Abstract
Background
Rhizopus stolonifer is an opportunistic fungus that causes respiratory infections, sinusitis, and otomycosis. Trichophyton mentagrophytes and Microsporum gypseum cause athlete’s foot, ringworm, and nail infections. Previous research has shown that some essential oils inhibit fungal growth.
Methods
We investigated the effects of 0.5% and 1% concentrations of Rosmarinus officinalis, Cinnamomum verum, Citrus paradisi, and Capsicum annuum extracts on the growth of R. stolonifer and T. mentagrophytes. The combined effects also were determined at 0.5% concentration for these fungal species and for M. gypseum. Fungal species were grown in flasks, along with growth media, for 7, 14, and 21 days, with each species of essential oil or combination of oils and mass determined and compared to its control.
Results
Rosmarinus officinalis, Cinnamomum verum, and Citrus paradisi had overall significant inhibitory effects (P ≤.05) on R. stolonifer and T. mentagrophytes. C. annuum significantly (P ≤.05) inhibited the growth of T. mentagrophytes and enhanced that of R. stolonifer. The combination of essential oils resulted in inhibition of growth (P ≤.05) at similar percentages as R. officinalis, C. verum, and C. paradisi only.
Conclusion
Essential oils such as Rosmarinus officinalis, Cinnamomum verum, and Citrus paradisi may be useful for treating opportunistic and dermatophytic fungal diseases.
Keywords: essential oils, Rhizopus stolonifer, Trichophyton mentagrophytes, Microsporum gypseum, opportunistic fungi, dermatophytes
Although some fungal species are beneficial to the environment, others cause diseases in humans and other animals. Rhizopus species are commonly visible as molds on breads, fruits, and vegetables.1 However, as opportunistic pathogens some species of Rhizopus cause life-threatening zygomycoses, most commonly in immunocompromised people and people with diabetes mellitus, diarrhea, or malnutrition. Manifestations may be pulmonary, rhinocerebral, gastrointestinal, or in the form of allergic diseases or cutaneous issues.2,3 Dermatophytes, such as Trichophyton and Microsporum species, can infect the skin, hair, and nails.4 Although Trichophyton spp cause a variety of skin and nail issues all over the body, T. mentagrophytes most commonly causes tinea pedis and tinea unguium. Microsporum spp, such as M. canis, commonly cause tinea corporis and tinea capitis in humans and animals, and strains of Microsporum gypseum more virulent than that found in soil can cause epidemics in humans. Opportunistic and dermatological diseases from Rhizopus, Trichophyton, and Microsporum spp are found worldwide.
The results of previous studies5,6 have indicated that certain essential oils inhibit the growth of specific fungal species in vitro. For example, Thymus vulgaris and Citrus aurantifolia essential oils were shown to inhibit the growth of R. stolonifer, whereas oils from plants such as Chenopodium ambrosiodes and Mentha arvensis, among others, have shown inhibitory effects against M. gypseum and T. rubrum. An ointment made of Artemisia oil was shown6 to cure ringworm in guinea pigs after 7 to 12 days. Also, Rosmarinus officinalis extract has been shown7 to inhibit the growth of Aspergillus parasiticus, as well as the aflatoxin that it produces, and is an extract that can be used in food preservation. A few Cinnamomum species, such as C. osmophloeum, have been tested against the growth of fungi, and have been shown to prevent fungal growth on wood. Although C. verum has been shown to inhibit the growth of Staphylococcus iniae, more testing is warranted regarding the effects of C. verum on human fungal pathogens.8,9 Similarly, because a few Citrus species have been tested against the growth of various fungi such as Aspergillus species and Penicillium species, more testing of Citrus spp with a greater variety of methodologies is warranted.10
Zygomycoses and tinea infections are worldwide issues, and more emphasis is needed on finding new methods to inhibit the growth of the fungal species that cause them. Hence, the focus of this study was to determine the inhibitory effect of various easily obtainable, safe, ingestible herbal extracts. Rosmarinus officinalis (rosemary), Cinnamomum verum (cinnamon), Citrus paradisi (grapefruit), and Capsicum annuum (cayenne) extracts were tested individually, at 0.5% and 1% concentrations, to determine their effects on the growth on R. stolonifer and T. mentagrophytes. We also determined the combined effects of R. officinalis, C. verum, and C. paradisi at an overall concentration of 0.5% on the growth of R. stolonifer, T. mentagrophytes, and M. gypseum. We hypothesized that each type of oil would inhibit the growth of each fungal pathogen, with 1.0% inhibiting more than 0.5%, and that the combination of oils would work synergistically to inhibit the fungi to a greater degree than any single oil.
Materials and Methods
Trichophyton mentagrophytes (ATCC18748), Microsporum gypseum (ATCC 24102), and Rhizopus stolonifer (ATTC6227a) were grown on Sabouraud Dextrose Agar (SDA).11 For each species, a methodology similar to that of Cvek et al12 was used to determine growth of fungi with each essential oil, compared to a control oil. We placed 106vegetative spores of one of the fungal species into 125 mL flasks with 0.5% dimethyl sulfoxide (DMSO), 0.5% or 1% filter sterilized essential oil or 0% for controls, and yeast media (20 g yeast extract and 20 g sucrose/L), for a total of 50.5 mL. Flasks were incubated at 30ºC for 7, 14, and 21 days in triplicate, and averages were determined for each time interval. The essential oils we tested were R. officinalis, C. verum, C. paradisi, and C. annuum. For the combination study, a mixture with overall 0.5% concentration of R. officinalis, C. verum, and C. paradisi was used. After the appropriate growth period, flask contents were vacuum filtered through a #4 Whatman 150 mm circle filter (Merck KGsA). The preweighed filters were dried in a 37ºC incubator for 10 days before final mass was determined. Statistical analyses were performed to ensure P values of .05 or less.
Results
Figure 1 indicates the growth in mg per mL at 7, 14, and 21 days for R. stolonifer for each essential oil at 1.0% concentration. Table 1 indicates that all essential oils at 1% concentration inhibit R. stolonifer except C. paradisi at 7 days and C. annuum at all time intervals. After 7 days, 1% C. annuum had 812.9% more growth than the control condition. C. paradisi inhibited growth of R. stolonifera by 70.1% after 21 days, and at the same time interval, C. verum and R. officinalis each inhibited growth by 79.4%.
Figure 1.
Growth at different time intervals for Rhizopus stolonifera for Rosmarinus officinalis, Cinnamomum verum, Citrus paradisi, and Capsicum annuum oils, each at a concentration of 1.0%.
Table 1.
Percentage Change in Growth of R. stolonifer and T. mentagrophytes for Each Essential Oil Tested, Compared with the Controla
| Fungus and Time Period | Concentration | |||||||
|---|---|---|---|---|---|---|---|---|
| Rhizopus stolonifer | 1% | 0.5% | ||||||
| RO | CV | CP | CA | RO | CV | CP | CA | |
| 7 d | −51.6 | −9.7 | 3.2 | 812.9 | −35.5 | −32.3 | −6.5 | 271.0 |
| 14 d | −15.8 | −18.4 | −39.5 | 315.8 | −42.1 | −28.9 | −39.5 | 260.5 |
| 21 d | −79.4 | −79.4 | −70.1 | 26.1 | −76.6 | −71.0 | −78.5 | 64.5 |
| Trichophyton mentagrophytes | ||||||||
| 7 d | −18.3 | −53.3 | −45.0 | −13.3 | −48.3 | −50.0 | −56.7 | −55.0 |
| 14 d | −63.8 | −54.4 | −65.1 | −28.9 | −67.8 | −66.4 | −70.5 | −26.2 |
| 21 d | −69.5 | −73.9 | −65.2 | −17.3 | −72.8 | −78.8 | −72.8 | −11.9 |
RO, Rosmarinus officinalis; CV, Cinnamomum verum; CP, Citrus paradisi; CA, Capsicum annuum.
a P ≤.05.
Figure 2 indicates the growth in mg per mL at 7, 14, and 21 days for R. stolonifer for each essential oil at 0.5% concentration. Table 1 indicates that all essential oils at 0.5% concentration, except C. annuum, inhibit R. stolonifer at all time intervals, including 271.0% more growth than the control after 7 days and 260.5% more after 14 days of growth. C. paradisi inhibited the growth of R. stolonifera by 78.5% after 21 days, and at the same time interval, C. verum and R. officinalis inhibited growth by 71.0% and 76.6%, respectively.
Figure 2.
Growth at different time intervals for Rhizopus stolonifera for Rosmarinus officinalis, Cinnamomum verum, Citrus paradisi, and Capsicum annuum oils, each at a concentration of 0.5%.
Figure 3 represents the growth of T. mentagrophytes for each essential oil at 1.0% concentration. Table 2 indicates that all oils at a concentration of 1.0% inhibited the growth of T. mentagrophytes at all time intervals, compared with the control. The greatest inhibitor was C. verum: at 21 days, by 73.9%.
Figure 3.
Growth of Trichophyton mentagrophytes for Rhizopus stolonifera for Rosmarinus officinalis, Cinnamomum verum, Citrus paradisi, and Capsicum annuum oils, each at a concentration of 1.0%.
Table 2.
Percentage Change in Growth of Combination of 0.5% RO, CV, and CP, Compared with the Controla
| Days | Rhizopus stolonifer | Trichophyton mentagrophytes | Microsporum gypseum |
|---|---|---|---|
| 7 | −12.9 | −55.0 | −64.2 |
| 14 | −28.9 | −63.8 | −75.5 |
| 21 | −68.2 | −71.7 | −67.4 |
RO, Rosmarinus officinalis; CV, Cinnamomum verum; CP, Citrus paradisi.
a P <.05.
Figure 4 represents the growth of T. mentagrophytes for each essential oil at 0.5% concentration. Table 1 indicates that C. paradisi had an inhibition of growth at 70.5% and 72.8% at 14 and 21 days, respectively, whereas C. verum and R. officinalis use resulted in inhibition, after 21 days, of 72.8% each.
Figure 4.
Growth of Trichophyton mentagrophytes for Rhizopus stolonifera after exposure to Rosmarinus officinalis, Cinnamomum verum, Citrus paradisi, and Capsicum annuum oils, each at a concentration of 0.5%.
Figure 5 indicates the growth of R. stolonifer, T. mentagrophytes, and M. gypseum at 7, 14, and 21 days after exposure of each to 0.5% concentration of R. officinalis, C. verum, and C. paradisi combined. Table 2 indicates that growth was inhibited for each fungal species at all time intervals.
Figure 5.
Growth of Rhizopus stolonifera, Trichophyton mentagrophytes, and Microsporum gypseum at different time intervals after each was exposed to 0.5% concentration of a combination of Rosmarinus officinalis, Cinnamomum verum, and Citrus paradisi oils.
Discussion
Although we hypothesized that 1.0% concentration of oils would inhibit growth of R. stolonifer and T. mentagrophytes more than 0.5%, inhibitions were similar at both concentrations. For example, the inhibition of the growth of R. stolonifer by R. officinalis after 21 days was significant (P ≤.05) at 79.4% for the 1% concentration and 76.6% for the 0.5% concentration. Using 1% C. verum, growth was inhibited significantly (P ≤.05) at 79.4% after 21 days, whereas 0.5% concentration inhibited growth by 71.0% after 21 days. C. paradisi inhibited growth by 70.1% and 78.5% (P ≤.05) at the 1% and 0.5% concentrations, respectively, at the same time interval.
However, C. annuum unexpectedly enhanced the growth of R. stolonifer significantly (P ≤.05) at all time intervals at both concentrations. We were intrigued by this finding because a previous test on the effects of spices on the growth and toxin production in Aspergillus species13 reported that Capsicum seeds had “relatively minor effects” on the growth of the fungal species and on toxin production. However, the findings from a separate study14 showed that some fractionated C. annum extracts were effective against the growth of bacterial species whereas other fractions were not. Cinnamic acid and m-coumaric acid contributed to the inhibition of bacteria, compared with the other fractions. It appears likely that various fractions within extracts of essential oils have varying effects on the growth of microorganisms. If the C. annum essential oil were fractionated, some of the extracts may be shown to inhibit fungal growth and others may have no effect or may even increase the growth.
The growth of T. mentagrophytes was inhibited significantly (P ≤.05) by R. officinalis, C. verum, and C. paradisi by 69.5%, 73.9%, and 65.2%, respectively, at the 1% concentration after 21 days and 72.8% for each of those essential oils at 0.5% concentration at the same time interval. These are comparable values for all oils at 21 days. Although C. annuum did not enhance the growth of T. mentagrophytes as it did for R. stolonifer, the inhibition was not as marked as it was with the other oils. C. annuum showed a significant inhibition of growth (P ≤.05)—55.0% at 7 days using 0.5% concentration; the other time intervals and concentrations inhibited the growth to a lesser degree.
The combination of R. officinalis, C. verum, and C. paradisi at 0.5% concentration did not inhibit the growths of R. stolonifer or T. mentagrophytes more than any single oil. R. stolonifer was inhibited significantly (P ≤.05) by 68.2% after 21 days, and T. mentogrophytes was inhibited by 71.7% using the combination. M. gypseum was also used in this study, and at 21 days, it was inhibited significantly (P ≤.05) by 67.4% after 21 days, 75.5% after 14 days, and 64.2% after 7 days using the combination of oils. These findings suggest that there is no resulting synergistic effect by using a combination of the oils that inhibit fungal growth when used separately. Inhibition was as high or higher using each of the inhibitory oils by itself.
Conclusion
Inhibition of the growth of R. stolonifer and T. mentagrophytes was similar at both concentrations used in this study of R. officinalis, C. verum, and C. paradisi, as well as the combination of those oils. M. gypseum was inhibited to a similar degree as R. stolonifer and T. mentagrophytes using the combination of oils. C. annuum markedly enhanced the growth of R. stolonifer and inhibited T. mentagrophytes to a lesser degree than the other oils. These study results indicate that R. officinalis, C. verum, and C. paradisi may be useful as inhibitors of zygomycoses and tinea infections. Further studies to determine the minimum concentrations that inhibit the growth of these fungal pathogens may be warranted. LM
Personal and Professional Conflicts of Interest
None reported.
Acknowledgements
We thank the National Institute of General Medical Sciences MARC U-STAR Program T34GM070416 for funding this research and the Clinical Laboratory Science Department at Winston-Salem State University, Winston-Salem, NC.
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