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Plant derived antifungals- trends and potential applications in veterinary

medicine: A mini-review
Nopamart Trakranrungsie
Department of Preclinical Sciences, Faculty of Veterinary Science, Mahidol University, Salaya, Nakhonpathom 73170,
Thailand
Due to increasing resistance of fungi against conventional drugs, as well as the observable side effects of the already
limited numbers of antifungals commonly used in veterinary practice, the fungal infections among animals, whether
superficial or systemic, often pose substantial management problems and are still a major concern. In this regard, the
alternative herbal formulations have become a renewed interest. Medicinal plants are in fact an integral part of
ethnoveterinary medicine and the practice is rather common in Asian countries, including China, India, Japan, Pakistan,
Sri Lanka and Thailand. Development in information technology has facilitated an explosion in the range and content of
electronic information concerning medicinal plants as a re-emergent health aid for both animals and humans. It has been
estimated that more than 100,000 plants/plant extracts have been assayed for their pharmacological properties. Based on
the current available data and the recently proposed IC
50
< 100 g/ml or MIC < 500 g/ml as a cut-off value if the extracts
would possess any promising antifungal effect, the potential candidates are summarized with their reported major
constituents. The indigenous plants with strong antifungal activity abundantly found in certain regions, the example of
their herbal products and the efficacy testing are also presented.
Keywords antifungal; plant extract; Piper betle; dermatophytes
1. Introduction
Plants have always served as essential sources of therapeutic agents for humans and animals. Records of medicinal
plants and their therapeutic values appear not only in the classic literature of traditional knowledge in all cultures of the
East and West, but also in numerous recent publications and databases developed by several institutes and organizations
such as the University of Illinois at Chicago (http://www.napralert.org/), Indian Institute of Ayurveda and Integrative
Medicine (http://www.frlht.org.in/), and Mahidol University (http://www.pharmacy.mahidol.ac.th/medplantdatabase/)
[1-6]. The return of interest in pharmacologic activity of plants, plant extracts and isolated plant compounds in the past
decades has markedly energized the establishment of standardized methods of preparation and extraction, as well as
standard assays and criteria for activity, as parts of the process in drug discovery and development [7].
Traditionally, the herbal remedies may be offered more or less in a holistic approach for maintenance and well-being
of health, enhanced various body processes, and some symptomatic relief. Although a cyclical phenomenon has been
described as a pattern of interest in herbal medicine in veterinary practice, the modern herbal formulation based on
scientific proven efficacy may provide a re-invented paradigm of treatment of choice for specific disease conditions
with a stronger reliability and predictability of clinical outcomes [8, 9].
In animals, dermatophytic infections are often challenging to manage and some do pose zoonotic potential. The
course of treatment particularly in companion animals requires long-term drug administration and hence is costly. The
application of plant-derived antifungal compounds, i.e. as topical therapy, could be one of the attractive alternatives.
This article provides an overview of fungal infections in animals and their clinical significance. The currently available
conventional antifungals and their clinical limitations in veterinary practice are summarized. In addition, due to the
renewed and increasing interest in the application of medicinal plants and plant-derived products as the treatment of
choice observed in recent years, plants with promising antifungal activity with reported IC
50
(concentration inhibiting
50% of fungal growth) < 100 g/ml or MIC (minimum concentration resulting in no visible fungal growth) < 500 g/ml
are listed exclusively. Finally, trends in herbal modalities and potential applications of plant-derived antifungals in
veterinary medicine are discussed.
2. Fungal infection in animals and their clinical significance
Major fungal infections in animals can be recognized in the forms of both systemic and superficial infections. Several
fungal species including Aspergillus spp., Cryptococcus neoformans, Histoplasma capsulatum and Candida spp. could
be involved in the pathogenesis of systemic mycoses. The common causes of superficial mycoses, on the other hand,
are dermatophytes, the keratinophilic fungi consisting of three genera: Microsporum, Trichophyton, and
Epidermophyton. In companion animals, Microsporum canis, Microsporum gypseum and Trichophyton
mentagrophytes are considered the main causative pathogens of skin infections, of which M. canis accounts for 50-70%
and 90-98% of the identified cases in dogs and cats, respectively [10]. M. persicolor, M. verrucosum, T. terrestre, T.
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rubrum, T. equinum, T. schoenleinee and E. floccosum can also be isolated from the animal skin, however, their clinical
significance is limited [11].
Fungal infections among animals are and will still be a major concern not only for veterinary but also public health.
In most cases, treatment of clinical mycoses often poses management problems as it requires regular and long-term
antifungal administration. The information concerning pharmacokinetic properties among animal species and guidance
for veterinary treatment regimen is however largely unavailable due to the fact that the majority of antifungal drugs
extensively used in animals are only approved for human use [12]. Often time, the treatment regimens resulting in
unfavorable clinical outcomes could lead to the development of resistant strains. Moreover, some fungal diseases
essentially cryptococcosis and dermatophytosis are also zoonosis [13].
3. Conventional antifungal drugs and their clinical limitations
Practically, the antifungals available in veterinary market are the same as those developed for the human markets but
with modified strengths [12]. Since an increasingly important role of fungal pathogens in human diseases such as
compromised immunity associated with organ transplantation and human immunodeficiency virus infection has become
more prevalent during past decades, the search for the more effective and less toxic compounds for treatment of fungal
diseases has been vigorous. Veterinarians have gained access to a rapidly expanding list of antifungal drugs with high
efficacy and low toxicity for the treatment of mycotic infections particularly in companion animals [14]. The summary
of currently available antifungals for systemic and topical treatment of animal mycotic diseases is presented in Tables 1
and 2. Nonetheless, it is noteworthy that the adverse effects among antifungals and among animal species have still
been evident mostly in systemic administration. This could be a result of a general lack of applicable clinical literature
for antifungal use in a given animal species, which leads to an extrapolation on the interpretation and use of MIC data.
In addition, different pharmacokinetic properties of the antifungal drugs across animal species as well as in the pregnant
and young animals may contribute to the observed side effects. Consequently, the veterinary practitioners and owners
concern over possible side effects of systemic and oral drugs has increased in recent years, resulting in more favourable
acceptance of topical therapy despite the greater requirement of the owner dedication, time and money [15].
4. Re-emergence of antifungal herbs as the treatment of choice
Fungi are able to improve resistance against conventional drugs rapidly, prompting the constant need to identify novel
antifungal agents. Among the natural sources for therapeutic substances, plants have always played a classic role for
such purposes since the dawn of human history. The application of medicinal plants has long been an integral part of
both human and veterinary medicine and the practice is still rather common in Asian countries, including China, India,
Japan, Pakistan, Sri Lanka and Thailand [1]. Reports from Latin and South America (Mexico, Brazil and Colombia),
Europe (Finland and Italy) and Africa (Tanzania and Ethiopia) also demonstrate long-stand values of ethnobotanical
remedy for treatment of mycotic diseases [2, 16-19]. The interest in antifungal activities of plants and plant-derived
compounds extensively noted from around the world during the past decades is therefore a renewed concept with
science-based approaches.
Thus far, it has been estimated that more than 10,000 species of plants are used medicinally and more than 100,000
plant products have been described for their pharmacologic properties [1, 19]. An exhaustive list of plants, plant
extracts, and isolated plant compounds shown to exhibit antifungal activity has been developed [19, 20]. According to
the recent proposed IC
50
< 100 g/ml or MIC < 500 g/ml to be a cut-off value to validate that the extracts possess any
promising therapeutic value [7, 21], the potential plant candidates with antifungal effect are tabulated in Table 3. It is
noted, however, that standard susceptibility testing has not been available for all tested organisms. In addition, the
majority of the preclinical data has been derived from testing of the extracts against Candida spp. and, to a lesser extent,
filamentous fungi. This is likely due to the comparative ease and reproducibility of yeast-based experiments, as well as
a significant prevalence and economic importance of Candidiasis in humans [19, 20].
As mentioned earlier, the increased incidence of dermatophytic infection in veterinary species and the growing
evidence of resistance has raised greater concern during the past decades due to its zoonotic potential, particularly
between children and companion animals such as cats, dogs, birds and small rodent/pocket pets [22-24]. The adverse
effects from systemic and oral application of conventional antifungal drugs have driven the owners favor toward
topical drugs, which requires regular and long-term administration. The expense burden from continuingly rising prices
of several conventional antifungals is therefore inevitable. In this regard, the re-emerging interest of natural products,
including plant-derived antifungals, could provide a niche for herbal formulations against dermatophytosis in animals
with possible better affordability.
In the Southeast Asian region, the commonly studied plants with antifungal activity include Allium sativum, Piper
betle, Rhinacanthus nasutus, and Senna alata (formerly Cassia alata) (Table 4). Among the outstanding candidates
with antidermatophytic activity, P. betle has been extensively investigated [8, 25, 26]. It is a tropical plant.
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Table 1 Antifungal drugs for systemic treatment in veterinary practice.
Antifungals Spectrum and Clinical applications Adverse effects
Polyenes: Amphotericin B Broad spectrum.
Systemic treatment of
filamentous fungal infections,
life-threatening yeast or
dimorphic fungal infections,
including blastomycosis,
histoplasmosis, cryptococcosis,
coccidioidomycosis, candidiasis,
sporotrichosis and pythiosis.
Treatment of systemic mycoses
that fail to respond to azole
therapy.
Dose-related renal toxicity due to
both vasoconstrictive and
tubulotoxic effects.
Pyrexia, tremors, nausea, malaise
and depression during intravenous
administration in some animals.
Possible thrombophlebitis,
hypokalemia, cardiac arrhythmias
and non-degenerative anemia.
Doses higher than 5 mg/kg of
conventional amphotericin B in
dogs could result in death due to
cardiac abnormalities.
Pyrimidine synthesis
inhibitors: Flucytosine
Effective against yeast and some
Aspergillus.
Used as an adjunct to
amphotericin B for
cryptococcosis treatment.
Reversible anorexia, nausea,
diarrhea and vomiting.
Dose-related bone marrow
suppression.
Skin eruptions characterized by
depigmentation, ulceration,
exudation and crust formation.
Seizures and abnormal behavior
may occur in cats.
Azole antifungals:
Imidazoles:
Clotrimazole and
Ketoconazole
Triazoles: Fluconazole,
Itraconazole and
Voriconazole

Broad spectrum.
Initial drug of choice for all but
the most rapidly progressing and
most severe systemic fungal
infections.

Dose-related gastrointestinal
irritation.
Hepatotoxicity (ketoconazole).
Suppression of adrenal and
testicular steroid synthesis as well
as embryotoxic and teratogenic
effects (ketoconazole).
Cataracts after long-term
ketoconazole therapy.
Thrombocytopenia (ketoconazole
and fluconazole).
Pharyngeal and upper airway
irritation as well as CNS irritation
and seizures (clotrimazole).
Dose-related local ulcerative
dermatitis (itraconazole).
Reversible visual distrurbances and
dermatopathies (voriconazole).
Allylamines: Terbinafine Broad spectrum.
Systemic treatment of
dermatophytosis and Malassezia
dermatitis.
Mild-to-moderate gastrointestinal
sign.
Hepatotoxicity (rare).
Neutropenia or pancytopenia (rare).
Echinocandins:
Caspofungin
Treatment against candidiasis
and aspergillosis.
Fever, nausea and phlebitis at the
infusion site.
Griseofulvin Systemic treatment of
dermatophytic infection.
Gastrointestinal irritation: diarrhea,
anorexia and vomiting.
Bone marrow suppression.
Hepatotoxicity and CNS signs.
Teratogenic effect in cats and the
use is contraindicated in pregnant
animals.
Not to be given to kittens less than
12 weeks of age or cats with FIV
positive.
References [10, 12, 14, 30-32].
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Table 2 Antifungal drugs for topical treatment.
Antifungals Spectrum and Clinical applications Adverse effects
Polyenes: Natamycin and
Nystatin

Filamentous and dimorphic fungi and
yeasts.
Local treatment against ringworm.
Cows with Candida mastitis
Filamentous fungal keratitis and
Candida metritis in horses.
Malassezia infections of the outer ear
in dogs
Mild and locally irritating.
Azole antifungals
Imidazoles:
Miconazole,
Enilconazole,
Clotrimazole and
Ketoconazole
Triazoles: Fluconazole
Broad spectrum
Topical treatment dermatophytes
infection.
Local application in mycotic keratitis
and endometritis in horses.
Local treatment for yeast mastitis and
mycotic endometritis in cows.
Canine nasal Aspergillosis.
Well tolerated.
Allylamines: Naftifine,
and Terbinafine
Treatment of dermatophytic and
Malassezia infections.
Gastrointestinal and skin
reactions, but transient and
mild.
Other class: Amorolfine,
Butenafine, Ciclopirox,
Haloprogin, Tolnaftate and
Undecylenic acid
Treatment of dermatophytosis. N/A
References [30, 33-36].

association with human culture can be traced back for more than 2,000 years and its role in ethnopharmacology has
been recorded in many parts of the world [25-29].
Previous phytochemical and pharmacological studies have suggested that the amides and cinnamoyl derivatives found
in the Piperaceae species might be responsible for their antimicrobial properties [37, 38]. Employing a direct analysis
real time mass spectrometric technique, Bajpai and colleagues have additionally revealed terpenes and phenols as major
constituents in betel leaves [25]. Based on the promising antidermatophytic value of P. betle extract, a 10% P. betle
cream has been formulated without the addition of benzoyl peroxide [8, 39, 40]. The P. betle cream, slightly acidic and
dark green in color, still maintained a characteristic odor of betel leaves. This preparation exhibited the
antidermatophytic activity comparable to that of ketoconazole cream up to 96 h after incubation as shown in Table 5 [8,
41]. Moreover, the study of gel and ointment preparations containing 4% of P. betle extracts revealed a remarkable
antifungal effect similar to clotrimazole cream (1%), but significantly higher than tolnaftate cream (1%). These
preparations induced no rash or irritation either before or after UV irradiation in the guinea pig and rabbit toxicity tests
[42, 43]. Nonetheless, due to less stability and rapid loss of activity of the P. betle preparation, further modification of
the cream formulation has been suggested so that it would be more clinically applicable.
Similarly, the potent antidermatophytic effect of A. sativum extracts has been well recognized and extensively
studied. Uses of the preparations of the garlic extract as 0.4% ajoene cream and 0.6% ajoene gel have been
documented. Trials of these products on fungal infections of the skin, including ringworm, jock itch and athletes foot,
yielded satisfactory outcomes comparable to terbinafine [44-46]. On the other hand, although the extracts of
Rhinacanthus nasutus, and Senna alata exhibit strong antifungal activities, the formulations of their products still need
much further research and development [47, 48].
5. Trends in herbal modalities and potential applications of plant-derived antifungals
in veterinary medicine
The World Health Organizations Commission on Intellectual Property and Innovation in Public Health has
continuingly recognized the promise and role of natural products and traditional medicines as health aid for both
humans and animals, and not only in developing nations but also in developed countries [1]. The re-emerging interest
in ethnopharmacological research on natural products, particularly the medicinal plants, as evidenced by numerous
publications in recent decades has additionally signified the clinical potential of medicinal plants beyond traditional
medical practices. It has been anticipated that the drug discovery and development may not necessarily be confined to
new molecule entities, but the rationally designed, carefully standardized, synergistic traditional herbal formulations
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A. Mndez-Vilas (Ed.) ______________________________________________________________________________



Table 3 Plants extracts with strong antifungal activity*
Plant Main components Test organism(s)
Kaempferia galanga polyphenols
flavonoids
Saprolignea parasitica H2.
Blepharispermum subsessile desmethyl isoencecalin
5-hydroxy-6-acetyl-2-hydroxymethyl-
2-methyl chromene
Cryptococcus neoformans
Candida albicans

Salvia texana flavanones
diterpenes

Aspergillus fumigatus
Histoplasma capsulatum
Coccidioides immitis
Candida albicans
Nigella sativa terpenes dermatophyte fungi
Pinellia ternata pinelloside Aspergillus niger
Candida albicans
Piper regnellii phenylpropanoids
neolignans
dermatophyte fungi
Inula viscosa sesquiterpene - tayunin dermatophyte fungi
Candida albicans
Elaeodendron schlechteranum triterpenoids
sterols
Candida albicans
Acacia tortilis terpenes
avonoids
cyclitols
essential oils
Candida albicans
Balanites aegyptiaca unsaturated fatty acids
sterols
alkaloids
Candida albicans
Ajania fruticulosa guaianolides
sesquiterpenes
Candida albicans
Xanthosoma sagittifolium polyphenols
flavonoids
dermatophyte fungi
Schinus molle monoterpenes
sesquiterpenes
Candida albicans
Anacardium occidentale phenolic lipids
flavonoids
tannins
Cryptococcus neoformans
Curtisia dentata triterpenoids Cryptococcus neoformans
Sporothrix schenckii
Aspergillus fumigatus
Microsporum canis
Candida albicans
Inula racemosa sesquiterpenes Aspergillus flavus
Aspergillus niger
Geotrichum candidum
Candida tropicalis
Candida albicans
Melaleuca alternifolia terpenes
sesquiterpenes
Aspergillus spp.
dermatophyte fungi
Candida albicans
Polygonum acuminatum sesquiterpenes Cryptococcus neoformans
Aspergillus spp.
dermatophyte fungi
Candida albicans
Toddalia asiatica flindersine dermatophyte fungi
Candida albicans
Alpinia conchigera phenylpropanoids
diarylheptanoids
avonoids
dermatophyte fungi
Daucus carota sesquiterpenes
phenylpropanoids
eugenol
Cryptococcus neoformans
dermatophyte fungi
* IC
50
< 100 g/ml or MIC < 500 g/ml [7, 21].
References [52-80].

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Science against microbial pathogens: communicating current research and technological advances
A. Mndez-Vilas (Ed.) _______________________________________________________________________________



Table 4 Plant species cited for antidermatiphytic remedy in the South East Asian region.


Botanical taxon
(Botanical family)
Preparation Active constituents MIC








Allium sativum L.
(Alliaceae)



Ethanolic or
water
extracts of
bulbs


allicin
allyl sulfide
allyl disulfide
ajoene


1.5-6.3 g/ml








Piper betle L.
(Piperaceae)



Ethanolic
extracts of
leaves


amides
cinnamoyl derivatives
terpenes
phenols


230.0 g/ml







Rhinacanthus
nasutus L.
(Acanthaceae)
Ethanolic or
chloroform
extracts of
leaves and
root

rhinacanthin- A, B, C,
D, and N
oxymethylanthraquinone


26.5-106 g/ml









Senna alata
(Fabaceae)


Ethanolic or
water
extracts of
leaves and
bark
rhein
emodin, aloe-emodin
4,5-dihydroxy-1-
hydroxymethylanthrone
chrysophanol


125 g/ml

References [8, 25-29, 37, 38, 81-87].


Table 5 In vitro antidermatiphytic activity of 10% P. betle cream and ketoconazole cream.
Compound M. canis T. mentagrophyte



10% P. betle cream





Inhibition zone:
28.00 0.12 mm





Inhibition zone:
32.00 0.15 mm




Ketoconazole cream






Inhibition zone:
29.30 0.03 mm





Inhibition zone:
35.70 0.23 mm


Modified from [41].

and botanical drug products with robust scientific evidence can also be the attractive options [3, 49]. In addition, the
global market of botanical and plant -derived compounds is expected to increase from $ 19.5 billion in 2008 to $ 32.9
billion in 2013 [50]. Although the recently observed upward trend in herbal and other alternative medical modalities in
human health relies in part on the social, economic and philosophical reasons, the rationales underlying the similar trend
in veterinary practice have not been extensively investigated. Nonetheless, it is undeniable that the accumulated
scientific data supporting pharmacologic activity of plants and plant extracts have provided medical professionals the
better knowledge, so that the application of plant-derived compounds and herbal formulation can be employed with a
more specific purpose, better efficacy, and reduced risk of toxicity [4, 9].
In veterinary practice, the market for animal antifungals is estimated to reach $110.7 million by 2012 [51]. The
treatment regimens for animal mycoses, especially dermatophytosis, with currently available antifungals resulting in
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Science against microbial pathogens: communicating current research and technological advances
A. Mndez-Vilas (Ed.) ______________________________________________________________________________



favorable clinical outcomes do not come in a one size fits all and oftentimes it depends rather on how much drug to
use and for how long in a given animal species, not the choice of drug [11, 14, 15, 30]. The growing favor towards the
topical antifungal therapy undoubtedly further enhances the need for longer-term drug administration, i.e. several
months, in most cases. As a consequence, some ingredients such as benzoyl peroxide usually found in conventional
formulations could create more risks of observable adverse effects in the patients [39, 40]. The attempts in developing
the antifungal herbal formulation for topical uses would then provide the possible safer treatment of choice, but also
generate the niche in the animal antifungal market and a more affordable animal health aid [8, 41, 44-46]. The hair coat
of animals, however, can limit the use of creams, gels and ointments, particularly when animals try to remove topical
agents by licking. Formulation as shampoos and moisturizers to be used as sole therapy to resolve clinical signs or as
an adjunct and applied at the right dosing schedule could also be effective [11, 14, 15, 30].
Despite the vast potential and possibilities associated with plant products with antifungal properties, drawbacks in
application remain, particularly in compound stability and bioavailability. Seeking strategies to prolong the product
shelf-life as well as searching for new synergistic combinations in order to improve bioavailability would be of interest
and could play a significant role in drug development. Such stability and bioavailability enhancing activity may have
numerous advantages in drug development including reduction in dose, toxicity and treatment costs [3, 49].
6. Conclusion
As it becomes necessary to identify and develop novel antifungal compounds and the shift in favor of topical
application particularly in dermatophytosis treatment in companion animals, the potential role of plants/ plant extracts
as sources for new antimycotics has never been more apparent. The recent and renewed interest in medicinal plants
inspired by traditional wisdom and the development in information technology have facilitated an explosion in the
range and content of electronic information concerning antifungal medicinal plants and herbal formulations as a re-
emergent treatment of choice with comparable efficacy, but perhaps safer and more affordable. Despite the great
opportunity for research and development of herbal formulation with antifungal effect, the long path between the robust
scientific approval of such activity and the entree of a drug onto the market still exists.
Acknowledgements The author expresses her gratitude to the Ministry of University Affairs of the Royal Thai Government for
research grant. Dr. James A. Will of University of Wisconsin-Madison, School of Veterinary Medicine is acknowledged for his kind
help in proved reading this manuscript.
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A. Mndez-Vilas (Ed.) ______________________________________________________________________________



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Science against microbial pathogens: communicating current research and technological advances
A. Mndez-Vilas (Ed.) _______________________________________________________________________________



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1204 FORMATEX 2011
Science against microbial pathogens: communicating current research and technological advances
A. Mndez-Vilas (Ed.) ______________________________________________________________________________