Poly-herbal medicine is a major part of treatment systems which is gaining popularity everyday due to clinical failures of conventional medicines due to drug resistance fungal strains [2]. The use of herbal medicine is based on practice and concepts emanating from ancient philosophies. In the current era of evidence-based medicine, it is important to accurately assess composition and efficacy of herbal medicine. In the present effort, chemical profiling of antifungal compounds of D. senecioides and D. mespiliformis was done using disc diffusion, poisoned food, p-iodonitrotetrazolium chloride and GC–MS/MS analyses. The study followed the poly-herbal formulation philosophy which has an advantage of achieving greater efficacy at low concentration that is likely to be safe to humans.

Preliminary anti-dermatophytic potency experiments were done using concentrations of 50, 25 and 1% made up of crude extracts of the 2 plants under study mixed in a 1:1 ratio. Radial growth inhibition of E. floccosum colonies was 80.00, 75.00, 20.00% at the aforementioned extract concentrations, respectively, and 73.30% for the positive control. T. rubrum was comparably inhibited at the same concentrations producing percentage inhibition of 73.82, 67.88 and 63.48, respectively, while the control inhibition was 57.50% (Table 2). At 1% concentration of extract, E. floccosum exhibited very low susceptibility compared to T. rubrum although the later had visibly disturbed mycelial growth (Figs. 2 and 3). E. floccosum was generally more susceptible to the plant extracts at higher concentrations than T. rubrum. Generally the extract proved to have potent activity against the test species. The results obtained in this study provide an impetus for further research as very little information is documented on antifungal activity of D. senecioides. Research done by [18], on D. mespiliformis leaves and bark extracts revealed efficacy in the range between 0.02 and 2.5 mg/ml against C. albicans and 0.02–0.16 mg/ml against T. rubrum. Little research has been documented on antifungal activity of raw fruit extracts despite their wide use in traditional practices.

In vitro antifungal activity of ethylacetate extracts of D. senecioides and D. mespiliformis against C. albicans is shown in Table 1, while Figs. 2 and 3 show inhibition of radial growth in E. floccosum and T. rubrum, respectively, by the same extracts. Table 1 gives a numerical summary of the percent inhibition of radial growth in E. floccosum and T. rubrum. In vitro antifungal analysis of the extracts showed that the extract consist of significant antifungal activity. The 1:1 mixture of D. senecioides and D. mespiliformis extracts exhibited greater antifungal activity against C. albicans (Fig. 1) than the standard drug miconazole. Figure 2 shows that at a concentration of 50 and 25%, the efficacy against E. floccosum of the polyherbal mixture was comparable to that of miconazole. No effect was observed at 1%. The polyherbal mixture was very effective against T. rubrum even at a low concentration of 1%. The efficacy was significantly greater (Fig. 3 and Table 2) than that of miconazole.

Using the p-iodonitrotetrazolium chloride assay to screen and isolate the active potions and GC–MS/MS analysis, many bioactive phytochemicals were identified (Fig. 5a–d). The list of compounds identified in the active bands of the extracts through GC–MS/MS are presented in Tables 4, 5, 6, and 7 for bands with Rf values of 0.98, 0.88 (D. senecioides) 0.64 and 0.78 (D. mespiliformis), respectively. Only compounds with match score ≥ 90% were considered. The 0.98 band of D. senecioides consisted of flavonoids and flavonoid glycosides amounting to 10 flavonoid compounds. From D. mespiliformis extract, a total of 6 flavonoid compounds were obtained from the 0.78 band. In a separate phyto-constituent study by Hawas et al. [19] of D. mespiliformis leaf extracts a new acylated flavone isoscutellarein 7-O-(4′′′-O-acetyl)-β-allo- pyranosyl(1′′′ → 2′′)-β-glucopyranoside, together with 8 known flavonoid metabolites, luteolin 3′,4′,6,8-tetramethyl ether, luteolin 4′-O-β-neohesperidoside, luteolin 7-O-β-glucoside, luteolin, quercetin, quercetin 3-O-β-glucoside, quercetin 3-O-α-rhamnoside, and rutin were isolated and identified using spectroscopic data (Ultra Violet, Nuclear Magnetic Resonance, and Mass Spectroscopy). The methylated flavone isolated from methanolic leaf extracts of the plant exhibited potent activity against C. albicans with an inhibition zone of 25 mm [19], while in preliminary antifungal assay in the current study potency of the crude extracts of the plants under study herein against C. albicans was between 15 and 17 mm at 6.25–50% extract concentration. No zones were obtained against C. albicans for both the positive and negative controls (Table 1). Stem bark extracts exhibited potency against C. albicans at 12.5 mg/ml producing a zone of inhibition of 13.00 ± 0.00 mm [10]. The current results further affirm the potency of D. mespiliformis against C. albicans. TLC chromatograms and bioautograms of the raw fruit extracts of D. mespiliformis and stem/leaves extracts of D. senecioides (1:1) mixture (Fig. 4a–d) revealed antifungal compounds with potency against E. floccosum and T. rubrum. Active compounds against E. floccosum had Rf value of 0.88 in D. senecioides and 0.64 in D. mespiliformis while potency against T. rubrum was observed at Rf values of 0.94 and 0.74 in D. senecioides and D. mespiliformis, respectively. GC spectra of these bands are shown in Fig. 5a–d. The 0.94 and 0.78 bands according to GC-MS/MS analysis consisted of flavonoid compounds (Tables 4 and 5). Flavonoids and flavonoid derivatives have been proved to have potent antifungal activity against many fungal pathogens including C. albicans and T. rubrum with efficacies ranging from concentrations as low as 1.95 µg/ml, [20]. The Rf 0.64 band was found to be a mixture of phenolic compounds, aromatic compounds and esters (Table 6). Phenolic compounds are known to provide resistance to plant pathogens, fungal species included [21, 22].

The band at Rf (0.88) was mainly made up of terpenoids, terpenes and methyl esters. Terpenes and terpenoids have antifungal potential. Previous studies have proved that crude terpenoids can inhibit mycelial growth in fungi [23], affect DNA (deoxyribonucleic acid), RNA (ribonucleic acid) and protein synthesis, disrupt cell membrane permeability and also disturb metabolic activity in fungi [23]. Other terpenoid compounds interfere with formation and viability of hyphae and induce morphological alterations in the envelopes of fungi [24]. The biological activity of these compounds in this study is indicative of the medicinal potential of the investigated plants. Inarguably, some of these compounds have different pharmacological activities; hence, the possibility of mutually advantageous conjunction in terms of bioactivity of the phytochemicals determined in the two plants cannot be ruled out as possible contributors to the observed activities [25].