Efficacy and phytochemical analysis of latex of Calotropis procera against selected dermatophytes.

BACKGROUND: Since ancient time, increased interest has been witnessed in the use of an alternative herbal medicine for managing, and the treatment of fungal diseases worldwide. This may be connected to the cost and relative toxicities of the available antifungal drugs. It has been a known tradition practiced in the northern part of Nigeria that parents and teachers use the white latex of Calotropis procera to treat Tinea capitis in children attending the local religious school in the area. This study was conducted in 2009 to ascertain the above claim.
MATERIALS AND METHODS: Fresh latex of C. procera was screened for their antifungal activity against species of dermatophytes: Trichophyton spp., Microsporum spp. and Epidermophyton spp. using the agar incorporation method.
RESULTS: The result shows that the latex inhibits the in vitro growth of these pathogenic fungi to varying extents with Trichophyton spp. being the most susceptible (P < 0.05) and thus highly inhibited by the latex followed by the Microsporum spp. and Epidermopyton spp. was least inhibited. These inhibitions followed a dose-dependent trend as undiluted latex (100%) gave the highest inhibitory impacts (P < 0.05) when compared to serially diluted latex. The phytochemical analysis of the fresh latex indicated the presence of alkaloids, saponin, tannins, steroids, flavonoids, anthraquinone, and triterpenoids.
CONCLUSION: The findings of this study confirmed the perceived usefulness of the latex in the treatment of T. capitis (ringworm) practiced in our society and therefore, its use topically in the treatment of dermatomycotic infection is encouraged.

INTRODUCTION

Dermatophytes are a group of three fungal genera that can invade keratinized tissues and therefore able to cause superficial infections of the skin, nails and hair, thereby producing a disease referred to as ringworm in human and animals [1]. The infection is mostly cutaneous, i.e. restricted to the non-living cornified layer of the skin due to the innate inability of these groups of fungi to penetrate beyond the keratinized tissues or organs of the immunocompetent host [2].

The genus Calotropis (Calotropis gigantea and Calotropis procera) belongs to the family of Asclepiadaceae. These shrubs have been reported to exhibit a lot of medicinal properties which includes the antimicrobial, antimycotic, and anti-inflammatory effect [3].

C. procera is commonly called calotrope; other names are King’s crown, kapok tree, Tumfafiya (in Hausa language), Bomubomu (Yoruba language). It is a spreading shrub with large grey-green leaves and large green inflated fruit similar in shape to a mango. A whitish sap (the latex) oozes out when the plant’s stem is broken. The plant is native to tropical Africa and Asia. Studies from phytochemical analysis of C. procera suggest the presence of biologically active compounds such as Alkaloids, steroids, triterpenes; others include madaralbun, madarfluavil, caoutchouc, and calotropin [4].

The use of plant extracts for medicinal purposes is very widespread in the world, Nigeria inclusive. Many of these medicinal plants were being used against infectious disease causing agents, which are frequent nowadays, due to the emergence and increase in antimicrobial resistance and poor hygienic condition of our environments. The increasing incidences of fungal infections coupled with the gradual rise in azole resistance and available antibiotics had highlighted the need to find more alternative antifungal agents from other sources [5]. Several plants have been shown to contain some significant amount of antifungal activity on a wide range of microorganisms [6]. The aqueous extract of the aerial part of C. procera is a prominent decoction used in Saudi Arabia for the treatment of varieties of diseases such as muscular spasm, joint pain, constipation, and fever [6]. Locally, the extract of C. procera is used topically for the treatment of ringworm [7]. In northern Nigeria, the latex, leaves, root, stem bark, and fresh follicles of C. procera were used in indigenous practice to treat topical fungal diseases, convulsion, asthma, cough and inflammation [8,9].

In Sokoto town (the area of the study), a study by Ameh and Okolo in 2004 [10] revealed the incidence of dermatomycosis among primary school pupils, and the study has attributed the observed incidence to the domestic animals as an important predisposing factor. It is evident from a survey (personal communication with the elderly people within the locality) that fresh latex of C. procera has been used from ancient time as a topical antimycotic treatment in the area.

This study was carried out to elucidate the antimycotic activity of the fresh latex of C. procera against selected representative species of the dermatophytes (the etiologic agent of dermatomycosis), and clarify the above claim by the local people.

MATERIALS AND METHODS

Plant Material

Fresh latex analysis of variance (the white liquid secretion) of C. procera was collected from the farmlands around Usmanu Danfodiyo University permanent site, Sokoto, Nigeria, in October 2009. The latex was collected into a sterile wide-necked screw-capped container by deliberately breaking the smooth stem and milky sap ooze out from the stem; this was repeated continuously until the required volume of the latex was tapped. The fresh latex was serially diluted 2-fold and 5-fold with sterile distilled water to give 50% and 20% of the original latex concentration respectively.

Phytochemical Analysis

The fresh latex of C. procera was sent to the Biochemistry laboratory of the department of Biochemistry, Faculty of Science, Usmanu Danfodiyo university, Sokoto for phytochemical analysis. The latex was analyzed for the presence of alkaloids, flavonoids, tannin, saponin, triterpenoids, anthraquinones, and glycosides compounds using the standard colorimetric procedures as described by Sofowora and Kennedy and Thorley [11,12].

The Fungal Species Used

Representative isolate each of Trichophyton spp., Microsporum spp. and Epidermophyton spp. were used in this study. The isolates were kindly provided by Mr. Abdulrahman Barau of the Mycology laboratory of the Biological science department of the Usmanu Danfodiyo University, Sokoto.

Antifungal Susceptibility Testing

Fresh latex of C. procera was examined for its antifungal properties against Trichophyton spp., Microsporum spp. and Epidermophyton spp. - the causative agents of dermatomycosis (ringworm). Four dilution groups were prepared for this study, this includes a negative control (sterile distilled water instead of latex), original, fresh latex (100%), 2-fold serially diluted latex (50%) and 5-fold serially diluted latex (20%) groups.

The antifungal assay of the latex was conducted using the agar incorporation method as described by Taudou and Dwivedi and Dubey [13,14]. Briefly, the aforementioned concentrations (sterile distilled water, 100%, 50% and 20% latex) were aseptically mixed in a ratio of 1:3 with sterile sabouraud dextrose agar (SDA) and poured in 150 mm × 30 mm petri dishes, allowed to solidify and seeded in duplicates with fungal isolates previously cultivated on SDA. The inocula was aseptically cut with a sterile 10 mm cork borer, seeded in the middle of the petri dishes and incubated at 28°C-30°C in the dark. The growth of the dermatophytes on each culture plate was measured linearly (growth diameter) by the use of transparent millimeter rule daily for 6 days.

Statistical Analysis

An SPSS 20®statistical software was used for statistical analysis. The data generated in the study were presented in the form of tables using frequency distribution. Average daily mycelial growth was analyzed using one-way (ANOVA) with Tukey’s multiple comparisons testing to determine the significant differences between the control and experimental groups. All comparisons were considered to be significant at P < 0.05.

RESULTS

The latex of C. procera was found to inhibit the in vitro growth of the three dermatophytic fungi studied to varying extents. Tables 1-3 show a measure of a diameter of mycelial spread for each of the fungi tested. Statistical analysis of the result shows that Trichophyton spp. was the most susceptible, and thus highly inhibited by the latex followed by the Microsporum spp. and Epidermophyton spp. was the least inhibited. It is observed that 100% (undiluted latex) of C. procera gave the highest inhibitory impact on the dermatophytes, whereas 20% latex recorded the lowest.

Table 3

Measure of mycelial spread of Epidermophyton spp. grown on SDA incorporated with varying concentration of C. procera latex

The diametric mycelial spread of the Trichophyton spp. ranges from 6.7 mm to 39.7 mm; the Microsporum spp. 7.3 mm to 65.1 mm and Epidermophyton spp., 8.3 mm to 72.7 mm. Statistical analysis shows that Trichophyton spp. was most susceptible, then Microsporum spp. and Epidermophyton spp. the least inhibited by the 100% latex (P < 0.05) [Table 1].

Table 1

Measure of mycelial spread of Trichophyton spp. grown on SDA incorporated with varying concentration of C. procera latex

Similar trends of growth of the dermatophytes were recorded at the other concentration of 50, and 20% latex. However, the inhibition of their growth by the latex reduced with decreasing concentrations. All the three concentrations of the latex tested (i.e. 100, 50 and 20%) were significantly (P < 0.05) better than the control (standard). The result of this study indicated that Trichophyton spp. was the most sensitive, followed by Microsporum spp. while the Epidrmophyton spp. was the most resistant ones.

The result of the phytochemical analysis of the fresh latex of C. procera shows the presence of alkaloids, saponin, tannins, steroids, flavonoids, anthraquinone and triterpenoids [Table 4].

Table 4

Results of the phytochemical analysis of the latex of C. procera

DISCUSSION

Various parts of C. procera has been reported to be used in many countries for the treatment of varieties of diseases, such as muscular spasm, joint pain, constipation, skin diseases and etc. [6]. The results of the present study indicated that the latex of C. procera has antifungal potentials against dermatophytes. This finding agreed with that of Kuta, 2008, who reported the same tradition of using the C. procera extracts in Gwari communities of Niger State, Nigeria, for the treatment of ringworm which stimulated his interest in evaluating the aqueous extracts of the plant and found it to display a significant inhibitory effect on the dermatophytes tested even at low concentration of the extracts.

The findings of this study are also in agreement with that of Halua and Vidyasagar 2012 who evaluated leaves extracts of two calotropis species (C. gigantea and C. procera) using three different solvents against dermatophytes and Aspergillus flavus with chloroform extract having the highest inhibition observed. Similarly, C. procera leaves extract was reported to have antifungal activity towards the tree dermatophytes genera: Microsporum spp., Trichophyton spp. and Epidermophyton spp. [15].

Furthermore, Iqbal et al. [3] reported the comparative efficacy of the chloroform and ethyl acetate C. procera leaf and latex extracts which proved active against some dermatophytes and other pathogenic fungi. However, the only dermatophytic fungi used in that study (Microsporum boulardii) was not inhibited by the extracts. This contrast with the present studies and could be explained by differences in the preparation of the plant products used and the methodology of the assay used to assess the efficacy of the plant latex.

Studies have reported several plant extracts to inhibit the growth of dermatophytes. Some of which include that of Alade and Irobi [16], who established that the ethanolic extract of Acalypha wilkesian had an antifungal effect on Trichophyton mentagrophytes, Trichophyton rubrum, A. flavus and Candida albicans. Verástegui et al. [17], showed that the alcoholic extract of Agave lecheguilla has an antifungal effect on Microsporum gypseum, Candida albicans, and Candida neoformans. Chevallier[18] described the use of extract of Ulmus campestris (Elm tree), Melissa ofinicalis (Balm tree), and Juglans duclouxiana (Walnut tree) against various dermatomycotic infections.

The result of the phytochemical analysis of the fresh latex of C. procera has indicated the presence of alkaloids, saponin, tannins, steroids, flavonoids, anthraquinone and triterpenoids. This result is similar to other reports on the leaves, stem and roots of C. procera in other studies [9,19,20]. Previous studies of phytochemical analysis of C. procera suggest the presence of biologically active compounds such as alkaloids, steroids, triterpenes; others include madaralbun, madarfluavil, caoutchouc, and calotropin, a very active poison of the digitalis type [21]. However, the chemical components responsible for the antifungal activity, and the mechanisms of action remain to be investigated. Though the mechanism of action of the drug is not known, but antimycotics generally inhibit fungal growth by either disrupting fungal membrane permeability, inhibiting sterol synthesis, inhibiting the nucleic acid synthesis, or protein synthesis [22].

The results of the present study show that the plant latex is an effective antimycotic agent against dermatomycosis in vitro. This finding shows that there is an element of truth in the claim of traditional healers on the medicinal value of this plant as an antidermatophytic agent. Therefore, the use of the plant latex in treating dermatomycotic infections should be encouraged and the government shall pay more attention to our local medicinal plants and help in processing them, which will create more job opportunities and will bring about a reduction in the cost of conventional antifungal drugs.

Table 2

Measure of mycelial spread of Microsporum spp. grown on SDA incorporated with varying concentration of C. procera latex