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Clinical Evaluation of Quantitative Differences in Ultraviolet Absorption of Compounds Containing the Substituted Benzoic Acid Nucleus
CLINICAL EVALUATION OF QUANTITATIVE DIFFERENCES IN ULTRAVIOLET ABSORPTION OF COMPOUNDS CONTAINING THE SUBSTITUTED BENZOIC ACID NUCLEUS* LEONARD C. HARBER, M.D.
The property of resonating compounds to absorb solar light has been hivestigated in the field of physics for at least fifty years. The relationship of the protection of skin surfaces from ultraviolet light rests upon the formula
E1—E0=hV where the energy of the compound is initially Eo; energy in the excited state is E1; and V is the frequency of light absorbed. h is Plank's constant. (Note: Frequency is inversely related to wavelength (A°).) Compounds which possess the physical property of absorbing energy in the range 2900A°—3170A° have previously been investigated for their advantages in preventing sunburn. They may also offer a prophylactic weapon in such diseases as lupus erythematosus and solar urticaria. Kesten and Slatkin have most recently offered a detailed classification and history of diseases related to sunlight and light sensitivity. The benzoic acid nucleus has been shown to have ultraviolet absorption qualities, and various attempts have been made to ascertain and evaluate the efficacy of substituted benzene ring compounds both in. vitro (flame spectrometer), and in. vivo (commercial sunscreen agents).
This paper describes attempts to clarify, by somewhat modified technics of quantitative comparison, the protection from light offered by various compounds
having the substituted benzoic acid nucleus. It is hoped that the list may be expanded at a later date, and that more studies of a quantitative nature may elucidate the presence of other compounds equally, if not more, effective than those mentioned here.
The purpose of the following investigation was to evaluate the relative sunscreening properties of five compounds containing the benzoic acid nucleus with various substituted side chains. These compounds are given in table on following page. A conscious and careful effort was made to establish quantitative differences in the sunscreening properties of the compounds themselves; consequently, in all of the following experiments a constant universal solvent was used to keep the
purified materials in solution. This solvent was 95 % ethyl alcohol, which in preliminary trials, had been found to have no significant ultraviolet absorption. * From the Department of Dermatology and Syphilology of the New York University Post-Graduate Medical School (Dr. Marion B. Sulzberger, Chairman) and the Skin and Cancer Unit of the New York University Hospital. Received for publication June 25, 1954. 427
428
ThE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Compound
1. para-aminobenzoic acid
Formula Structure
MocuIar
137.2
—'-00011
NH2C6H4000H
—NH2
2. menthyl anthralinate
151.16
NH2C6H4COOCH0
3. phenyl salicylate
214.12
C6ILOC5H4000H
C C'r00011 4. glyceryl para-aniinobenzoate
5. tannie acid
196.0
010111204
1701
C76He045
C—OR
/_oR
/ C—OR 01 C—OR.
\::R In all procedures efforts were also made to keep constant the time of testing as well as the method of evaluating results. PROCEDURE
The fifty volunteers used in this experiment, who were chosen completely at random, included 32 females, ages 14—65, and 18 males, ages 18—68. The sole pre-
requisite was that they had no skin lesion on their back (test site used). Analysis of results revealed no significant difference in data that could be correlated with age or sex.
429
ULTRAVIOLET ABSORPTION OP COMPOUNDS
The volunteers were exposed to either ultraviolet rays from an artificial source or to direct sunlight. Throughout each experiment, a sheet of tinfoil was applied to protect the back of the subject from irradiation, except where six circular apertures, (2.9 cm. in diam.) had been cut in the tinfoil in order to permit the radia-
tion to reach the skin (Figure 1). In each subject, compounds were placed at random in the apertures, thus insuring no undue repetition of a particular site for any given compound.
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®
®
®
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®
Fxa. 1
In experiment 1, a cylindrical quartz cup was used as a container for the test materials. In experiments 2 and ft 3, the compounds to be tested were placed directly on the skin; two drops, approximately 0.4 cc., being gently spread
over each test site. Volunteers who were irradiated with ultraviolet light from the artificial source were placed under the Hanovia D.C. lamp at a distance of 30 inches and were exposed for sixty seconds. (On the particular machine used, this approximated 13/i times the empirical minimal erythema dose.) The volunteers who were exposed to natural sunlight were placed in the open courtyard of the New York Skin and Cancer Unit during the month of June 1953, for a period of 2 hours. The time of day being that of maximum sunlight, between 10:00—1:00 D.S.T. The subjects' responses to the above exposures were evaluated at 6 and at 24 hours later, or both. The degree of erythema was evaluated by a single constant observer1 who had no knowledge of which compound had been placed in each
test site. The same method of grading erythema was used in all experiments (Fig. 2). Score (arbitrary figure Symbol
InterpretaSio.
indicating netue)
0
No erythema Questionable erythema Minimal erythema Moderate erythema Considerable erythema Maximal erythema
+1—
+ ++
---+
1 2 4
FIG. 2. Key
In experiments *1 and * 2, groups of at least three persons were exposed to ultra violet light. The time of exposure was 60 seconds, and the results were evaluated 6 hours later. 1 Without the aid and generous patience of Miss Sylvia Buchanan, these experiments would not have been possible.
430
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
In the data of experiment 3, where the subjects were exposed to natural sunlight, groups of at least three persons were tested simultaneously. The time of exposure was 2 hours, and the results were evaluated 24 hours later.
As illustrated in Figure 1, each volunteer was used for an evaluation of five compounds compared to each other and to a control. Each reading represented a comparison of equimolar or equipercentage solutions. DATA
Note
In order to facilitate reading of the following data, certain arbitrary abbreviations have been employed. They are: a) Initials have been substituted for the names of the volunteers; b) Compounds tested have been designated by the following abbreviations: para-aminobeuzoic acid menthyl anthralinate phenyl salicylate glyceryl para-aminobenzoate tannic acid control (95% ethyl alcohol)
PABA Menth. Ant. Phen. Sal. Glyc. PAB Tan. Ac. Contr. (eth. ale.).
c) For interpretation of symbols used in grading erythema see Key (Figure 2). RESULTS
Quartz Cup tests for protection against U.V. lamp In experiment 1, the compounds were not in contact with the skin surface.
and a D.C. Hanovia Lamp was used as the source of ultraviolet irradiation, EXPERIMENT %1
Part a CuP
U.V.-E.D. 1 3.10—2 M sol.
3 ml. Solution
Volunteer
S.R.
B.M. C.D.
D.F. R.P. Totals
PABA
MenUs, ant.
Plien. sal.
Glyc. PAB
Tan. ac.
Contr. (eth. aic.)
0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
++++ ++++ +++ ++++ ++++
0
0
0
0
0
19
431
ULTRAVIOLET ABSORPTION OF COMPOUNDS
EXPERIMENT Part S CuP TJ.V.-E.D. =
'A 1
3.l0 M sol. 3 ml. Solution Volunteer
H. R.
L.T.
B. G.
F.D. R.K. B.S.
J.T.
H. L.
M.N. N. D. Totals
PA3A
Menth. ant.
Phen. sal.
Glyc. PAB
+1++ + ++ ++ + +
++ ++ ++ +++ + +++ ++ +++ ++ ++
+++ ++ +++ +++ +++ +++ ++ +++ +++ +++
++ + ++ ++ ++ ++ + ++ + ++
9.5
22
28
17
Tan. ac.
Contr. (eth. aic.)
+ 0 +10 +1-
++++ +++ ++++ ++++ +++ ++++ +++ +++ +++ ++++
2.5
35
+1-
EXPERIMENT f 1 Part c CuP U.V.-E.D. = M sol. 3 ml.
3.10—s
Solution Volunteer
B.R. J. S. T. K.
F.T. Totals
Tan. ac.
PABA
Menth. ant.
Phen. sal.
Glyc. PAB
+ +1++ +
+++ +++ +++ +++
++++ +++ ++++ ++++
+++ ++ +++ ++
0
+10
++++ +++
4.5
12
15
10
1.5
15
+
++++ ++++
Equi.molar solutions of the following concentrations were employed as the testing
standards.
3 X 102M (3 ml.), 3 X lOM (3 ml.), 3 X 105M (3 ml.), 3 X 1OM (2 ml.) 1) At 3 X 102M, all of the sunscreening agents were effective in preventing erythema, and no significant difference in effectiveness of erythema protection could be ascertained (see part a).
432
ThE JOURNAL OF INVESTIGATIVE DERMA.TOLOGY
EXPERIMENT f, 1
Part d Cup
U.V.-E.D. 1 3.1O M sol. 2 ml. Solution Volunteer
MBA
Menth. ant.
Phen. sal.
Giyc. PAB
Tan. ac.
Contr.
(eth sic.)
++++ ++++ ++++ ++++ ++++ ++++
L.H.
4
Totals
'
4
4
2) At 3 X 10—4M, tannic acid was excellent and para-aminobenzoic acid was also superior to the other agents. The remaining compounds listed in decreasing order with respect to preventing erythema were glyceryl para-aminobenzoate, menthyl anthralinate, and phenyl salicylate. It is important to note that per unit molecule, tannic acid has 10 moles of benzene nuclei per one of para-aininobenzoic acid (see part b).
3) At 3 X 10M, the compounds had only limited sunscreening ability, and with the exception of para-aminobenzoic acid and tannic acid, the degree of erythema produced through the remaining compounds closely paralleled that of the control (see part c). EXPERIMENT f2 Skin U.V.-E.D. 5% so). 2 drops Solution
Volunteer
Menth. ant.
Phen. sat.
Otyc. PAB
Tan SC.
(eth.alc.)
+++ ++ +++ ++ +++ +++ ++ ++ +++ ++
+
++ + +++ ++ + ++ + ++
+1-
++++ +++ ++++ +++ ++++ +++ +++ +++
+
+++ ++ ++ ++ +++ +++ ++ +++ +++ ++
4.5
25
25
16
PABA
A. K.
R.W.
I.w.
M. E.
J.D.
M.G. S. B.
B.H. L.D. R.H. Totals
+1-
+ +1+
+1-
+1-
+1-
+++ +++
+
1.5
33
433
ULTRAVIOLET ABSORPTION OF COMPOUNDS
EXPERIMENT J(3 Skin—2 Hrs. 5% aol. Solution
- ________ ____________
Volunteer
A.R. B.L. H.B. J. P.
B. R.
s.v.
PADA
Menth. ant.
Phen. sal.
Glyc. PAD
Tan. ac.
(eth.alc.)
++ +1++ +1-
+++ ++ +++ ++++
+++ + ++ +++
+++ + ++ +++
+
+/-
++++ +++ ++++
+ ++ +1-
+1- ++++
+
++ ++
6
24
15
J.s.
R. R.
Totals
+
++
++ +++ +++
R.L.
D.K.
+++
++
+1—
15
+
+++ ++++ ++ ++ +1— +++
4
29.5
+
4) Using only 2 ml. of solution, in contrast to 3 ml., as in part c, none of the compounds exhibited any erythema protection when the concentration was 3 X 1OM (see part d). Direct skin application and protection against U.V. lamp
The second aspect of this investigation was to evaluate the compounds in direct contact with the skin surface and when they were not kept from contact by a cup. Therefore, equal amounts of a 5% solution of each compound placed on the skin were evaluated. The source of irradiation again was the Hanovia D.C.
lamp asusedinExp. f1. 1) In experiment 2,5% concentrations of tannic and para-aminobenzoic acid were excellent when compared to the control.
2) Glyceryl para-aininobenzoate was good, whereas phenyl salicylate and menthyl anthralinate showed only minimal differences from the control. Direct skin application and protection against natural sunlight
In experiment * 3, natural conditions were utilized, i.e. subjects were tested by being exposed to natural sunlight, and equipercentage, (5%), solutions were applied directly to the skin. 1) Tannic acid and para-aminobenzoic acid were excellent in preventing erythema. 2) Glyceryl para-aminobenzoate and phenyl salicylate had fair sunscreening activity. 3) Menthyl anthralinate was poor. For convenience in analyzing data a tabular summary of the three previously described experiments is presented here.
434
Cup 3.104 M
9.5
22
28
17
' 2.5
35
Ranked in order of protective
II
IV
V
III
I
VI
U.V.-5% Solution
4.5
25
25
16
1.5
33
Ranked in order of protective
II
V
IV
III
I
VI
Sun-5% Solution
6
24
15
15
4
29.5
Ranked in order of protective
II
V
IV
III
I
VI
ThE JOURNAL OF INVESTIGATIVE DERMATOLOGY Solution Phen. SaL
PABA
iic.)
capacity
capacity
capacity CONCLUSIONS
Experiments designed to evaluate the sunscreening properties of a group of five compounds containing the substituted benzoic acid nucleus were performed.
The compounds were tested for their capacity to prevent erythema caused by exposure to the Hanovia D.C. ultraviolet lamp and to natural sunlight. The findings indicate a relationship between their chemical properties and biologic effects as postulated in Blum's monograph on light sensitivity (see p. 177). Although all compounds tested exhibited erythema-preventing properties, the degree of their effectiveness fell into two groups.
Group I: Tannic acid and para-aminobenzoic acid Both tarmic acid and para-aminobenzoic acid were superior to all other agents
tested. The superiority of tannic acid above all agents tested was particularly evident when comparisons with other compounds were on an equimolar basis. Para-aminobenzoic acid was almost as effective as tannic acid in preventing erythema when tested in equipercentage solutions. Group II: Glyceryt para-aminobenzoate, phenyt saticyt ate, and menthyt ant hratinate
Under rigid statistical analysis, no significant differences could be established in the sunscreening properties of phenyl salicylate, menthyl anthralinate,
or glyceryl para-aminobenzoate. It is the author's belief that further studies may demonstrate that menthyl anthralinate is the poorest erythema-protecting agent of all compounds tested in this study. REFERENCES 1. BLUM, H. F.: Photodynamic action and diseases caused by light. Amer. Chem. Soc. Mono. Series 85, 1941. 2. FRIEDL AND OBCHIN: Ultraviolet Spectra of Aromatic Compounds, pp. 1—36. New York,
J. Wiley and Sons, Inc., 1945.
ULTRAVIOLET ABSORPTION OF COMPOUNDS
435
3. GIESE, A. C. AND WELLs, J. M.: Sunburn protection, natural and artificial. Scient.
Monthly, 62: 458, 1946. 4. KESTEN, B. M. AND SLATEIN, M.: Diseases related to light sensitivity. Arch. Dermat. & Syph., 67: 3, 1953. 5. KLINE, P. AND BABE, R. L.: Pyribenzamine inhibition of sunburn. J. Invest. Dermat., 10: 397, 1948. 6. LUCKIESH, TAYLOR, COLE AND SOLLMAN: Protective skin coatings for prevention of
sunburn. J.A.M.A., 130: 1, 1946. 7. ROTHMAN, S. AND HENNINGSEN, A. B.: The sunburn protecting effect of PABA. J. Invest. Dermat., 9: 307, 1947. 8. ROTHMAN, S. AND RUBIN, J.: Sunburn and PABA. J. Invest. Dermat., 6: 445, 1942. 9. SHARLIT, H.: Salol investigation. Arch. Dermat. & Syph., 32: 290, 1935. 10. SULZBEBGER, M. B. AND WoLF, J.: Section II on Sunburn, Dermatology; Essentials of Diagnosis and Treatment, p. 481. Chicago, Year Book, Inc., 1952. 11. SUTTON, R. L. AND Surrow, R. L., Jn.: Handbook of Diseases of the Skin, p. 73. St. Louis, C. V. Mosby, 1949.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.
The property of resonating compounds to absorb solar light has been hivestigated in the field of physics for at least fifty years. The relationship of the protection of skin surfaces from ultraviolet light rests upon the formula
E1—E0=hV where the energy of the compound is initially Eo; energy in the excited state is E1; and V is the frequency of light absorbed. h is Plank's constant. (Note: Frequency is inversely related to wavelength (A°).) Compounds which possess the physical property of absorbing energy in the range 2900A°—3170A° have previously been investigated for their advantages in preventing sunburn. They may also offer a prophylactic weapon in such diseases as lupus erythematosus and solar urticaria. Kesten and Slatkin have most recently offered a detailed classification and history of diseases related to sunlight and light sensitivity. The benzoic acid nucleus has been shown to have ultraviolet absorption qualities, and various attempts have been made to ascertain and evaluate the efficacy of substituted benzene ring compounds both in. vitro (flame spectrometer), and in. vivo (commercial sunscreen agents).
This paper describes attempts to clarify, by somewhat modified technics of quantitative comparison, the protection from light offered by various compounds
having the substituted benzoic acid nucleus. It is hoped that the list may be expanded at a later date, and that more studies of a quantitative nature may elucidate the presence of other compounds equally, if not more, effective than those mentioned here.
The purpose of the following investigation was to evaluate the relative sunscreening properties of five compounds containing the benzoic acid nucleus with various substituted side chains. These compounds are given in table on following page. A conscious and careful effort was made to establish quantitative differences in the sunscreening properties of the compounds themselves; consequently, in all of the following experiments a constant universal solvent was used to keep the
purified materials in solution. This solvent was 95 % ethyl alcohol, which in preliminary trials, had been found to have no significant ultraviolet absorption. * From the Department of Dermatology and Syphilology of the New York University Post-Graduate Medical School (Dr. Marion B. Sulzberger, Chairman) and the Skin and Cancer Unit of the New York University Hospital. Received for publication June 25, 1954. 427
428
ThE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Compound
1. para-aminobenzoic acid
Formula Structure
MocuIar
137.2
—'-00011
NH2C6H4000H
—NH2
2. menthyl anthralinate
151.16
NH2C6H4COOCH0
3. phenyl salicylate
214.12
C6ILOC5H4000H
C C'r00011 4. glyceryl para-aniinobenzoate
5. tannie acid
196.0
010111204
1701
C76He045
C—OR
/_oR
/ C—OR 01 C—OR.
\::R In all procedures efforts were also made to keep constant the time of testing as well as the method of evaluating results. PROCEDURE
The fifty volunteers used in this experiment, who were chosen completely at random, included 32 females, ages 14—65, and 18 males, ages 18—68. The sole pre-
requisite was that they had no skin lesion on their back (test site used). Analysis of results revealed no significant difference in data that could be correlated with age or sex.
429
ULTRAVIOLET ABSORPTION OP COMPOUNDS
The volunteers were exposed to either ultraviolet rays from an artificial source or to direct sunlight. Throughout each experiment, a sheet of tinfoil was applied to protect the back of the subject from irradiation, except where six circular apertures, (2.9 cm. in diam.) had been cut in the tinfoil in order to permit the radia-
tion to reach the skin (Figure 1). In each subject, compounds were placed at random in the apertures, thus insuring no undue repetition of a particular site for any given compound.
®
®
®
®
®
®
Fxa. 1
In experiment 1, a cylindrical quartz cup was used as a container for the test materials. In experiments 2 and ft 3, the compounds to be tested were placed directly on the skin; two drops, approximately 0.4 cc., being gently spread
over each test site. Volunteers who were irradiated with ultraviolet light from the artificial source were placed under the Hanovia D.C. lamp at a distance of 30 inches and were exposed for sixty seconds. (On the particular machine used, this approximated 13/i times the empirical minimal erythema dose.) The volunteers who were exposed to natural sunlight were placed in the open courtyard of the New York Skin and Cancer Unit during the month of June 1953, for a period of 2 hours. The time of day being that of maximum sunlight, between 10:00—1:00 D.S.T. The subjects' responses to the above exposures were evaluated at 6 and at 24 hours later, or both. The degree of erythema was evaluated by a single constant observer1 who had no knowledge of which compound had been placed in each
test site. The same method of grading erythema was used in all experiments (Fig. 2). Score (arbitrary figure Symbol
InterpretaSio.
indicating netue)
0
No erythema Questionable erythema Minimal erythema Moderate erythema Considerable erythema Maximal erythema
+1—
+ ++
---+
1 2 4
FIG. 2. Key
In experiments *1 and * 2, groups of at least three persons were exposed to ultra violet light. The time of exposure was 60 seconds, and the results were evaluated 6 hours later. 1 Without the aid and generous patience of Miss Sylvia Buchanan, these experiments would not have been possible.
430
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
In the data of experiment 3, where the subjects were exposed to natural sunlight, groups of at least three persons were tested simultaneously. The time of exposure was 2 hours, and the results were evaluated 24 hours later.
As illustrated in Figure 1, each volunteer was used for an evaluation of five compounds compared to each other and to a control. Each reading represented a comparison of equimolar or equipercentage solutions. DATA
Note
In order to facilitate reading of the following data, certain arbitrary abbreviations have been employed. They are: a) Initials have been substituted for the names of the volunteers; b) Compounds tested have been designated by the following abbreviations: para-aminobeuzoic acid menthyl anthralinate phenyl salicylate glyceryl para-aminobenzoate tannic acid control (95% ethyl alcohol)
PABA Menth. Ant. Phen. Sal. Glyc. PAB Tan. Ac. Contr. (eth. ale.).
c) For interpretation of symbols used in grading erythema see Key (Figure 2). RESULTS
Quartz Cup tests for protection against U.V. lamp In experiment 1, the compounds were not in contact with the skin surface.
and a D.C. Hanovia Lamp was used as the source of ultraviolet irradiation, EXPERIMENT %1
Part a CuP
U.V.-E.D. 1 3.10—2 M sol.
3 ml. Solution
Volunteer
S.R.
B.M. C.D.
D.F. R.P. Totals
PABA
MenUs, ant.
Plien. sal.
Glyc. PAB
Tan. ac.
Contr. (eth. aic.)
0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
++++ ++++ +++ ++++ ++++
0
0
0
0
0
19
431
ULTRAVIOLET ABSORPTION OF COMPOUNDS
EXPERIMENT Part S CuP TJ.V.-E.D. =
'A 1
3.l0 M sol. 3 ml. Solution Volunteer
H. R.
L.T.
B. G.
F.D. R.K. B.S.
J.T.
H. L.
M.N. N. D. Totals
PA3A
Menth. ant.
Phen. sal.
Glyc. PAB
+1++ + ++ ++ + +
++ ++ ++ +++ + +++ ++ +++ ++ ++
+++ ++ +++ +++ +++ +++ ++ +++ +++ +++
++ + ++ ++ ++ ++ + ++ + ++
9.5
22
28
17
Tan. ac.
Contr. (eth. aic.)
+ 0 +10 +1-
++++ +++ ++++ ++++ +++ ++++ +++ +++ +++ ++++
2.5
35
+1-
EXPERIMENT f 1 Part c CuP U.V.-E.D. = M sol. 3 ml.
3.10—s
Solution Volunteer
B.R. J. S. T. K.
F.T. Totals
Tan. ac.
PABA
Menth. ant.
Phen. sal.
Glyc. PAB
+ +1++ +
+++ +++ +++ +++
++++ +++ ++++ ++++
+++ ++ +++ ++
0
+10
++++ +++
4.5
12
15
10
1.5
15
+
++++ ++++
Equi.molar solutions of the following concentrations were employed as the testing
standards.
3 X 102M (3 ml.), 3 X lOM (3 ml.), 3 X 105M (3 ml.), 3 X 1OM (2 ml.) 1) At 3 X 102M, all of the sunscreening agents were effective in preventing erythema, and no significant difference in effectiveness of erythema protection could be ascertained (see part a).
432
ThE JOURNAL OF INVESTIGATIVE DERMA.TOLOGY
EXPERIMENT f, 1
Part d Cup
U.V.-E.D. 1 3.1O M sol. 2 ml. Solution Volunteer
MBA
Menth. ant.
Phen. sal.
Giyc. PAB
Tan. ac.
Contr.
(eth sic.)
++++ ++++ ++++ ++++ ++++ ++++
L.H.
4
Totals
'
4
4
2) At 3 X 10—4M, tannic acid was excellent and para-aminobenzoic acid was also superior to the other agents. The remaining compounds listed in decreasing order with respect to preventing erythema were glyceryl para-aminobenzoate, menthyl anthralinate, and phenyl salicylate. It is important to note that per unit molecule, tannic acid has 10 moles of benzene nuclei per one of para-aininobenzoic acid (see part b).
3) At 3 X 10M, the compounds had only limited sunscreening ability, and with the exception of para-aminobenzoic acid and tannic acid, the degree of erythema produced through the remaining compounds closely paralleled that of the control (see part c). EXPERIMENT f2 Skin U.V.-E.D. 5% so). 2 drops Solution
Volunteer
Menth. ant.
Phen. sat.
Otyc. PAB
Tan SC.
(eth.alc.)
+++ ++ +++ ++ +++ +++ ++ ++ +++ ++
+
++ + +++ ++ + ++ + ++
+1-
++++ +++ ++++ +++ ++++ +++ +++ +++
+
+++ ++ ++ ++ +++ +++ ++ +++ +++ ++
4.5
25
25
16
PABA
A. K.
R.W.
I.w.
M. E.
J.D.
M.G. S. B.
B.H. L.D. R.H. Totals
+1-
+ +1+
+1-
+1-
+1-
+++ +++
+
1.5
33
433
ULTRAVIOLET ABSORPTION OF COMPOUNDS
EXPERIMENT J(3 Skin—2 Hrs. 5% aol. Solution
- ________ ____________
Volunteer
A.R. B.L. H.B. J. P.
B. R.
s.v.
PADA
Menth. ant.
Phen. sal.
Glyc. PAD
Tan. ac.
(eth.alc.)
++ +1++ +1-
+++ ++ +++ ++++
+++ + ++ +++
+++ + ++ +++
+
+/-
++++ +++ ++++
+ ++ +1-
+1- ++++
+
++ ++
6
24
15
J.s.
R. R.
Totals
+
++
++ +++ +++
R.L.
D.K.
+++
++
+1—
15
+
+++ ++++ ++ ++ +1— +++
4
29.5
+
4) Using only 2 ml. of solution, in contrast to 3 ml., as in part c, none of the compounds exhibited any erythema protection when the concentration was 3 X 1OM (see part d). Direct skin application and protection against U.V. lamp
The second aspect of this investigation was to evaluate the compounds in direct contact with the skin surface and when they were not kept from contact by a cup. Therefore, equal amounts of a 5% solution of each compound placed on the skin were evaluated. The source of irradiation again was the Hanovia D.C.
lamp asusedinExp. f1. 1) In experiment 2,5% concentrations of tannic and para-aminobenzoic acid were excellent when compared to the control.
2) Glyceryl para-aininobenzoate was good, whereas phenyl salicylate and menthyl anthralinate showed only minimal differences from the control. Direct skin application and protection against natural sunlight
In experiment * 3, natural conditions were utilized, i.e. subjects were tested by being exposed to natural sunlight, and equipercentage, (5%), solutions were applied directly to the skin. 1) Tannic acid and para-aminobenzoic acid were excellent in preventing erythema. 2) Glyceryl para-aminobenzoate and phenyl salicylate had fair sunscreening activity. 3) Menthyl anthralinate was poor. For convenience in analyzing data a tabular summary of the three previously described experiments is presented here.
434
Cup 3.104 M
9.5
22
28
17
' 2.5
35
Ranked in order of protective
II
IV
V
III
I
VI
U.V.-5% Solution
4.5
25
25
16
1.5
33
Ranked in order of protective
II
V
IV
III
I
VI
Sun-5% Solution
6
24
15
15
4
29.5
Ranked in order of protective
II
V
IV
III
I
VI
ThE JOURNAL OF INVESTIGATIVE DERMATOLOGY Solution Phen. SaL
PABA
iic.)
capacity
capacity
capacity CONCLUSIONS
Experiments designed to evaluate the sunscreening properties of a group of five compounds containing the substituted benzoic acid nucleus were performed.
The compounds were tested for their capacity to prevent erythema caused by exposure to the Hanovia D.C. ultraviolet lamp and to natural sunlight. The findings indicate a relationship between their chemical properties and biologic effects as postulated in Blum's monograph on light sensitivity (see p. 177). Although all compounds tested exhibited erythema-preventing properties, the degree of their effectiveness fell into two groups.
Group I: Tannic acid and para-aminobenzoic acid Both tarmic acid and para-aminobenzoic acid were superior to all other agents
tested. The superiority of tannic acid above all agents tested was particularly evident when comparisons with other compounds were on an equimolar basis. Para-aminobenzoic acid was almost as effective as tannic acid in preventing erythema when tested in equipercentage solutions. Group II: Glyceryt para-aminobenzoate, phenyt saticyt ate, and menthyt ant hratinate
Under rigid statistical analysis, no significant differences could be established in the sunscreening properties of phenyl salicylate, menthyl anthralinate,
or glyceryl para-aminobenzoate. It is the author's belief that further studies may demonstrate that menthyl anthralinate is the poorest erythema-protecting agent of all compounds tested in this study. REFERENCES 1. BLUM, H. F.: Photodynamic action and diseases caused by light. Amer. Chem. Soc. Mono. Series 85, 1941. 2. FRIEDL AND OBCHIN: Ultraviolet Spectra of Aromatic Compounds, pp. 1—36. New York,
J. Wiley and Sons, Inc., 1945.
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3. GIESE, A. C. AND WELLs, J. M.: Sunburn protection, natural and artificial. Scient.
Monthly, 62: 458, 1946. 4. KESTEN, B. M. AND SLATEIN, M.: Diseases related to light sensitivity. Arch. Dermat. & Syph., 67: 3, 1953. 5. KLINE, P. AND BABE, R. L.: Pyribenzamine inhibition of sunburn. J. Invest. Dermat., 10: 397, 1948. 6. LUCKIESH, TAYLOR, COLE AND SOLLMAN: Protective skin coatings for prevention of
sunburn. J.A.M.A., 130: 1, 1946. 7. ROTHMAN, S. AND HENNINGSEN, A. B.: The sunburn protecting effect of PABA. J. Invest. Dermat., 9: 307, 1947. 8. ROTHMAN, S. AND RUBIN, J.: Sunburn and PABA. J. Invest. Dermat., 6: 445, 1942. 9. SHARLIT, H.: Salol investigation. Arch. Dermat. & Syph., 32: 290, 1935. 10. SULZBEBGER, M. B. AND WoLF, J.: Section II on Sunburn, Dermatology; Essentials of Diagnosis and Treatment, p. 481. Chicago, Year Book, Inc., 1952. 11. SUTTON, R. L. AND Surrow, R. L., Jn.: Handbook of Diseases of the Skin, p. 73. St. Louis, C. V. Mosby, 1949.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
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linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
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skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
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No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
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