Introduction

In recent years, proliferation of new classes of drugs such as, allylamines (e.g terbinafine) and orally active triazoles (e.g. itraconazole), has represented the most noteworthy trend in dermatophytosis therapy (Weitzman and Summerbell, 1995). However, treatment with both itraconazole and terbinafine for prolonged times requires periodic laboratory monitoring of liver function (Zapata et al., 2003). Moreover, these antifungal agents may have drug interactions with other medications (Huang et al., 2004). Griseofulvin, which had been for many years the only antifungal available for the treatment of dermatophytoses is still the longstanding drug of choice for tinea capitis, but there are concerns with resistance and toxicities with this agent (Huang et al., 2004). Taken together, the improved cure rates, reduced adverse effects; decreased drug interactions and lower cost of topical agents make therapy with these drugs a favourable choice with management of superficial fungal infections including dermatophytoses. The spread of drug resistant pathogens is one of the most serious threats to successful treatment of microbial diseases.

In this context, new antifungals of plant derivatives could be useful alternatives for the treatment of dermatophytoses where a topical therapy is required. These plant compounds can have different structures and different action when compared with antimicrobials conventionally used to control the microbial growth and survival they also have a reduced risk of side effects and lower cost (Nascimento et al., 2000). It is thus not surprising that in recent years, attention has turned to natural antimicrobial agents (antibacterials and antifungls), particularly, there is a rapid growth of interest in the use of medicinal plants to cure skin diseases. There are many reports on the antimicrobial activity of the active the compounds which represent various groups including alkaloids, flavonoids, isoflavonoids, tannins, cumarins, glycosides, terpenes, phenylpropannes, organic acids (Nychas, 1996).

Ceasalpinia bonducella (L.) Flem. (Caesalpiniaceae) grows wild in the scrub forests, of the Eastern Himalayas, in Assam and West Bengal. It is also found in the Eastern Ghats in Andhra Pradesh and Madhya Pradesh, commonly known as fever nut. The leaves and seeds of this plant are used as a folk medicine to treat asthma, tumor, inflammation, liver disorders and chronic fever (Nadkarni, 1976). It is found to be very potent as an antiperiodic, antipyretic anti-inflammatory, antidiabetic, antifungal and utilized as febrifuge, antispasmodic, an antirheumatic and mild purgative (Nadkarni, 1976; Satiyavati et al., 1976). Since, there are very few reports on antimicrobial activity of C. bonducella, thus keeping this in view, attempts have been made to isolate the active the compound from the ethyl acetate leaf extract of C. bonducella and evaluate its antifungal efficacy against clinical isolates of dermatophytes.

Materials and Methods

Agar dilution method

Germination test

Effect of pH

Effect of inoculum size

Broth dilution method

Mycelial dry weight

Effect of pH

Effect of inoculum size

Statistical analysis

Experimental results

In IR spectrum of the compound (Fig. 1) the absorption band at 346 cm-1 was observed due to the presence of OH group. The absorption peak at 2920 cm-1 corresponds to the stretching of C-C bond. The peak was observed at 1459 cm-1 due to –C=C– stretching. The peak at 1651 cm-1due –C-O- stretching. In the 1HNMR spectrum (Fig.2) the singlet was observed at 0.85 δ due to six protons of two methyl groups attached to C-18. Two singlets at 1.5- and 1.7 δ due to the protons of two methyl groups attached to C-1. The four protons of –2CH₂- groups present at C-3 and C-17 were resonated as singlet at 2.05 δ and the signals due to of thirteen methylene groups were resonated at 1.1 – 1.4 δ. The peak due to two –OH groups observed at 5.2 δ as broad singlet. In the mass spectrum, it showed the molecular ion peak at m/z 339 due to [M-1]+ ion. These data confirm the structure of the compound as ά-(2-hydroxy-2-methylpropyl)-ω-[2-hydroxy-3-methylbut-2-en-1-yl] polymethylene with the structural formula C₂₂H₄₄O₂.

Figure 1

Fig.1: IR spectrum

Figure 2

Fig. 2: 13CNMR spectrum Agar dilution method

Figure 3

Fig. 3: 1HNMR spectrum

Effect on conidial germination

Effect of pH on activity

The antifungal activity of the compound was assessed at different pH levels viz., 6.0, 6.5, 7.0 and 7.5 with an inoculum consisting of a standard spore suspension fig. 1.2). The compound could potentially inhibit the growth of T. mentagrophytes, T. rubrum, M. gypseum and E. floccosum completely at all the pH tested, whereas more than 70% growth of T. tonsurans was inhibited at pH 6.0 and above that the organism did not grow. M. gypseum proved to be resistant at pH 7.5 where at 100, 200, 300 and 400 μg/ml of the compound could inhibit 10.20±0.43, 9.05±0.57, 8.20±0.43 and 8.40±0.40 percent of mycelial growth of M. gypseum, respectively (Fig. 1.2). The activity of the compound at different pH differed significantly (P<0.05).

Figure 4

Effect of inoculum size on radial growth of colony

The effect of inoculum size on radial growth of colonies of test dermatophytes after 7 days of incubation on SDA medium containing 100, 200, 300 and 400 μg/ml was studied. The size of the colonies decreased when inoculum size was decreased from 107 to 104 spores/ml with the increase in the compound concentration from 100 to 400 μg/ml, but decreased colony growth was exhibited by T. tonsurans only with 104 spores/ml at 100, 200 and 300 μg/ml, whereas other fungi could not grow even at 100 μg/ml of the compound (Fig. 1.3). The surfaces of the colonies of T. tonsurans in the presence of MICs of the compound were granular, irregular in outline, and highly wrinkled (Plate-I).

Figure 5

Plate I: Effect of compound on the colony growth of

A= 100 µg/ml; B= 200 µg/ml; C= 300 µg/ml;

-> Indicates the colony with granular, irregular in outline and highly wrinkled

Broth dilution method

Effect of on mycelial dry weight

The effect of the compound in SD broth on mycelial dry weight is presented in figure 2.1. The mycelial dry weight of all test dermatophytes decreased with the increase in the concentration of the compound, which could inhibit all the fungi, except T. tonsurans at 100, 200, 300 μg/ml. The mycelial dry weight differed significantly at all concentrations (P< 0.05).

Effect of pH on activity

Various pH i.e. 6.0, 6.5, 7.0 and 7.5 of broth medium were tested for the growth of dermatophytes. The compound could inhibit all organisms except T. tonsurans and pH 6.5 was found suitable (Fig. 2.2).

Effect of inoculum size on activity

The biomass concentrations of the test dermatophytes were assessed with varying sizes of inoculum i.e., 104, 105, 106 and 107 spores/ml. Only T. tonsurans could grow at 100, 200 and 300 μg/ml concentrations (Fig. 2.3). Decline in biomass of all the fungi along with increase in concentrations of the compound were observed which were significantly different (P<0.05).

Figure 6

Discussion

Recently, the use of medicinal plants covering the basic health needs in developing countries is increasing, which offer a new source of antibacterial, antifungal and antiviral agents with significant activity against infective microorganisms.

The antifungal effect of the isolated compound from ethyl acetate leaf extract of Caesalpinia bonducella (L.) Flem. was tested on all the dermatophytes of clinical origin by agar and broth dilution methods. This provided the in vitro assessment of the MICs of the compound. The results show that the compound has an interesting activity even if it differed between the test fungi. In agar dilution method, the conidia germinated less along with the increase in concentrations. At various concentrations of the compound a few were germinated, but had been inhibitory after 7 days. This was probably due to the normal decomposition of the compound, but the small number of spores that germinated at high concentration could not continue to develop into normal hyphae. The high activity of the compound was found at pH 6.0 and as the inoculum size increased the colony growth increased with the decrease in concentration of the compound. Whereas, in broth dilution, optimum pH was 6.5 with the same inoculum size. The biomass concentration of fungi decreased with increase in concentration i.e. from 100 μg/ml–400 μg/ml in broth medium. The MIC of the compound was considerably affected by inoculum size and medium pH. Also, there were some differences in MIC with to whether solid or liquid medium used, i.e. more concentration of the compound was required to inhibit growth in liquid medium than in solid medium.

The compound, except T. tonsurans, inhibited the germination of all the fungi tested. Concentrations approaching the inhibitory level resulted from the delay in swelling and germination of spores, and the restriction of the radial growth of fungal colony was noticeable inhibition of longitudinal extension of hyphae.

The dermatophytes used in this study are the most common and widespread of this group. They represent the three genera of dermatophytes and their respective ecological types geophilic (Microsporum gypseum), zoophilic (Microsporum canis and Trichophyton mentagrophytes) and anthropophilic (Trichophyton tonsurans, Trichophyton mentagrophytes and Epidermophyton floccosum). The morphological nature of these fungi, which grow as hyphae containing various numbers of micro and macroconidia, accompanied by thick-walled chlamydospores, causes difficulty, when attempting to standardize inocula. Hence, to produce evenly dispersed suspensions, they were subjected to mechanical fragmentation and homogenization. This process resulted in homogenous suspensions containing small septate fragments and gave reproducible results. All the dermatophytes tested, proved to be more susceptible to the compound. The less biomass concentration in broth at all concentrations tested, may probably be due to homogenous dispersion of particles in a liquid medium which enabled closer contact between the fungal elements and the active agent.

At a particular concentration, the potency of a fungitoxicity of the compound can thus depend on the inoculum density of the test fungus. For example, the antifungal activities of citral and menthol were found to vary with inoculum density (Venkataraman and Pattisapu, 1987). This suggests that a high inoculum density of a fungus may either degrade or convert the compound into detoxified forms. The nature of detoxification of the fungitoxic products will also depend on the detoxifying enzymes produced by them. Thus, the detoxification of fungitoxic products at varying inoculum densities could be one of the main factors limiting their efficiency as ideal antifungal agents, leading to the minimum inhibitory concentration of the compound depending on inoculum density. However, an increased inoculum density did not affect the antifungal potency of the compound in the present study.

Although it is desirable to develop the compound having a broad spectrum of activity, it is also important to bear in mind that the treatment of chronic different tineas with the same broad spectrum antifungal agent leads sometimes to a high resistance to the available antifungal agents (Ghannoum and Rice ,1999). Thus, one of the strategies for overcoming this problem is the treatment of fungal infections with the appropriate narrow spectrum agent when the ethiological agent is known (Di Domenico, 1999).

Tinea pedis is one of the most frequent mycoses; it occurs in most classes of patients, especially in immunosuppressed patients (Soares, et al., 1995). T. rubrum is the most common superficial fungus, accounting for at least 60% of all superficial fungal infections in humans. This organism may remain viable in environment for over six months, thus accounting for widespread infection. In this study, this organism has been inhibited by the compound. Therefore, antifungal activity of the compound reported in this study against the dermatophytes open the avenues that it could be helpful for developing the new antifungal agents for treating dermatomycoses caused by T. tonsurans, T. mentagrophytes, T. rubrum, M. gypseum and E. floccosum. Since, there is no scientific evidence to support the medical use of ά-(2-hydroxy-2-methylpropyl)-ω-[2-hydroxy-3-methylbut-2-en-1-yl] polymethylene} [the compound used in the present study], further studies are needed in order to elucidate the mechanism(s) of action of these compounds and their derivatives, as well as the antimicrobial activity against other microbial strain in particularly antibiotic resistant dermatophytes.

Acknowledgement