Antifungal Properties of Cranberry Juice

Transcription

1 APPLIED MICROBIOLOGY, OCt. 1968, p American Society for Microbiology Vol. 16, No. 10 Printed in U.S.A. Antifungal Properties of Cranberry Juice JACOB H. SWARTZ AND THEODORE F. MEDREK Departments ofdermatology and Bacteriology, Massachusetts General Hospital, Boston, Massachusetts Received for publication 22 May 1968 Cranberry juice exerts a significant in vitro antifungal effect on eight representative species of dermatophytes, whereas it has no apparent effect on Candida albicans. The antifungal effect is fungistatic. Benzoic acid or other small molecular weight components, or both, were responsible for the fungistatic action. Studies with C. albicans on the effect of ph alone and the effect of ph on the ionization of benzoic acid indicate that cranberry juice would exert an even more significant antifungal action if the ph were left at its native value of 2.8; not adjusted to 5.6. This would probably be due to ph and a larger amount of free benzoic acid. Further investigation suggested that benzoic acid loses some of its antifungal properties in cranberry juice atph 5.6. This investigation suggests that the dermatophytes may have a higher sensitivity to benzoic acid or other small molecular weight components of cranberry juice, or to both. Many years ago, J. H. Swartz prescribed empirically an ointment then known as Vaccinol that had cranberry as the active ingredient. It proved surprisingly effective in some dermatoses, including some alleged to be caused by fungi. A search of the literature on the antifungal properties of cranberries failed to locate a single paper dealing with cranberry juice in its natural state. However, Ujvary et al. (6) reported the antifungal properties of an ethyl alcohol extract of cranberry juice. The authors reported the antifungal effect of the varying concentrations (2 to 30%) of cranberry juice extract. They postulated that the antifungal action might be due to (i) a pigment known as idaein, (ii) various organic acids, (iii) 6-benzyl-D-glucose, or (iv) other unidentified substances. The purpose of this investigation was to determine (i) whether cranberry juice in its natural state has antifungal properties and, if so, whether it is fungistatic or fungicidal, and (ii) which component(s) is involved. MATERIALS AND METHODS Test strains included C. albicans and representative species of dermatophytes. The experimental material was unpasteurized and unadulterated cranberry juice (454 g of the berries yielded approximately 340 ml of juice). Because the cranberry juice from a commercial source (Ocean Spray Cranberries, Inc., Hanson, Mass.) had a ph value of approximately 2.8, and thus could at least partially inhibit growth of the test fungi, it was decided to adjust the ph of the juice to one optimal for growth of fungi in order to determine whether the antifungal activity of cranberry juice is due to a factor(s) other than ph. Because Sabouraud's dextrose agar is widely used for growing fungi and has a ph value of approximately 5.6, the ph value of cranberry juice was adjusted to this level. A second reason for adjusting the low ph of the juice was to permit the use of a solidified agar surface for testing. After adjusting the ph with 10 N NaOH, the cranberry juice was centrifuged at approximately 2,250 X g for 1 hr to eliminate particulate matter. The juice was then sterilized by passing it through a membrane filter (pore diameter, 0.45,zm; Millipore Corp., Bedford, Mass.); it was then incorporated in Sabouraud's dextrose agar (Difco) in appropriate concentrations. Growth of the fungi on Sabouraud's dextrose agar without additives was used as a control. C. albicans and eight representative species of dermatophytes (Table I) were tested in duplicate. Existing literature (2, 4, 5) revealed some ingredients which could account for any fungistatic or fungicidal activity that cranberries might have. Two possible components were benzoic acid and iodine. These were tested in parallel with cranberry juice in double the mean concentration (benzoic acid, 0.087% and iodine, 40 ppm) of the widely published values. The stock solution of benzoic acid was prepared in 95% ethyl alcohol because of its limited solubility in water. RESULTS Benzoic acid, alone and in combination with iodine, completely inhibited all the organisms tested. Iodine alone had little or no antifungal effect. Cranberry juice (40%) in Sabouraud's dextrose agar had no effect on the growth of C. albicans. It completely inhibited all the other organisms tested except Trichophyton tonsurans, in which minimal growth was noted, and Tri Downloaded from on September 16, 2018 by guest

2 VOL. 16, 1968 ANTIFUNGAL PROPERTIES OF CRANBERRY JUICE 1525 TABLE 1. Effect of cranberry juice, iodine, benzoic acid, and benzoic acid plus iodine on C. albicans and on some dermatophytesa Fungus Controib Cranberry juice Cranberry juice I2 (final concn, (2:3 dilution) (1: 2 dilution) 40 ppm) Candida albicanis Abundant growth Abundant growth Abundant growth Abundant growth Microsporum canis 35.5 mmc No growth on one 8.8 mm 32.0 mm plate; dubious growth on second plate Microsporum audouini 21.5 mm No growth Dubious growth 18.5 mm Microsporum gypseum 39.0 mm No growth Dubious growth 40.0 mm Trichophyton schoenleinii 5.0 mm No growth No growth 6.0 mm Trichophyton rubrum 7.3 mm No growth No growth 7.8 mm Trichophyton tonsurans 16.0 mm Dubious growth 4.0 mm Not done Trichophyton mentagrophytes 32.5 mm 7.8 mm 7.8 mm 32.0 mm Epidermophyton floccosum 10.8 mm No growth No growth 9.5 mm a None of the fungi grew in benzoic acid (final concentration, 0.087%) or in benzoic acid plus I2. b Sabouraud's dextrose agar. c Quantities of growth expressed as mean colony diameter. chophyton mentagrophytes, which showed about 1.0 _ 80% reduction in mean colony diameter. The 33% dilution of cranberry juice showed a decrease in inhibition with some of the organisms 0.8 tested (Table 1). Because the 40% and 33%0 concentrations of t 0.6 A cranberry juice showed significant fungistatic action, it was of interest to determine the minimal inhibitory concentration of the juice on the test 0.4 organisms. There was a gradual decrease of B / fungistatic action as the dilutions of cranberry cj juice were increased (1:4, 1:8, 1:16, and 1:32). 0.2 Higher dilutions showed minimal or no inhibition. The lack of inhibitory effect of the 40% cranberry juice on C. albicans, as compared to the ph demonstrated inhibition of 0.087% benzoic acid, may result from the smaller amount of benzoic FIG. 1. Effect of ph value and benzoic acid on the acid in the cranberry juice tested. If our cranberry juice, in fact, contained only the mean pub- Symbols: (A), broth alone; (B), broth with 0.044% growth of C. albicans in Sabouraud's dextrose broth. lished value of benzoic acid rather than double benzoic acid; (C), broth with 0.087% benzoic acid. the mean published value, we should expect the growth of C. albicans to be inhibited by 40% control (Sabouraud's dextrose agar without benzoic acid). These results are interpreted to mean cranberry juice to which 0.044% benzoic acid had been added. This experiment was done with that cranberry juice contains a factor that diminishes the antifungal effect of the benzoic acid. appropriate controls. The result was that 40% cranberry juice to which 0.044% benzoic acid had In an attempt to distinguish the effect of ph been added showed slightly reduced growth compared to the controls (Sabouraud's dextrose agar benzoic acid to the salt sodium benzoate, C. alone and the effect of ph on the ionization of with no additives and 40% cranberry juice with no albicans was grown in Sabouraud's dextrose additives). To investigate the role of benzoic acid broth that had been adjusted from ph 2.5 to 8.0 as the most important possible antifungal agent at ph unit increments of 0.5. Similar broths were of cranberry juice, tests were carried out with prepared containing 0.044% and 0.087% benzoic 0.044% benzoic acid in Sabouraud's dextrose acid. Tubes were incubated at room temperature agar. The growth of C. albicans was diminished for 5 days prior to determining optical density at by approximately two-thirds, compared to the 540 nm. (Fig. 1). At ph 2.5 to 4.5, ph is not the

3 1526 SWARTZ AND MEDREK APPL. MICROBIOL. only factor in the complete inhibition of C. albicans in broth that contains benzoic acid. At the 0.044% level of benzoic acid, ph 2.5 was fungicidal, whereas ph 3.0 to 4.5 was fungistatic. At the 0.087% level of benzoic acid, ph 2.5 to 4.0 was fungicidal, whereas ph 4.5 was fungistatic. Fungicidal and fungistatic levels were determined by streaking tubes that had no observable turbidity on blood-agar plates. The first observable turbidity of tubes that contained benzoic acid occurred at ph 5.0. At higher values, the antifungal effect of benzoic acid continued to decrease. Broths that did not contain benzoic acid showed a trend toward increased optical density. To gain further insight into the nature of the antifungal properties of cranberry juice, it was of interest to determine whether the factor(s) is dialyzable. All the dermatophytes tested, with the exception of Trichophyton rubrum, showed insignificant differences in mean colony diameters between the control (Sabouraud's dextrose agar) and the dialyzed cranberry juice. Thus, it was concluded that benzoic acid or other dialyzable components, or both, were responsible for the antifungal action against the dermatophytes. Because it was of interest to determine whether the observed complete inhibition of the test organisms was fungistatic or fungicidal in nature, we attempted to culture the organisms in Sabouraud's dextrose broth after contact for 3 weeks with 40% and 33% concentrations of cranberry juice. The results indicated that the inhibition of growth was due to a fungistatic rather than to a fungicidal action. DISCUSSION The results of the antifungal studies reported in this paper are not as pronounced as those reported by Ujivary et al. (6). However, these differences could be attributed to (i) the difference in the variety of the cranberries used, (ii) their use of an extraction procedure, (iii) their use of an extract about four times the concentration used in our experiments, (iv) their failure to adjust the low ph of the extract used, and (v) use of different test strains of the same species. Ujjvary et al. stated that if the extract were neutralized, its antifungal action was reduced; however, in spite of it, an extract of S to 10%7 of "neutral ph" was found to have an even greater effect on two dermatophytes. In our experiments, the ph value of the cranberry juice used was adjusted to one generally accepted to be optimal for growth of fungi (5.6). However, the preservative action of benzoic acid and sodium benzoate that is present naturally in cranberry juice may be expected to be not as active as it would be in a strongly acid solution; furthermore, the more active form of free benzoic acid is reduced in concentration as the ph is increased, whereas the more inactive form of sodium benzoate is increased (1, 3). Thus, we believe that our results on the antifungal properties of cranberry juice are less pronounced than we would expect to obtain if the ph value were unadjusted. The results (Fig. 1) with C. albicans have shown that at ph 2.5 to 4.5, a complete inhibition of growth occurred with the concentrations of benzoic acid used, and that this was not due to ph alone, because significant growth occurred in broth without benzoic acid at this ph range. At higher ph values, there was a continued decrease of antifungal action of benzoic acid as the ph increased. This is probably related to the decreased concentration of free benzoic acid and to ph. The change from no turbidity to turbidity occurred near the pk of benzoic acid, which is At ph 5.6, the value we selected for our studies, the ratio of sodium benzoate to free benzoic acid is 27 to 1. Thus, the benzoic acid present in our experimental cranberry juice was mainly in the relatively inactive form of sodium benzoate. It is reasonable to assume, therefore, that cranberry juice with an unmodified ph value would be even more potent as an antifungal agent than when conducted at ph 5.6. Studies concerned with a more detailed investigation of the role of benzoic acid in cranberry juice as possibly the most important antifungal agent revealed that benzoic acid loses some of its antifungal properties when incorporated in cranberry juice at ph 5.6, as shown by studies on C. albicans. If benzoic acid in cranberry juice is an important factor in the inhibition of the dermatophytes, it may be due to their higher sensitivity to benzoic acid as compared to C. albicans. ACKNOWLEDGMENTS This investigation was supported by grants from Ocean Spray Cranberries, Inc., and by the Leon Newton Foundation. LITERATURE CITED 1. Block, S. S Industrial preservatives, p In G. F. Reddish (ed.), Antiseptics, disinfectants, fungicides, and chemical and physical sterilization, 2nd ed. Lea & Febiger, Philadelphia. 2. Clague, J. A., and C. R. Fellers Relation of benzoic acid content and other constituents of cranberries to keeping quality. Plant Physiol. 9: Dunn, C. G Food preservatives, p In G. F. Reddish (ed.), Antiseptics, disinfec-

4 VOL. 16, 1968 ANTIFUNGAL PROPERTIES OF CRANBERRY JUICE 1527 tants, fungicides, and chemical and physical 5. Morse, F. W The iodine content of Cape sterilization, 2nd ed. Lea & Febiger, Phila- Cod cranberries. J. Biol. Chem. 79: delphia. 6. {Cjvary, I., J. Orlik, G. RAcz, and A. D6nith. 4. Fellers, C. R. and W. B. Esselen Cranberries On the fungistatic effect of the cranberry's and cranberry products. Univ. Mass. Agr. Exp. (Vaccinium Vitis Idoea L.) crop-extract. Sta. Bull (Translation). Orv. Szemle 4:

5 ERRATA Production of Nuclease-forming 5'-Nucleotide by Aspergillus quercinus in a Low Phosphate Medium YOSHIYUKI OHTA AND SEINOSUKE UEDA Department of Agricultural Chemistry, Faculty of Agriculture, Kyushu University, Fukuoka, Japan Volume 16, no. 9, page 1293, in article heading under authors' address: insert "Received for publication 17 June 1968." Antifungal Properties of Cranberry Juice JACOB H. SWARTZ AND THEODORE F. MEDREK Departments ofdermatology and Bacteriology, Massachusetts General Hospital, Boston, Massachusetts Volume 16, no. 10, p : change "40%" to "67%" *herever "40%" occurs in the text; page 1525, Table 1: change "(2:3 dilution)" to "(67%)" and change "(1:2 dilution)" to "(33%)." 192