of the hydrochloride solutions on bacteria.
Source of Crude Bases I n refining transformer-oil stock with liquid sulfur dioxide, this solvent withdraws the small amount of contained nitrogen bases, and after its recovery the residual volume of hydrocarbon oil constitutes what is known as an Edeleanu extract. At their Oleum refinery the Union Oil Company processed 500 barrels of this extract as follows: I n the usual way the bases were extracted with 15 per cent sulfuric acid, liberated with sodium hydroxide, and finally divided into three fractions (I, 11, 111) by vacuum distillation from a laboratory shell still. The yield of bases was 3.75 barrels. (Acknowledgment is made for the generous supply of 30 gallons of these bases.) TABLEI. PHYSICAL CONSTANTS OF TRANSFORMER-OIL BASES AS RECEIVED FROM THE REFINERY Fraction
I
I1
I11
n 1.5.131 1.5695 1.5864
dlb.6
0.984 1.005 1.039
Test Organisms
B
Stock laboratory cultures of E. @phi (Rawlings strain) and Staph. aureus (a laboratory strain) were used in all tests. Although the Rawlings strain is not the standard recommended in the Food and Drug Administration (F. D. A.) method (11) for the determination of phenol coefficients, the remarkable constancy observed by other investigators (7), and verified in this work, justifies its use. The resistance of the E. typhhi culture employed fell within the prescribedlimits in every instance over a period of 6 months. The minimal resistance usually prescribed for Staph. aureus is survival of the bacteria for 5 minutes in a 1 to 60 dilution of phenol, whereas the particular culture used in these tests showed a slightly weaker but constant resistance. Growth after contact for 5 minutes in a 1to 70 dilution of phenol was observed in each control run. At 37" C . a resistance similar to the conditions stated above appeared with phenol dilutions of 1 t o 100, whereas the F. D. A. standard resistance limit should be found with a dilution of 1to 80.
ASES from petroleum distillates are of unusual phar-
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macological interest because, unlike volatile bases from other natural sources, they contain in decidedly preponderant amount nonaromatic types of unknown structure. For many of these products a cyclopentane, or so-called naphthenic structure, is highly probable. If these products were of plant occurrence, they would be classed as alkaloids; however, they differ from the most important plant alkaloids in being volatile and oxygen-free. During the exhaustive investigation of petroleum bases in t h e Texas Laboratory ( I , 2, 3, 6,6, 8, 9, IO), their apparent low toxicity has suggested the possible discovery of products of therapeutic value; therefore, in order to determine the particular fractions and types of these bases of greatest promise for a pharmacological study, a determination of their germicidal action appeared desirable. Since experiment has indicated that the higher boiling bases contain the most potent bactericidal material, a study of transformer-oil bases was 503
INDUSTRIAL A N D E N G I N E E R I N G CHEMISTRY
504
Nature of Germicidal Solutions Since transformer-oil bases are insoluble in water, a readily soluble salt was selected for this work. Experiment showed that the bases emulsified in soap solution possessed no germicidal activity. On the other hand, solutions in dilute hydrochloric acid exhibited considerable bactericidal efficiency. However, under the experimental conditions employed, the peptone in the culture medium, by virtue of its buffer action, precipitates the petroleum bases in a high state of dispersion; accordingly, it shouid be emphasized that the measurements reported here apply to free bases liberated in situ as a fine emulsion. The stock solutions of the bases were made up with standard 0.1 N acid; hence the acidity of any particular dilution was known quite accurately. Although the wide variation in phenol coefficients of a series of fractions with the same acid content apparently precluded the possibility that the acid was the active germicidal agent, this conclusion was confirmed by control tests with the bases omitted.
Processing of Crude Bases and Determination of Phenol Coefficients Fraction I (Table I) in 6 N hydrochloric acid was carried through cumulative extraction (8) with chloroform. In this way the aromatic hydrochlorides were segregated in the aqueous layer and the nonaromatic hydrochlorides in the chloroform layer. The liberated aromatic bases (6.5 per cent of the total) had a refractive index (n”) of 1.6020; for the nonaromatics this value was 1.5364. The aromatic bases were separated into twenty-seven fractions by distillation through a reflux column under diminished pressure. Stock solutions of, (a) the crude bases, (b) the unfractionated nonaromatics, and (c) aromatic fractions 2 and 27 were prepared by dissolving 1-gram samples in just the requisite amount of 0.1 N hydrochloric acid. The results obtained from these preparations are compiled in Table 11. COEFFICIENTSOF TRANSFORMER-OIL BASES TABLE 11. PHENOL FROM FRACTION I B. P.
Fraction
c. ..... .....
n??
Phenol Coefficient (F. D. A . )
I (orude bases) 1.5431 5.0 I N A (crude nonaromatic) 1.5364 2.0 276-279 1.5614 2.0 IAZ (aromatic) IAZ?(aromatic) 340 (decomposed) 1.6604 7.0° This value is assumed t o be the highest for t h e aromatio portion of fraction I inasmuch as a run using identical dilutions of fratotion IAls (b. p., 306309O C.; ass, 1.5842) showed no germicidal activity. -
COEFFICIENTSOF MIXED TYPEBASES TABLE 111. PHENOL FROM FRACTIONS I1 AND I11 Fraction 11, 11; 111, 1112
Phenol Coefficient (F. D. A.) 6.4 8.0 9.0
10.0
In order to determine whether any exaltation in germicidal power occurs in the higher boiling bases, the original fractions I1 and I11 (Table I) were distilled under reduced pressure from a Claisen flask, the distillate from each sample being collected in two equal portions. Stock hydrochloride solutions were prepared as before and used in arriving a t the phenol coefficients given in Table 111. The phenol coefficients of the preceding roughly fractionated bases indicated that a more thorough examination of fraction I11 would be profitable. Consequently a portion of bases from this latter source was carried through cumulative
VOI.. 29, NO. 5
extraction with the conditions the same as those observed in processing fraction I. The regained aromatics representing 12 per cent of the total were resolved into five fractions by distillation a t 12 mm. pressure from a Claisen flask, and the usual hydrochloride solutions were prepared (Table IV). TABLE Iv.
DATA O F AROMATICTRANSFORMEROIL BASESFROM FRACTION I11
DISTILL-4TION
Fraction
Distilling Temp. (23 Mm.)
n L5
232-236 236-238 238-240 240-247 247-265
1.6470 1.6600 1.6620 1.6630 1.6666
c.
IIIAl IIIA2 IIIAa 1x1~4 IIIAL
Since dilutions of these fractions showed no exaltation in germicidal power over the better define’d aromatic cuts of fraction I, their exact coefficients were not determined. From 100 cc. of regained nonaromatic bases (111) distilled under 23 mm. pressure, 80 cc. were collected in 10-cc. portions, and the phenol coefficients of fractions 1, 4, 5, 6, and 8 in dilute hydrochloric acid were determined. ~~~~
TABLEV. DISTILLATION DATAAND PHENOL COEFFICIENTSOF NONAROMATIC TRANSFORMER-OIL BASES FROM FRACTION I11 Distilling Temp. Fraction (23 Mm.)
c.
1 1 1 1
1 1 ~ ~210-220 1 1 1 ~ ~ 220-224 2 1 1 ~ ~ 224-227 8 1 1 ~ ~ 227-232 4
ny
Phenol CoeffiDistilling cient Temp. (F. D. A.) Fraction (23 Mm.)
1.5564 1.5622 1.5662 1.5682
c.
13 0
. .. ,
15.0
1 1 1 ~ ~ 232-237 6 1 1 1 ~ ~ 237-240 6 I I I N A ~ 240-249 1 1 1 ~ ~ 249-270 8
Phenol Coefficient n”D”(F. D. A.) 1.5740 1.5786 1.5826 1.5910
17.0 14.0
..
13.0
Since the data presented in Tables I1 and V were derived from bases of unknown composition, two petroleum bases of known structure, 2, 3, 8-trimethylquinoline (S),and a nonaromatic base of the formula ClaH26N (IO), were tested to provide a basis of comparison : Base 2 3 8-Trimethylquinoline
Phenol Coefficient (F. D. A.) 1.0 1.8
d ~ k dbase l The preceding tables show that the transformer-oil bases are structurally different from the two control bases studied, and this view is confirmed by an investigation now in progress in the Texas Laboratory. The phenol coefficients in Table V suggest that certain individual nonaromatic components in the higher boiling ranges possess exceptional germicidal properties. However, it is worthy of note that fraction 5 has the maximum coefficient, whereas, the first and last cuts show identical values.
Phenol Coefficients of Fractions Inn and 1 1 1 ~ ~ s Stock 1 : l O O dilutions of fractions I A 2 1 (Table 11) and 111~~5 (Table V) were made as follows: One gram of bases was dissolved by the dropwise addition of standard 0.1 N hydrochloric acid. The acid required for Ian7 and IIINAS solutions was 62.46 and 65.82 cc., respectively. After removal of tarry matter and hydrocarbon oil, the solutions were diluted to 100 cc. with sterile distilled water. Although this procedure does not reveal the exact amount of bases in solution, reproducible results were possible. Furthermore, since all of the subsequent data are based on the original one gram of material, the inherent error favors the resistant action of the bacteria rather than the germicidal effect of the bases. The pH values of the stock solutions,
MAY, 1937
INDUSTRIAL AND E N G I N E E R I N G CHEMISTRY
as estimated colorimetrically, were 3.8 and 4 for I A 2 j and IIINAs, respectively. Phenol coefficients of the two fractions under consideration were redetermined with care, using E . typhi as the test organism a t 20’ C., and were found to check the values previously listed. In these tests dilutions were prepared in volumetric flasks rather than in the medication tubes: Conditions
a
Aqueous dilutions Aqueous dilutione Aqueous dilutions Aqueous dilutions 20% horse serum G r o n t h observed in
Temp.
c.
Test Organism
Phenol Coefficient
llrnAr
20 E . typha 7 37 E. typhz 7 20 Staph. aureus” 37 Staph. aureus 0 37 E. typhi 6 tubes containing 1: 25 dilution
0 5
27 9
17.0 11.9 4 3 44
8 8 of the base.
Inhibition Tests Employing Solid Media The mycostatic tests described were carried out according to standard procedure (4). Preliminary tests were made emulsified in agar medium. The using the free 1.427 and IIINA~ tubes, cooled in slants, were streaked with a 4-mm. loop holding a broth culture. Under these conditions growth of E. typhi and Staph. aureus was not inhibited. The inhibiting action of base emulsions formed in situ from the hydrochloride solutions was also studied. One cubic centimeter of various dilutions was added to sterile test tubes with subsequent addition of 5 cc. of beef extract agar. The content of the tubes was thoroughy mixed and then allowed to harden in slants. Phenol dilutions were made up in an analogous manner, thus affording a basis of comparison. Blank tubes containing only agar medium served as controls. The slants were streaked with a 4-mm. loop as uniformly as possible. The results in Table VI refer to a 48-hour incubation period. TABLEVI. Stock Dilution
INHIBITION TESTSUSINGE. typhi AND Staph. aureu~ Final Dilution
E. ?uphi
Slaph. aureu8
1:600 1:7200 1:7800 1 :8400 1 :9000 1 : 10,800 1:12,000
Complete inhibition Normal growth Normal nrowth Normal growth Normal growth Normal growth Normal growth
Complete inhibition Complete inhibition Comnlete inhibition Complete inhibition Slight growth Slight growth Slight growth
1 :360 1:420 1 :480 1 :540 1:600 1:660
Complete inhibition Complete inhibition Complete inhibition Slight growth Slight growth Normal growth
Slight growth Slight growth Slight growth Normal growth Normal growth Normal growth
IAZ?:
1:lOO 1:300 1:400 1:500 11rNA6: 1:lOO 1:1200 1:1300 1 :1400 1:1500 1 :1800 1:2000 Phenol: 1:60 1:70 1:80 1:90 1:100 1:llO
1:600 1:800 1:2400 1:3000
505
filter paper 6 mm. in diameter. Again 1 ~ 2 was 7 without effect showed a clear zone in dilution of 1to 100. However, IIINAL in 1 to 100 dilution and a faint clear zone a t 1 to 1400 dilution.
Discussion of Results Under the conditions of the tests carried out in this general survey, the nitrogen bases compare favorably with phenol. A resume of the inhibition experiments brings out the fact that growth of E. typhi of standard virulence is completely inhibited by I A 2 j and IIINA~ in dilutions of 1 to 600, as com~ ineffective 7 pared with 1 to 480 for phenol. Although 1 ~ was against Staph. aureu8 in the usual germicide tests, it inhibits the growth of the organism in dilution of 1to 600. Likewise, 1 1 1 ~ is~ highly 5 efficient in this respect, inhibiting completely the growth of Staph. aureus in a dilution of 1 to 8400. The exact nature of the action of the solutions against bacteria was not investigated. Experiments with ordinary emulsions of the free bases showed no appreciable germicidal action. Also it may be pointed out that emulsions of free bases in agar medium possess no inhibiting action against the bacteria used. However, in all of the phenol coefficient determinations a very pronounced turbidity, due to liberation of free bases, takes place upon addition of the broth culture. The buffer action of the peptone evidently takes up the hydrochloric acid, liberating the bases in a finely dispersed state. With respect to the inhibition tests, a similar action occurs with the broth-agar medium. The molten agar becomes perfectly opaque because of the liberated bases, yet inhibitory action is quite pronounced. Thus it would seem that an extremely fine emulsion is germicidal, whereas the macrodroplets produced by agitating the bases with soap solution are without effect.
Literature Cited (1) Armendt and Bailey, J. Am. Chem. Soc., 55, 4145 (1933). (2) Biggs and Bailey, Ibid., 55, 4141 (1933). ( 3 ) King and Bailey, Ibid., 52, 1245 (1930). (4) Kolmer and Boerner, “Approved Laboratory Technic,” p. 427, New York, Appleton-Century Co., 1931. (5) Lackey and Bailey, J. Am. Chem. Soc., 56, 2741 (1934). (6) Lake and Bailey, Ibid., 55, 4143 (1933). ( 7 ) Patterson and Frederick, AnaEyst, 56, 93 (1931). ( 8 ) Perrin and Bailey, J. Am. Chem. SOC.,55, 4136 (1933). (9) Poth, Schulze, King, Thompson, Slagle, Floyd, and Bailey, Ibid., 52, 1239 (1930). (10) Thompson and Bailey, Ibid.. 53, 1002 (1931). (11) Zinsser and Bayne-Jones, “Textbook of Bacteriology,” p. 1144, New York, Appleton-Century Co., 1934. RECEIVEDDecember 21, 1936.
Correction Tests on Heavily Seeded Agar Plates The usual beef extract peptone agar was employed. Each plate contained 0.2 cc. of a 24-hour broth culture and 15 cc. of the agar medium. The base dilutions were applied by means of a single streak of a 2-mm. loop, and the readings were taken after a 48-hour incubation period. The plate containing E. typhi streaked with 1 . 2 7 showed only a faint clear zone, and the Staph. aureus plate streaked with the same dilution remained unchanged; in contrast, the 1 to 100 dilution of I I I N A 6 gave well-defined clear zones in both the E. typhi and Staph. aureus plates. Phenol dilutions of 1 to 90, 1 to 100, and 1 to 110 were applied in like manner without any observable effect on the bacterial growth. In an additional test with Staph. aureus the germicides were applied to the heavily seeded plates through a saturated circular
In the February, 1937, number of INDUSTRIAL AND ENGINEERING CHEMISTRY a drawing of the famous Count Rumford, by James Gillray, appeared as No. 74 in the Berolzheimer Series of Alchemical and Historical Reproductions. To the notes on page 166 accompanying this picture of Count Rumford and his stove, the following correction should be made: That phase of the versatile count’s scientific and technical work which has greatest significance is his study of the nature of heat; his classic determination of the amount of heat produced in boring cannon demolished at one stroke the theory of the materiality of heat and, aved the way for the first law of thermodynamics. The famiEar Rumford phosphate baking powder is so called because the inventor, Eben Horsford, was a Rumford Professor at Harvard University, t o which the count bequeathed the bulk of his estate. C. D. LOWRY, JR. UNIVERSAL OIL PRODUCTS COMPANY CHIOAQO,ILL. March 5, 1937
