mycoses
Diagnosis,Therapy and Prophylaxis of Fungal Diseases
Original article
Treatment of dermatophytosis by a new antifungal agent ‘apigenin’ Geeta Singh,1 Padma Kumar2 and Suresh Chandra Joshi2 1 Laboratory of Plant Tissue Culture and Secondary Metabolites, Department of Botany, University of Rajasthan, Rajasthan, India and 2Laboratory of Reproductive Toxicology Unit, Department of Zoology, University of Rajasthan, Rajasthan, India
Summary
Dermatophytes are the most common causative agents of cutaneous mycosis and remain a major public health problem in spite of the availability of an increasing number of antifungal drugs. It was, therefore considered necessary to pursue the screening of different extracts (compounds) of selected traditional medicinal plants reportedly having antidermatophyte potential. The aim of this study was to isolate and identify specific compound from the most active extract (free flavonoid) of stem of Terminalia chebula of the selected plants to treat dermatophytosis induced on experimental mice. Mice which were experimentally induced with Trichophyton mentagrophytes were grouped in six of five animals each. To treat the lesions on infected mice, two concentrations of isolated apigenin ointment, i.e. 2.5 mg g1 (Api I) and 5 mg g1 (Api II), and terbinafine (standard) of concentration 5 mg g1 were used. Complete recovery from the infection was recorded on 12th day of treatment for reference drug Terbinafine and Api II (5 mg g1) concentration of ointment, whereas Api I (2.5 mg g1) ointment showed complete cure on 16th day of treatment. Fungal burden was also calculated by culturing skin scraping from infected mice’s of different groups. Apigenin has shown potency as the infected animals recover completely by Api II comparable to the standard drug in 12th day. So Apigenin can be explored as an antifungal agent in the clinical treatment of dermatophytosis in future.
Key words: Dermatophytosis, Trichophyton mentagrophytes, apigenin, ointment, terbinafine, fungal burden.
Introduction Dermatomycoses are infections of the skin, hair and nail caused as a result of colonisation of the keratinised layers of the body. This colonisation is brought about by the organisms belonging to the three genera, namely Trichophyton, Microsporum and Epidermophyton.1,2 Infection may also be caused rarely by the members of the genus Candida and by non-dermatophyte moulds belonging to the genera Fusarium, Scopulariopsis and Aspergillus.3,4 Interestingly dermatophyte
Correspondence: G. Singh, Department of Botany, University of Rajasthan, Rajasthan, India. E-mail: [email protected] Submitted for publication 18 November 2013 Revised 21 February 2014 Accepted for publication 23 February 2014
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infections are predominant in both tropical and temperate climate, but the condition of tropical climate facilitates dermatophytes more as compared to temperate climate which is manifested by the number of patients of dermatophytoses being reported from tropical climates, e.g. India,5,6 and currently no race is totally free from dermatophytoses. T. mentagrophytes is a zoophilic dermatophyte of wild and domestic rodents which is occasionally transmitted to humans and animals by direct contact with an infected animal or asymptomatic carrier or with contaminated material (hair and scales) from the environment.7,8 The present line of treatment involves use of antifungal medication, topical such as tolnaftate, terbinafine hydrochloride and imidazoles such as ketoconazole, miconazole nitrate and clotrimazole. Fungal infections need long-term therapy involving several weeks. But the patient discontinues the application, due to cost factor, when the clinical manifestations
doi:10.1111/myc.12188
G. Singh et al.
subside resulting in the recurrence of the disease. As a result superficial fungal infections become chronic, causing enormous physical and psychological distress to the sufferers.9 This study prepared a novel herbal composition in an attempt to detect their chemical compositions and to demonstrate the antifungal activity of them. Flavonoids constitute one of the most characteristic classes of secondary metabolites. They are known to be synthesised by plants in response to microbial infection.10 They have been found to be effective antimicrobial substances against a wide array of microorganisms. Flavonoid compounds exhibit inhibitory effects against multiple viruses, bacteria and other microorganisms. Owing to the widespread ability of flavonoids to inhibit spore germination on of plant pathogens, they have been proposed for use against fungal pathogens of human.11,12 Among flavonoids rutin and quercetin were estimated from fruits of T. chebula by high-performance liquid chromatography (HPLC).13 Although several studies have been carried out regarding biochemical evaluation, but perusal of literature reveals that antidermatophyte activity of apigenin from stem of T. chebula has not been reported so far. Therefore, plants were selected after reviewing the literature where by the three plants, viz. Euphorbia hirta, Terminalia chebula and Withania somnifera were found to have medicinal importance specifically for skin diseases. Of the three plants selected, Withania somnifera and Euphorbia hirta were not showing as much activity as was shown by T. chebula. Hence, T. chebula was selected for in vivo study.
Materials and methods Flavonoid extraction
Different parts of selected plants were taken for flavonoids extraction following the well-established method.14 Each extract was dried in vacuo and stored at 4 °C in airtight vials for further use.
dermatophyte). Each extract was twofold serially diluted in 96-well microtitre plates, thereafter fungal suspension (inoculum size 106 CFU ml1) was added to each well and incubated at 27 2 °C for 7 days. The MIC values were taken as the lowest concentration of the extracts in the well of the microtitre plate that showed no turbidity after incubation. The turbidity of the wells in the microtitre plate was interpreted as visible growth of microorganisms. Here, turbidity is used to show the presence of fungal mycelium which can be seen by naked eyes and ‘No turbidity’ means the absence of fungal mycelium which can be not seen by naked eyes, which has to be further cultured to see the actual presence or absence of fungus. The minimum fungicidal concentration (MFC) was determined by subculturing the content of each well (Table 1). Thin-layer chromatography (TLC)
Selected extract (free flavonoids) from stem of T. chebula which showed excellent activity against T. mentagrophytes tested was dissolved in ethyl acetate and applied on silica gel-coated (0.2–0.3 mm) and -activated glass plates (20 9 20 cm) in an oven at 100 °C for 30 min along with the standard reference compound of apigenin 1 cm above the edge of the plates. These plates were developed in an organic solvent mixture of benzene, acetic acid and water (125 : 72 : 3), air dried and visualised under UV light. One spot (Rf 0.65) was observed which was further confirmed by spraying the plates with 5% ethanolic ferric chloride solution (Table 2, Fig. 1). A few other solvent systems (n-butanol, acetic acid and water, 4 : 1 : 5; n-butanol, water 1 : 1; n-butanol, acetic acid and water 6 : 1 : 2) were used, but in the present investigation the solvent system of benzene, acetic acid and water (125 : 72 : 3) gave excellent results.16 Rf value 0.65 obtained indicate the presence of apigenin in the free flavonoids of stem extract subjected to TLC. Preparative thin-layer chromatography (PTLC)
In vitro antidermatophyte activity
Test dermatophyte T. mentagrophytes (MTCC No. 7687) was procured from IMTECH, Chandigarh (India), and maintained on Sabouraud medium at 27 2 °C. Minimum inhibitory concentration (MIC) was determined for each extract by ‘Microbroth Dilution’ method.15 Stock solution (10 mg ml1) of each extract was prepared in acetone (inactive against test
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Preparative TLC of the free flavonods from stem of T. chebula was carried out on silica gel-coated and activated (0.4–0.5 mm thick) glass plates in the selected solvent system (B: A: W). Spot of Rf value 0.65 was marked in each plate and was collected and eluted with ethyl acetate. Elutes were pooled, completely dried and rechromatographed to test the purity of the isolated compound.
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Treatment of dermatophytosis
Table 1 Antidermatophytic activity of selected plants. Antidermatophytic activity Plants
Plant parts
Flavonoids
IZ
W. somnifera
Leaf
E1 E2 E1 E2 E1 E2 E1 E2 E1 E2 E1 E2 E1 E2 E1 E2 E1 E2 E1 E2 E1 E2 E1 E2
13.00 19.00 9.00 8.00 – – – – – – – – – – – – 20.75 – 41.50 31.00 21.25 – 18.83 15.25
Stem Root Fruits E. hirta
Leaf Stem Root Fruits
Terminalia chebula
Leaf Stem Stem bark Fruits
AI
0.250 0.500 0.289 0.289
0.750 0.500 1.000 0.750 0.250 0.250
0.288 0.422 0.200 0.177 – – – – – – – – – – – – 0.461 – 1.011 0.689 0.472 – 0.406 0.339
MIC/MFC
0.006 0.061 0.021 0.012
0.017 0.011 0.023 0.017 0.005 0.005
0.156 0.078 0.312 0.625 – – – – – – – – – – 0.078 – 0.039 0.039 0.039 – 0.078 0.156
0.156 0.156 1.25 0.625 – – – – – –– – – – – – – 0.156 – 0.039 0.078 0.078 – 0.156 0.312
E1, Free Flavonoid; E2, Bound Flavonoid.
Table 2 Characteristics of isolated flavonoid (apigenin) from stem of Terminalia chebula. Rf
FeCl3 spray
Compound isolated
Be. A. W
B. A. W
Forestal system
Under UV lamp
Ammonia fumes
Iodine vapours
Visible
UV
Apigenin
0.65
0.89
0.83
Flourescent blue
Bright yellowish green
Yellowish brown
Brown
Black
Infrared spectral study of eluted compound
The isolated compound was crystallised, weighed and subjected to melting point and infrared spectral studies on Perkins Elmer model 555 spectrophotometer in KRr pellets. High-performance liquid chromatography
A method HPLC with LC2010 CHT Auto Sampler (UVVIS detector) SHIMADZU was optimised for the separation and identification of isolated compound from free flavonoids of stem of T. chebula through PTLC. Mobile phase composition was screened to obtain chromatograms with good resolution within an acceptable time of analysis. Ten mmol l1 Ammonium formate in water–
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acetonitrile 50 : 50 v/v adjusted with formic acid, as solvent was chosen for the gradient elution (Table 3). Changes in the pH value of the mobile phase had a significant effect on the resolution of compounds, especially the phenolic acids. Formic acid, acetic acid, trifluoroacetic acid, ammonium acetate and ammonium formate are volatile and thus compatible with LC/MS system. Because acetic acid was found to have weak ion-pairing capacity, ammonium formate (10 mmol l1) was used to buffer the mobile phase at pH 4.0. The higher concentration of acid in mobile phase (lower pH values) ensures better sample separation, but shortens the HPLC column lifetime and affects ESI ionisation.17 325 nm was chosen as monitoring wavelength according to absorption maximum of analytes.
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the present investigation. Mice were first immunosuppressed by subcutaneous injection of 500 lg of estradiol valerate. Estradiol pretreatment is known to inhibit innate and acquired immune defences.19 After 3 days of immunosuppression, flanks of mice were shaved with an electric razor and the exposed area was lightly abraded with a sterile scalpel blade. Hundred microlitre of standard inoculum size (1 9 106 cfu ml1) was applied on the shaved site and was gently rubbed with the flat part of a sterile blade.20 Experimental design
Figure 1 Thin-layer chromatography (TLC). E, extract; A, apigenin.
Table 3 Chromatographic conditions for high-performance liquid chromatography. Wave length Flow rate Injection volume Column Column Temp. Mobile phase
325 nm 1 ml min1 20 µl Hypersil BDS C18 250 9 4.6 mm, 5 Micron Ambient 10 mmol l1 ammonium formate in water–acetonitrile 50 : 50 v/v, pH 4.0
Inoculum preparation
Trichophyton mentagrophytes (MTCC No. 7687) procured from IMTECH Chandigarh was selected for experimental induction of dermatophytic infection on mice. Cultures of T. mentagrophytes were subcultured on ‘Sabouraud Dextrose Agar’ media and incubated at 27 2 °C for 7 days. Thereafter, a suspension of conidia/spores was prepared by washing the surface of the plate with sterile distilled water. The suspension was adjusted to bring the inoculum size to 1 9 106 conidia per ml, and conidial viability was checked by subcultured different inoculums sizes.18 Infection on mice model
Male Swiss albino mice (Mus musculus) of approximately 8 weeks old and 25–30 g weight were used for
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Six experimental groups (A, B, C, D, E and F) were made containing five animals each. Animals of groups B, C, D, E and F were immunosuppressed before inoculating with T. mentagrophytes, whereas animals of group A were inoculated without immunosuppression. Groups A and B were not treated to see the lesion severity, time course of disease and considered as control groups. The lesions were visually examined daily throughout the experiment to determine the severity and recovery of lesion. On seventh day of infection skin lesions were scored as follows: 0 (absence of lesion), 1 (appearance of erythema at infected site or new hair growth on the bald exposed area), 2 (moderate erythema spreading over entire infected site), 3 (intense erythema with abrasions, swelling and scaling) and 4 (severely erythematous lesion with crusting spreading over the entire exposed area). Isolated and identified compound (apigenin) from most active extract (free flavonoids of stem of T. chebula) by considering lowest MIC/MFC values against test dermatophyte was used for preparing test ointment for preclinical animal studies. Ointment for topical treatment was prepared by mixing 25 and 50 mg of test extract in 10 g inert petroleum jelly to make 2.5 and 5 mg g1 concentrations of test extract. Terbinafine (5 mg) was used as reference drug. On eighth day of infection all animals of groups C, D and E were topically treated by applying 0.2 g of the reference drug (5 mg g1), test ointment 2.5 and 5 mg g1, respectively, once in a day at the infected site. Treatment continued until complete recovery was observed. To find out any effect of petroleum jelly on lesion, 0.2 g of petroleum jelly was applied once in a day at the infected site of the animals of group F. Infected site of each animal was carefully examined daily throughout the treatment period to determine per cent recovery of infected site, and treatment scores were given to animals as follows: 0 (not cured), 1 (25% cured), 2 (50% cured), 3 (75% cured) and 4 (100% cured). The
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Treatment of dermatophytosis
average treatment scores for an experimental group were determined by dividing the treatment score by number of animals. Complete cure defined as mycological and clinical cure or improvement which was determined by fungal burden. Fungal burden was calculated by culturing infected skin tissues. Five skin samples (skin scrapings, hair stubs) were collected on 15th day of infection (control groups A and B) and 20th day of treatment (treated groups C, D and E) from the infected site of each animal of different groups. Each sample was cultured on SDA medium at 27 2 °C for 7 days and samples showed that fungal growth was considered as culture positive. Fungal burden was assessed with scores ranging from 0 to 5 based on the number of culture positive skin samples of each animal. The average fungal burden of a group was determined by dividing the sum of the burden scores by number of animals.21
Results
BDS C18 250 9 4.6 mm, 5 micron and detecting at 325 nm. Figure 3a represented the chronograms of the standard apigenin and diluent with retention times 4.030 and 2.582 min respectively. While the Fig. 3b represented the chromatograms of the sample (isolated compound by PTLC) and diluent with retention time 4.032 and 2.578, respectively, comparison of the chromatograms of the sample with that of the standard indicated the presence of apigenin in the extract of free flavonoid of stem of T. chebula, depending upon the comparison of the retention times of the standard of apigenin and the compound present in the sample (Tables 4 and 5). Content and solubility of apigenin:
4.5 mg g1 apigenin was eluted from 25 mg g1 free flavonoids crude extract of stem (100 gm) of T. chebula. Apigenin itself is almost insoluble in water, moderately soluble in hot alcohol and it is soluble in dilute sodium (or potassium).
Identification of apigenin by TLC
Single spot was observed in TLC of the free flavonoids from stem of T. chebula (most potent extract with antidermatophyte activity in vitro) with Rf 0.65, 0.89 and 0.83 in Be: A: W, B: A: W and forestal solvent systems, respectively, and was identified as apigenin (UV flourescent – blue; ammonia – bright yellow; iodine vapours – yellowish brown; 5% FeCl3 solution – brown in colour; melting point – 340 °C) (Table 2, Fig. 1). The characteristic IR spectral peaks were also found to be superimposable with the reference compound (Fig. 2). MS analysis and identification by HPLC
The separation and identification of the isolated compound in PTLC were carried out by HPLC with Hpersil
Antidermatophyte activity
In vitro Antifungal Activity. It is evident from the results obtained (Table 1) that of the 24 extracts of different parts of selected three plants, viz. W. somnifera, E. hirta and T. chebula, ten extracts showed their antidermatophyte activity against test dermatophyte (T. mentagrophytes). Three extracts were found to be fungicidal where same values of MIC and MFC were recorded; whereas seven extracts were recorded as fungistatic (MFC values were higher than their MIC values). For animal studies, most active plant extract, free flavonoids from stem of T. chebula which showed excellent inhibition zone (IZ 41.50 0.500 mm) with the lowest MIC and MFC values 0.039 mg/ml, was selected. Isolated apigenin was also screened against T.
Figure 2 Infrared spectra of isolated com-
pound ‘Apigenin’.
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(a)
(b)
Figure 3 (a) High-performance liquid
chromatography (HPLC) chromatogram of standard (apigenin), and (b) HPLC chromatogram of isolated apigenin. Table 4 Peak table for standard apigenin. 0
Peak
Ret. time
Area
Height
Area %
Height %
K
1 2 Total
2.582 4.030
40577 916739 957316
2188 134048 136236
4.239 95.761 100.000
1.606 98.394 100.000
0.000 0.561
Table 5 Peak table for isolated compound from free flavonoids of stem of Terminalia chebula. Peak
Ret. time
Area
Height
Area %
Height %
K0
1 2 Total
2.578 4.032
40962 916845 957807
2070 133941 136010
4.277 90.723 100.000
1.522 98.478 100.000
0.000 0.564
mentagrophytes which showed inhibition zone of IZ 38.50 0.250 mm (Fig. 4). Induction of dermatophyte infection on mice
The severity of skin lesions in all groups of animals was assessed every day after inoculation of test
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dermatophyte. Time course of changes in mean lesion score is shown in Table 6. Infection of mice model resulted lesion in 100% of the animals. First signs of infection were observed on second and third day of inoculation and lesion reached at its maximum at the infected site on eighth and tenth day of inoculation in all animals of groups B (immunosuppressed infected animal group) and A (infected animal group without immunosuppression) respectively. Lesion scores started decreasing as a result of spontaneous healing after 20 and 25 days of inoculation, whereas complete spontaneous recovery was observed on 26th and 32nd day of inoculation in groups A and B respectively. Treatment of induced dermatophytosis on mice
Recovery of skin lesions of animals of treated groups (C, D and E) was assessed by giving them treatment scores depending upon per cent resolution of infected site (Table 7). First signs of recovery was seen on second day of treatment in animals of groups C (group
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Treatment of dermatophytosis
treated with terbinafine 5 mg g1 ointment) and E (test extract ointment 5 mg g1), whereas group D (test extract ointment 2.5 mg g1) showed recovery signs on third day of treatment (Figs 5, 6 and 7). On 12th day of treatment 100% animals of groups C and E were completely cured and new hairs were observed on the bald exposed area of infected site. In case of group D, 100% recovery (i.e. treatment score 4) of dermatophyte infection was observed on 16th day of treatment in all animals of the group. Course of infection in animals of group F (treated with petroleum jelly) was found similar as in untreated groups (A and B) and lesion became more severe with time indicating ineffectiveness of petroleum jelly. Skin irritation was observed in 45% animals of the group treated with terbinafine, whereas such side effects were not observed in both the concentrations (2.5 and 5 mg g1) of test ointment. The fungal burden (FB) of infected loci in animals of untreated groups (A and B) and group F (treated with petroleum jelly) was recorded relatively high, i.e. 3.30 0.510, 3.60 0.330 and 3.80 0.240 respectively. Fungal burden was calculated 0 for groups C, D and E on 20th day of treatment, thus proved complete eradication of the fungal mycelia/conidia from the infected loci (Table 8).
(a)
(b)
Figure 4 (a) Inhibition zone of isolated apigenin against T. ment-
agrophyte. (b) Inhibition zone of free Flavonoids of stem against T. mentagrophytes.
Table 7 Treatment scores of infected animal groups. Average treatment score1
Table 6 Time course of changes in lesion in mice model of dermatophytosis. Animal group
Days
Normal infected Group (A) Average lesion score
Immunosuppressed infected Group (B) Average lesion score
1 2 3 4 5 6 7 8 9 10
0 0 0.6 0.8 1.8 2.4 2.8 3.4 3.8 4
0 0.8 2.2 2.6 2.8 3.2 3.8 4 4 4
Average lesion score: Sum of lesion score of the group/total number of animals in the group. 0, Absence of lesion; 1, Appearance of erythema at infected site; 2, Moderate erythema spreading over entire infected site; 3, Intense erythema with abrasions, swelling and scaling; 4, Severely erythematous lesion with crusting spreading over the entire exposed area.
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Days
Group C
Group D
Group E
Group F
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
0 0.2 0.8 1.2 1.8 2.2 2.6 3.0 3.4 3.6 3.8 4.0 4.0 4.0 4.0 4.0
0 0 0.2 0.6 0.8 1.2 1.6 1.8 2.0 2.4 2.8 3.0 3.2 3.6 3.8 4.0
0 0.2 0.6 1.0 1.6 2.0 2.4 2.8 3.0 3.2 3.6 4.0 4.0 4.0 4.0 4.0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Group Group Group Group
C, treated with standard drug terbinafine (5 mg g1); D, treated with test ointment Api I (apigenin 2.5 mg g1); E, treated with test ointment Api II (apigenin 5 mg g1); F, treated with petroleum jelly (placebo treated).
1
Average treatment score: Sum of treatment score of each animal/total number of animals in group; (0): not cured, (1): 25% cured, (2): 50% cured, (3): 75% cured, (4): 100% cured.
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Lesion on 1st day of treatment
Lesion on 9th day of treatment
Lesion on 3rd day of treatment
Lesion on 6th day of treatment
Lesion on 12th day of treatment
Figure 5 Recovery of skin lesion in animal group of C (treated with standard drug terbinafine).
Lesion on 1st day of treatment
Lesion on 12th day of treatment
Lesion on 4th day of treatment
Lesion on 8th day of treatment
Lesion on 16th day of treatment
Figure 6 Recovery of skin lesion in animal group of D (treated with test ointment Api I).
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Treatment of dermatophytosis
Lesion on 1st day of treatment
Lesion on 9th day of treatment
Lesion on 3rd day of treatment
Lesion on 6th day of treatment
Lesion on 12th day of treatment
Figure 7 Recovery of skin lesion in animal group of E (treated with test ointment Api II).
Table 8 Determination of fungal burden (FB) of animal groups.
Animal group
Days
Score of FB FB SEM
A B C D E F
151 151 202 202 202 151
3.30 0.510 3.60 0.330 0 0 0 3.80 0.240
SEM, standard error mean; A, animal group inoculated without immunosuppression; B, animal group immunosuppressed before inoculation; C, animal group treated with terbinafine cream (5 mg); D, animal group treated with test ointment (5 mg); E, animal group treated with test ointment (10 mg); F, animal group treated with petroleum jelly. 1 Days after inoculation. 2
Days after treatment.
Discussion Results indicate antidermatophyte potency of both the concentrations of apigenin (Api I and Api II) on the infection, as results were comparable with reference drug terbinafine. In addition, fungal burden results also showed excellent antidermatophyte activity of test ointments (Api I and Api II) as none of the epidermal flank was found culture positive, thus giving
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FB score 0 and depicted complete cure of dermatophytosis. Results revealed that the cure pattern of placebo group (petroleum jelly–treated) mice and that of negative control (with no application) was same which showed that jelly had no effect to cure dermatophytoses. ‘In vivo’ fungicidal activity of free flavonoids (from stem of T. chebula) against T. mentagrophytes can be assigned to apigenin because thin-layer chromatography results showed that free flavonoids (from stem of T. chebula) considered of only one compound as single spot was observed in all TLC plates and later this compound was identified as apigenin. Thus, it can be concluded that apigenin isolated from stem of T. chebula was found with significant antidermatophyte activity in ‘in vivo’ and ‘in vitro’ studies. Terbinafine (which was taken as a reference drug in this study) is a potent fungicidal agent against dermatophytes and is often preferred over other dermatophytes have also reported.22,23 Trichophyton spp may develop resistance against terbinafine in future, which indicates limited life span of the drug. Hence, continuous search for therapeutic alternatives should always be encouraged.24 In the present investigation, apigenin gave encouraging results in the tropical treatment of dermatophytosis
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in mice. As like other topical antidermatophyte agents, apigenin when applied topically on the skin, penetrate the stratum corneum and interfered with the growth of fungi, leading to their structural disintegration which make them unable to grow or divide. Structural disintegration is possibly known to be due to interference of cytoplasmic membrane synthesis and by blocking sterol biosynthesis.25 Although systemic therapy is often suggested for Tinea/dermatophytosis when infection condition is very serious and lesions involve large area which could not be cured by topical treatment, but systemic therapy besides being expensive may cause certain side effects as drugs taken orally are bound to affect physiology of the host. Thus, lower cost of plant-based topical agent’s makes therapy a favourable choice in the management of superficial dermatophyte infections in its initial stage which prevent the serious stage of disease26 Present investigation suggests clinical trials of apigenin for dermatophytosis and advocates the use of T. chebula for isolating apigenin which is established as an antidermatophyte compound in this study and also to exploit it as an alternative plant-based drug for dermatophytosis in future.
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Acknowledgments Authors are thankful to the ‘Head’ Department of Botany, University of Rajasthan for providing all necessary facilities and to UGC for providing financial assistance for this work.
Conflict of interest None.
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Emmons CW, Bindford CH, Utz JP, Kwon-Chung KL. Dermatophytoses. Chapter 10. In: Emmons CW, Bindford CH, Utz JP, Kwon-Chung KL (eds), Medical Mycology, 3rd edn. Philadelphia: Lea & Febiger, 1977; 117–67. Gurgel LA, Sidrim JJ, Martins DT, Cechinel Filho V, Rao VS. In vitro antifungal activity of dragon’s blood from Croton urucurana against dermatophytes. J Ethnopharmacol, 2005; 97: 409–12. Pinto E, Pina-Vaz C, Salgueiro L et al. Antifungal activity of the essential oil of Thymus pulegioides on Candida, Aspergillus and dermatophyte species. J Med Microbiol 2006; 55: 1367–73. Naveed AM, Naeem R, Nasiruddin. Nondermatophyte moulds and yeasts as causative agents in onychomycosis. J Pak Assoc Dermatol 2009; 19: 74–8.
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Rippon JW. The Pathogenic Fungi and Pathogenic Actionmycetes, 3rd edn. Philadelphia: WB Saunders, 1988. Rao A. Mycotic diseases in India – a critical review. Bulletin Sch Trop Med Calcutta 1959; 13–22. Kristensen S, Viggo Krogh H. A study of skin diseases in dogs and cats. Nord Vet Med 1981; 33: 134–40. Hajsig M, Nagliæ T, Hajsig D et al. Dermatopytoses in rodents. IV. Trichophyton mentagrophytes transmission from ringworm rabbits to swine. Vet Arhiv 1983; 53: 251–7. Weinstein A, Berman B. Topical treatment of common superficial Tinea infections. Amer Family Physician Rev 2002; 65: 2095–103. Dixon RA, Dey PM, Lamb CJ. Phytoalexins: enzymology and molecular biology. Adv Enzymol 1983; 55: 1–69. Kumar A, Lakshman K, Jayaveera K et al. Estimation of rutin and quercetin from Terminalia chebula by HPLC. Internet J Aesthetic Antiaging Med 2009; 2: p3. Harborne JB, Williams CA. Advances in flavonoid research since 1992. Phytochemistry 2000; 55: 481–504. Cushnie TPT, Lamb AJ. Antimicrobial activity of flavonoids. Int J Antimicrob Agents 2005; 26: 343–56. Subramanian SS, Nagarjan S. Flavonoids of the seeds of Crotolaria retusa and Crotolaria striata. Curr Sci 1969; 38: 65. Basri DF, Fan SH. The potential of aqueous and acetone extracts of galls of Quercus infectoria as antibacterial agents. Indian J Pharmacol 2005; 37: 26–29. Wong E, Francis CM. Flavonoids in genotypes of Trifolium subterraneum. I. The normal flavonoids pattern of the geraldton variety. Phytochemistry 1968; 7: 2123–9. Plazonic A, Bucar F, Males Z et al. Identification and quantification of flavonoids and phenolic acids in burr parsley (Caucalis platycarpos L.), using high-performance liquid chromatography with diode array detection and electrospray ionization mass spectrometry. Molecules 2009; 14: 2466–90. Hay RJ, Raquel AA, Collins JC. Experimental dermatophytosis: the clinical and histopathologic features of a mouse model using Trichophyton quinckeanum (mouse favus). J Invest Dermatol 1983; 81: 270– 4. Carlsten H, Holmdahl R, Tarkowski A. Analysis of the genetic encoding of oestradiol suppression of delayed type hypersensitivity in (NZB 9 NZW) F1 mice. Immunology 1991; 73: 186–90. Odds F, Ausma J, Gerven FV et al. In vitro and in vivo activities of the novel azole antifungal agent R 12 6638. Antimicrob Agents Chemother 2004; 48: 388–91. Uchida K, Tanaka T, Yamaguchi H. Achievement of complete mycological cure by topical antifungal agent NND-502 in guinea pig model of tinea pedis. Microbiol Immunol 2003; 47: 143–6. Favre B, Ghannoum MA, Ryder NS. Biochemical characterization of terbinafine resistant Trichophyton rubrum isolates. Med Mycol 2004; 42: 525–9. Mukharjee PK, Leidich SD, Isham N et al. Clinical Trichophyton rubrum strain exhibiting primary resistance to terbinafine. Antimicrob Agents Chemother 2004; 47: 82–86. Osborne CS, Leitner I, Hofbauer B et al. Biological, biochemical and characterization of new clinical Trichophyton rubrum isolates resistant to terbinafine. Antimicrobial Agents Chemother 2006; 50: 2234–6. Ryder NS. The mechanism of action of terbinafine. Clin Exp Dermatol 1989; 14: 98–100. Huang DB, Ostrosky-Zeichner L, Wu JJ. Therapy of common superficial fungal infections. Dermatol Ther 2004; 2: 517–22.
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Diagnosis,Therapy and Prophylaxis of Fungal Diseases
Original article
Treatment of dermatophytosis by a new antifungal agent ‘apigenin’ Geeta Singh,1 Padma Kumar2 and Suresh Chandra Joshi2 1 Laboratory of Plant Tissue Culture and Secondary Metabolites, Department of Botany, University of Rajasthan, Rajasthan, India and 2Laboratory of Reproductive Toxicology Unit, Department of Zoology, University of Rajasthan, Rajasthan, India
Summary
Dermatophytes are the most common causative agents of cutaneous mycosis and remain a major public health problem in spite of the availability of an increasing number of antifungal drugs. It was, therefore considered necessary to pursue the screening of different extracts (compounds) of selected traditional medicinal plants reportedly having antidermatophyte potential. The aim of this study was to isolate and identify specific compound from the most active extract (free flavonoid) of stem of Terminalia chebula of the selected plants to treat dermatophytosis induced on experimental mice. Mice which were experimentally induced with Trichophyton mentagrophytes were grouped in six of five animals each. To treat the lesions on infected mice, two concentrations of isolated apigenin ointment, i.e. 2.5 mg g1 (Api I) and 5 mg g1 (Api II), and terbinafine (standard) of concentration 5 mg g1 were used. Complete recovery from the infection was recorded on 12th day of treatment for reference drug Terbinafine and Api II (5 mg g1) concentration of ointment, whereas Api I (2.5 mg g1) ointment showed complete cure on 16th day of treatment. Fungal burden was also calculated by culturing skin scraping from infected mice’s of different groups. Apigenin has shown potency as the infected animals recover completely by Api II comparable to the standard drug in 12th day. So Apigenin can be explored as an antifungal agent in the clinical treatment of dermatophytosis in future.
Key words: Dermatophytosis, Trichophyton mentagrophytes, apigenin, ointment, terbinafine, fungal burden.
Introduction Dermatomycoses are infections of the skin, hair and nail caused as a result of colonisation of the keratinised layers of the body. This colonisation is brought about by the organisms belonging to the three genera, namely Trichophyton, Microsporum and Epidermophyton.1,2 Infection may also be caused rarely by the members of the genus Candida and by non-dermatophyte moulds belonging to the genera Fusarium, Scopulariopsis and Aspergillus.3,4 Interestingly dermatophyte
Correspondence: G. Singh, Department of Botany, University of Rajasthan, Rajasthan, India. E-mail: [email protected] Submitted for publication 18 November 2013 Revised 21 February 2014 Accepted for publication 23 February 2014
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infections are predominant in both tropical and temperate climate, but the condition of tropical climate facilitates dermatophytes more as compared to temperate climate which is manifested by the number of patients of dermatophytoses being reported from tropical climates, e.g. India,5,6 and currently no race is totally free from dermatophytoses. T. mentagrophytes is a zoophilic dermatophyte of wild and domestic rodents which is occasionally transmitted to humans and animals by direct contact with an infected animal or asymptomatic carrier or with contaminated material (hair and scales) from the environment.7,8 The present line of treatment involves use of antifungal medication, topical such as tolnaftate, terbinafine hydrochloride and imidazoles such as ketoconazole, miconazole nitrate and clotrimazole. Fungal infections need long-term therapy involving several weeks. But the patient discontinues the application, due to cost factor, when the clinical manifestations
doi:10.1111/myc.12188
G. Singh et al.
subside resulting in the recurrence of the disease. As a result superficial fungal infections become chronic, causing enormous physical and psychological distress to the sufferers.9 This study prepared a novel herbal composition in an attempt to detect their chemical compositions and to demonstrate the antifungal activity of them. Flavonoids constitute one of the most characteristic classes of secondary metabolites. They are known to be synthesised by plants in response to microbial infection.10 They have been found to be effective antimicrobial substances against a wide array of microorganisms. Flavonoid compounds exhibit inhibitory effects against multiple viruses, bacteria and other microorganisms. Owing to the widespread ability of flavonoids to inhibit spore germination on of plant pathogens, they have been proposed for use against fungal pathogens of human.11,12 Among flavonoids rutin and quercetin were estimated from fruits of T. chebula by high-performance liquid chromatography (HPLC).13 Although several studies have been carried out regarding biochemical evaluation, but perusal of literature reveals that antidermatophyte activity of apigenin from stem of T. chebula has not been reported so far. Therefore, plants were selected after reviewing the literature where by the three plants, viz. Euphorbia hirta, Terminalia chebula and Withania somnifera were found to have medicinal importance specifically for skin diseases. Of the three plants selected, Withania somnifera and Euphorbia hirta were not showing as much activity as was shown by T. chebula. Hence, T. chebula was selected for in vivo study.
Materials and methods Flavonoid extraction
Different parts of selected plants were taken for flavonoids extraction following the well-established method.14 Each extract was dried in vacuo and stored at 4 °C in airtight vials for further use.
dermatophyte). Each extract was twofold serially diluted in 96-well microtitre plates, thereafter fungal suspension (inoculum size 106 CFU ml1) was added to each well and incubated at 27 2 °C for 7 days. The MIC values were taken as the lowest concentration of the extracts in the well of the microtitre plate that showed no turbidity after incubation. The turbidity of the wells in the microtitre plate was interpreted as visible growth of microorganisms. Here, turbidity is used to show the presence of fungal mycelium which can be seen by naked eyes and ‘No turbidity’ means the absence of fungal mycelium which can be not seen by naked eyes, which has to be further cultured to see the actual presence or absence of fungus. The minimum fungicidal concentration (MFC) was determined by subculturing the content of each well (Table 1). Thin-layer chromatography (TLC)
Selected extract (free flavonoids) from stem of T. chebula which showed excellent activity against T. mentagrophytes tested was dissolved in ethyl acetate and applied on silica gel-coated (0.2–0.3 mm) and -activated glass plates (20 9 20 cm) in an oven at 100 °C for 30 min along with the standard reference compound of apigenin 1 cm above the edge of the plates. These plates were developed in an organic solvent mixture of benzene, acetic acid and water (125 : 72 : 3), air dried and visualised under UV light. One spot (Rf 0.65) was observed which was further confirmed by spraying the plates with 5% ethanolic ferric chloride solution (Table 2, Fig. 1). A few other solvent systems (n-butanol, acetic acid and water, 4 : 1 : 5; n-butanol, water 1 : 1; n-butanol, acetic acid and water 6 : 1 : 2) were used, but in the present investigation the solvent system of benzene, acetic acid and water (125 : 72 : 3) gave excellent results.16 Rf value 0.65 obtained indicate the presence of apigenin in the free flavonoids of stem extract subjected to TLC. Preparative thin-layer chromatography (PTLC)
In vitro antidermatophyte activity
Test dermatophyte T. mentagrophytes (MTCC No. 7687) was procured from IMTECH, Chandigarh (India), and maintained on Sabouraud medium at 27 2 °C. Minimum inhibitory concentration (MIC) was determined for each extract by ‘Microbroth Dilution’ method.15 Stock solution (10 mg ml1) of each extract was prepared in acetone (inactive against test
2
Preparative TLC of the free flavonods from stem of T. chebula was carried out on silica gel-coated and activated (0.4–0.5 mm thick) glass plates in the selected solvent system (B: A: W). Spot of Rf value 0.65 was marked in each plate and was collected and eluted with ethyl acetate. Elutes were pooled, completely dried and rechromatographed to test the purity of the isolated compound.
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Treatment of dermatophytosis
Table 1 Antidermatophytic activity of selected plants. Antidermatophytic activity Plants
Plant parts
Flavonoids
IZ
W. somnifera
Leaf
E1 E2 E1 E2 E1 E2 E1 E2 E1 E2 E1 E2 E1 E2 E1 E2 E1 E2 E1 E2 E1 E2 E1 E2
13.00 19.00 9.00 8.00 – – – – – – – – – – – – 20.75 – 41.50 31.00 21.25 – 18.83 15.25
Stem Root Fruits E. hirta
Leaf Stem Root Fruits
Terminalia chebula
Leaf Stem Stem bark Fruits
AI
0.250 0.500 0.289 0.289
0.750 0.500 1.000 0.750 0.250 0.250
0.288 0.422 0.200 0.177 – – – – – – – – – – – – 0.461 – 1.011 0.689 0.472 – 0.406 0.339
MIC/MFC
0.006 0.061 0.021 0.012
0.017 0.011 0.023 0.017 0.005 0.005
0.156 0.078 0.312 0.625 – – – – – – – – – – 0.078 – 0.039 0.039 0.039 – 0.078 0.156
0.156 0.156 1.25 0.625 – – – – – –– – – – – – – 0.156 – 0.039 0.078 0.078 – 0.156 0.312
E1, Free Flavonoid; E2, Bound Flavonoid.
Table 2 Characteristics of isolated flavonoid (apigenin) from stem of Terminalia chebula. Rf
FeCl3 spray
Compound isolated
Be. A. W
B. A. W
Forestal system
Under UV lamp
Ammonia fumes
Iodine vapours
Visible
UV
Apigenin
0.65
0.89
0.83
Flourescent blue
Bright yellowish green
Yellowish brown
Brown
Black
Infrared spectral study of eluted compound
The isolated compound was crystallised, weighed and subjected to melting point and infrared spectral studies on Perkins Elmer model 555 spectrophotometer in KRr pellets. High-performance liquid chromatography
A method HPLC with LC2010 CHT Auto Sampler (UVVIS detector) SHIMADZU was optimised for the separation and identification of isolated compound from free flavonoids of stem of T. chebula through PTLC. Mobile phase composition was screened to obtain chromatograms with good resolution within an acceptable time of analysis. Ten mmol l1 Ammonium formate in water–
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acetonitrile 50 : 50 v/v adjusted with formic acid, as solvent was chosen for the gradient elution (Table 3). Changes in the pH value of the mobile phase had a significant effect on the resolution of compounds, especially the phenolic acids. Formic acid, acetic acid, trifluoroacetic acid, ammonium acetate and ammonium formate are volatile and thus compatible with LC/MS system. Because acetic acid was found to have weak ion-pairing capacity, ammonium formate (10 mmol l1) was used to buffer the mobile phase at pH 4.0. The higher concentration of acid in mobile phase (lower pH values) ensures better sample separation, but shortens the HPLC column lifetime and affects ESI ionisation.17 325 nm was chosen as monitoring wavelength according to absorption maximum of analytes.
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the present investigation. Mice were first immunosuppressed by subcutaneous injection of 500 lg of estradiol valerate. Estradiol pretreatment is known to inhibit innate and acquired immune defences.19 After 3 days of immunosuppression, flanks of mice were shaved with an electric razor and the exposed area was lightly abraded with a sterile scalpel blade. Hundred microlitre of standard inoculum size (1 9 106 cfu ml1) was applied on the shaved site and was gently rubbed with the flat part of a sterile blade.20 Experimental design
Figure 1 Thin-layer chromatography (TLC). E, extract; A, apigenin.
Table 3 Chromatographic conditions for high-performance liquid chromatography. Wave length Flow rate Injection volume Column Column Temp. Mobile phase
325 nm 1 ml min1 20 µl Hypersil BDS C18 250 9 4.6 mm, 5 Micron Ambient 10 mmol l1 ammonium formate in water–acetonitrile 50 : 50 v/v, pH 4.0
Inoculum preparation
Trichophyton mentagrophytes (MTCC No. 7687) procured from IMTECH Chandigarh was selected for experimental induction of dermatophytic infection on mice. Cultures of T. mentagrophytes were subcultured on ‘Sabouraud Dextrose Agar’ media and incubated at 27 2 °C for 7 days. Thereafter, a suspension of conidia/spores was prepared by washing the surface of the plate with sterile distilled water. The suspension was adjusted to bring the inoculum size to 1 9 106 conidia per ml, and conidial viability was checked by subcultured different inoculums sizes.18 Infection on mice model
Male Swiss albino mice (Mus musculus) of approximately 8 weeks old and 25–30 g weight were used for
4
Six experimental groups (A, B, C, D, E and F) were made containing five animals each. Animals of groups B, C, D, E and F were immunosuppressed before inoculating with T. mentagrophytes, whereas animals of group A were inoculated without immunosuppression. Groups A and B were not treated to see the lesion severity, time course of disease and considered as control groups. The lesions were visually examined daily throughout the experiment to determine the severity and recovery of lesion. On seventh day of infection skin lesions were scored as follows: 0 (absence of lesion), 1 (appearance of erythema at infected site or new hair growth on the bald exposed area), 2 (moderate erythema spreading over entire infected site), 3 (intense erythema with abrasions, swelling and scaling) and 4 (severely erythematous lesion with crusting spreading over the entire exposed area). Isolated and identified compound (apigenin) from most active extract (free flavonoids of stem of T. chebula) by considering lowest MIC/MFC values against test dermatophyte was used for preparing test ointment for preclinical animal studies. Ointment for topical treatment was prepared by mixing 25 and 50 mg of test extract in 10 g inert petroleum jelly to make 2.5 and 5 mg g1 concentrations of test extract. Terbinafine (5 mg) was used as reference drug. On eighth day of infection all animals of groups C, D and E were topically treated by applying 0.2 g of the reference drug (5 mg g1), test ointment 2.5 and 5 mg g1, respectively, once in a day at the infected site. Treatment continued until complete recovery was observed. To find out any effect of petroleum jelly on lesion, 0.2 g of petroleum jelly was applied once in a day at the infected site of the animals of group F. Infected site of each animal was carefully examined daily throughout the treatment period to determine per cent recovery of infected site, and treatment scores were given to animals as follows: 0 (not cured), 1 (25% cured), 2 (50% cured), 3 (75% cured) and 4 (100% cured). The
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Treatment of dermatophytosis
average treatment scores for an experimental group were determined by dividing the treatment score by number of animals. Complete cure defined as mycological and clinical cure or improvement which was determined by fungal burden. Fungal burden was calculated by culturing infected skin tissues. Five skin samples (skin scrapings, hair stubs) were collected on 15th day of infection (control groups A and B) and 20th day of treatment (treated groups C, D and E) from the infected site of each animal of different groups. Each sample was cultured on SDA medium at 27 2 °C for 7 days and samples showed that fungal growth was considered as culture positive. Fungal burden was assessed with scores ranging from 0 to 5 based on the number of culture positive skin samples of each animal. The average fungal burden of a group was determined by dividing the sum of the burden scores by number of animals.21
Results
BDS C18 250 9 4.6 mm, 5 micron and detecting at 325 nm. Figure 3a represented the chronograms of the standard apigenin and diluent with retention times 4.030 and 2.582 min respectively. While the Fig. 3b represented the chromatograms of the sample (isolated compound by PTLC) and diluent with retention time 4.032 and 2.578, respectively, comparison of the chromatograms of the sample with that of the standard indicated the presence of apigenin in the extract of free flavonoid of stem of T. chebula, depending upon the comparison of the retention times of the standard of apigenin and the compound present in the sample (Tables 4 and 5). Content and solubility of apigenin:
4.5 mg g1 apigenin was eluted from 25 mg g1 free flavonoids crude extract of stem (100 gm) of T. chebula. Apigenin itself is almost insoluble in water, moderately soluble in hot alcohol and it is soluble in dilute sodium (or potassium).
Identification of apigenin by TLC
Single spot was observed in TLC of the free flavonoids from stem of T. chebula (most potent extract with antidermatophyte activity in vitro) with Rf 0.65, 0.89 and 0.83 in Be: A: W, B: A: W and forestal solvent systems, respectively, and was identified as apigenin (UV flourescent – blue; ammonia – bright yellow; iodine vapours – yellowish brown; 5% FeCl3 solution – brown in colour; melting point – 340 °C) (Table 2, Fig. 1). The characteristic IR spectral peaks were also found to be superimposable with the reference compound (Fig. 2). MS analysis and identification by HPLC
The separation and identification of the isolated compound in PTLC were carried out by HPLC with Hpersil
Antidermatophyte activity
In vitro Antifungal Activity. It is evident from the results obtained (Table 1) that of the 24 extracts of different parts of selected three plants, viz. W. somnifera, E. hirta and T. chebula, ten extracts showed their antidermatophyte activity against test dermatophyte (T. mentagrophytes). Three extracts were found to be fungicidal where same values of MIC and MFC were recorded; whereas seven extracts were recorded as fungistatic (MFC values were higher than their MIC values). For animal studies, most active plant extract, free flavonoids from stem of T. chebula which showed excellent inhibition zone (IZ 41.50 0.500 mm) with the lowest MIC and MFC values 0.039 mg/ml, was selected. Isolated apigenin was also screened against T.
Figure 2 Infrared spectra of isolated com-
pound ‘Apigenin’.
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G. Singh et al.
(a)
(b)
Figure 3 (a) High-performance liquid
chromatography (HPLC) chromatogram of standard (apigenin), and (b) HPLC chromatogram of isolated apigenin. Table 4 Peak table for standard apigenin. 0
Peak
Ret. time
Area
Height
Area %
Height %
K
1 2 Total
2.582 4.030
40577 916739 957316
2188 134048 136236
4.239 95.761 100.000
1.606 98.394 100.000
0.000 0.561
Table 5 Peak table for isolated compound from free flavonoids of stem of Terminalia chebula. Peak
Ret. time
Area
Height
Area %
Height %
K0
1 2 Total
2.578 4.032
40962 916845 957807
2070 133941 136010
4.277 90.723 100.000
1.522 98.478 100.000
0.000 0.564
mentagrophytes which showed inhibition zone of IZ 38.50 0.250 mm (Fig. 4). Induction of dermatophyte infection on mice
The severity of skin lesions in all groups of animals was assessed every day after inoculation of test
6
dermatophyte. Time course of changes in mean lesion score is shown in Table 6. Infection of mice model resulted lesion in 100% of the animals. First signs of infection were observed on second and third day of inoculation and lesion reached at its maximum at the infected site on eighth and tenth day of inoculation in all animals of groups B (immunosuppressed infected animal group) and A (infected animal group without immunosuppression) respectively. Lesion scores started decreasing as a result of spontaneous healing after 20 and 25 days of inoculation, whereas complete spontaneous recovery was observed on 26th and 32nd day of inoculation in groups A and B respectively. Treatment of induced dermatophytosis on mice
Recovery of skin lesions of animals of treated groups (C, D and E) was assessed by giving them treatment scores depending upon per cent resolution of infected site (Table 7). First signs of recovery was seen on second day of treatment in animals of groups C (group
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Treatment of dermatophytosis
treated with terbinafine 5 mg g1 ointment) and E (test extract ointment 5 mg g1), whereas group D (test extract ointment 2.5 mg g1) showed recovery signs on third day of treatment (Figs 5, 6 and 7). On 12th day of treatment 100% animals of groups C and E were completely cured and new hairs were observed on the bald exposed area of infected site. In case of group D, 100% recovery (i.e. treatment score 4) of dermatophyte infection was observed on 16th day of treatment in all animals of the group. Course of infection in animals of group F (treated with petroleum jelly) was found similar as in untreated groups (A and B) and lesion became more severe with time indicating ineffectiveness of petroleum jelly. Skin irritation was observed in 45% animals of the group treated with terbinafine, whereas such side effects were not observed in both the concentrations (2.5 and 5 mg g1) of test ointment. The fungal burden (FB) of infected loci in animals of untreated groups (A and B) and group F (treated with petroleum jelly) was recorded relatively high, i.e. 3.30 0.510, 3.60 0.330 and 3.80 0.240 respectively. Fungal burden was calculated 0 for groups C, D and E on 20th day of treatment, thus proved complete eradication of the fungal mycelia/conidia from the infected loci (Table 8).
(a)
(b)
Figure 4 (a) Inhibition zone of isolated apigenin against T. ment-
agrophyte. (b) Inhibition zone of free Flavonoids of stem against T. mentagrophytes.
Table 7 Treatment scores of infected animal groups. Average treatment score1
Table 6 Time course of changes in lesion in mice model of dermatophytosis. Animal group
Days
Normal infected Group (A) Average lesion score
Immunosuppressed infected Group (B) Average lesion score
1 2 3 4 5 6 7 8 9 10
0 0 0.6 0.8 1.8 2.4 2.8 3.4 3.8 4
0 0.8 2.2 2.6 2.8 3.2 3.8 4 4 4
Average lesion score: Sum of lesion score of the group/total number of animals in the group. 0, Absence of lesion; 1, Appearance of erythema at infected site; 2, Moderate erythema spreading over entire infected site; 3, Intense erythema with abrasions, swelling and scaling; 4, Severely erythematous lesion with crusting spreading over the entire exposed area.
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Days
Group C
Group D
Group E
Group F
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
0 0.2 0.8 1.2 1.8 2.2 2.6 3.0 3.4 3.6 3.8 4.0 4.0 4.0 4.0 4.0
0 0 0.2 0.6 0.8 1.2 1.6 1.8 2.0 2.4 2.8 3.0 3.2 3.6 3.8 4.0
0 0.2 0.6 1.0 1.6 2.0 2.4 2.8 3.0 3.2 3.6 4.0 4.0 4.0 4.0 4.0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Group Group Group Group
C, treated with standard drug terbinafine (5 mg g1); D, treated with test ointment Api I (apigenin 2.5 mg g1); E, treated with test ointment Api II (apigenin 5 mg g1); F, treated with petroleum jelly (placebo treated).
1
Average treatment score: Sum of treatment score of each animal/total number of animals in group; (0): not cured, (1): 25% cured, (2): 50% cured, (3): 75% cured, (4): 100% cured.
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G. Singh et al.
Lesion on 1st day of treatment
Lesion on 9th day of treatment
Lesion on 3rd day of treatment
Lesion on 6th day of treatment
Lesion on 12th day of treatment
Figure 5 Recovery of skin lesion in animal group of C (treated with standard drug terbinafine).
Lesion on 1st day of treatment
Lesion on 12th day of treatment
Lesion on 4th day of treatment
Lesion on 8th day of treatment
Lesion on 16th day of treatment
Figure 6 Recovery of skin lesion in animal group of D (treated with test ointment Api I).
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Treatment of dermatophytosis
Lesion on 1st day of treatment
Lesion on 9th day of treatment
Lesion on 3rd day of treatment
Lesion on 6th day of treatment
Lesion on 12th day of treatment
Figure 7 Recovery of skin lesion in animal group of E (treated with test ointment Api II).
Table 8 Determination of fungal burden (FB) of animal groups.
Animal group
Days
Score of FB FB SEM
A B C D E F
151 151 202 202 202 151
3.30 0.510 3.60 0.330 0 0 0 3.80 0.240
SEM, standard error mean; A, animal group inoculated without immunosuppression; B, animal group immunosuppressed before inoculation; C, animal group treated with terbinafine cream (5 mg); D, animal group treated with test ointment (5 mg); E, animal group treated with test ointment (10 mg); F, animal group treated with petroleum jelly. 1 Days after inoculation. 2
Days after treatment.
Discussion Results indicate antidermatophyte potency of both the concentrations of apigenin (Api I and Api II) on the infection, as results were comparable with reference drug terbinafine. In addition, fungal burden results also showed excellent antidermatophyte activity of test ointments (Api I and Api II) as none of the epidermal flank was found culture positive, thus giving
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FB score 0 and depicted complete cure of dermatophytosis. Results revealed that the cure pattern of placebo group (petroleum jelly–treated) mice and that of negative control (with no application) was same which showed that jelly had no effect to cure dermatophytoses. ‘In vivo’ fungicidal activity of free flavonoids (from stem of T. chebula) against T. mentagrophytes can be assigned to apigenin because thin-layer chromatography results showed that free flavonoids (from stem of T. chebula) considered of only one compound as single spot was observed in all TLC plates and later this compound was identified as apigenin. Thus, it can be concluded that apigenin isolated from stem of T. chebula was found with significant antidermatophyte activity in ‘in vivo’ and ‘in vitro’ studies. Terbinafine (which was taken as a reference drug in this study) is a potent fungicidal agent against dermatophytes and is often preferred over other dermatophytes have also reported.22,23 Trichophyton spp may develop resistance against terbinafine in future, which indicates limited life span of the drug. Hence, continuous search for therapeutic alternatives should always be encouraged.24 In the present investigation, apigenin gave encouraging results in the tropical treatment of dermatophytosis
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in mice. As like other topical antidermatophyte agents, apigenin when applied topically on the skin, penetrate the stratum corneum and interfered with the growth of fungi, leading to their structural disintegration which make them unable to grow or divide. Structural disintegration is possibly known to be due to interference of cytoplasmic membrane synthesis and by blocking sterol biosynthesis.25 Although systemic therapy is often suggested for Tinea/dermatophytosis when infection condition is very serious and lesions involve large area which could not be cured by topical treatment, but systemic therapy besides being expensive may cause certain side effects as drugs taken orally are bound to affect physiology of the host. Thus, lower cost of plant-based topical agent’s makes therapy a favourable choice in the management of superficial dermatophyte infections in its initial stage which prevent the serious stage of disease26 Present investigation suggests clinical trials of apigenin for dermatophytosis and advocates the use of T. chebula for isolating apigenin which is established as an antidermatophyte compound in this study and also to exploit it as an alternative plant-based drug for dermatophytosis in future.
5 6 7 8
9 10 11
12 13 14 15
16
17
18
Acknowledgments Authors are thankful to the ‘Head’ Department of Botany, University of Rajasthan for providing all necessary facilities and to UGC for providing financial assistance for this work.
Conflict of interest None.
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2
3
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