Evaluation of antidermatophytic activity of ά-(2-hydroxy-2-methylpropyl)-ω-[2-hydroxy-3-methylbut-2-en-1-yl] polymethylene - the compound isolated from Caesalpinia bonducella (L.) Flem.
K Sagar, G Vidyasagar
K Sagar, G Vidyasagar. Evaluation of antidermatophytic activity of ά-(2-hydroxy-2-methylpropyl)-ω-[2-hydroxy-3-methylbut-2-en-1-yl] polymethylene - the compound isolated from Caesalpinia bonducella (L.) Flem.. The Internet Journal of Microbiology. 2008 Volume 7 Number 1.
A compound was isolated from ethyl acetate leaf extract of
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
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
Materials and Methods
Agar dilution method
End points for determining MICs and MFCs.
The MIC of the compound was prepared viz., 100, 200, 300 and 400 μg/ml tested for the dermatophytes was defined as lowest drug concentrations that yielded visible growth of the fungi. The minimal fungicidal concentrations (MFCs) was determining by sub culturing 0.01 ml from each tube with no visible growth onto SDA plate. The plates were incubated at 30oC for 7 days. Afterward, the colonies were counted, and the MFC was defined as the lowest concentration of drug which yielded a negative subculture or less than two colonies (killing of more than 98% of the fungal inoculum).
The test organisms used were clinical isolates namely,
Isolation of the compound
The leaves of
Fungal inoculum preparation
All the dermatophytes grown on SDA after a week, spores were flooded with 0.85% saline (Ghannoum
Different concentrations of the compound were mixed with 20 ml SDA and poured onto petriplates, immediately added 0.1 ml standard spore suspension of the test dermatophytes on the medium and left for solidification. After 24 h of incubation, square samples (10 by 10 mm) cut off from the plates were mounted in lactophenol cotton blue and examined microscopically. A spore was considered to be germinated when the germ tube was a long as it was wide.
Effect of pH
The activity of and the compound against test dermatophytes on medium with different pH values was determined by the agar dilution method on SDA medium adjusted to a final pH value of 5.6, 6.0, 6.5 and 7.5 with inoculum sizes of 0.01 ml of standard spore suspension.
Effect of inoculum size
The activity of and the compound against test dermatophytes with different inoculum sizes was determined by agar dilution method on SDA medium (pH 5.6) with an inoculum size of 0.01 ml of 104, 105, 106 and 107 spores per ml.
Broth dilution method
For antidermatophytic assay in broth, 5 ml of the medium (SDA) was added to each screw-capped test tube and were autoclaved at 121oC for 15 min. Tubes with 5 ml sterile SDA were inoculated with 10, 20, 30 and 40 mg/ml concentrations of the compound in DMF. Tubes were kept in the salutation position overnight for checking the sterility. The next day, the tubes were inoculated with 20 µl of fungal suspension on the salutation position and all the test tubes were incubated at 37oC. After one week, the results were noted. The visible mycelial growth in the test tubes expressed the degree of activity of the compound.
Mycelial dry weight
Fungal mycelia from the above culture were separated passing through Whatman filter No.1. A known amount of thoroughly washed mycelia was placed on preweighed petriplates and allowed to dry at 60oC for 6 h to reach a constant weight. Fungal growth inhibition was calculated by considering the control’ and sample’ mycelial dry weights by following the method of Rasooli and Razzaghi Abyaneh (2004).
Effect of pH
The activity of the compound against test dermatophytes in medium with different pH values was determined by the broth dilution method on SD medium adjusted to a final pH value of 5.6, 6.0, 6.5 and 7.5 with inoculum sizes of 0.01 ml of standard spore suspension.
Effect of inoculum size
The activity of the compound against test dermatophytes with different inoculum sizes was determined by broth dilution method in SD medium (pH 5.6) with an inoculum size of 0.01 ml of 104,105, 106 and 107 spores per ml.
All data are expressed as mean ± standard errors of the mean (SEM) were analyzed by the analysis of variance by students ‘t’ test. A P value of <0.05 was considered to be significant.
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.
Effect on conidial germination
The percent germination of conidia of all the test dermatophytes after one week in the presence of the compound (incorporated into SD Agar medium) was determined by counting the number of germinating spores microscopically. The result is plotted in figure 1.1.
The compound completely inhibited swelling and germination of spores of
After 7 days of incubation, the numbers of spores that germinated at sub inhibitory concentrations of the compound (only of
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
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
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
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
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
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
The compound, except
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 (
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,
The author is thankful to Dr. M.G.Purohit, Emeritus Professor, Luqman college of Pharmacy, Gulbarga, Karnataka for providing the results of spectras of the compound and Dr. R. R. Rao, CSIR Emeritus Scientist, CIMAP, Bangalore for his critical suggestions and The Director, Central Institute of Medicinal and Aromatic Plants, Lucknow for providing facilities.