Glycosylated heterocyles emulsified with antifungal fraction of Moringa oleifera for potentiation of mycolytic activity

Abstract To enhance the clinical effect of antifungal medications for treating deadly fungal infections there is an increasing demand for novel treatments. Exploration of multiple-drug targeting in antifungal therapeutics is the need of the present era. In this pursuit, we identified potent antifungal compounds that were directed towards the multiple virulent targets in Rhizopus arrhizus. Quinoxaline di-N-oxide and piperazine derivatives were identified to exhibit antifungal activities. 03 bioactive compounds were identified from the docking results and antifungal activity. Furthermore, these compounds which were combined with the alkaline extract of M. olifera to make the aqueous phase, an oil phase containing cinnamon oil or clove oil and a combination of surfactants was made to prepare a bioactive composite emulsion. A significant antimycotic activity was seen for the bioactive composite emulsion when compared with the clinically used antifungal drugs. Our results indicate the synergy and potentiation of antimycotic drugs based on integrative medicine. Graphical Abstract


Introduction
Fungal infections are considered a severe health hazard in humans, as they induce a substantial damage to the immune system in immuno-compromised and neutropenic patients, thereby causing a significant morbidity and mortality (Saeed et al. 2017).Recently in the CoVid pandemic, a virulent mucormycosis infection rose drastically however, and the treatment of this infection has not still been fully understood (Jha and Kumar 2019).Rhizopus is the most studied and the common species of the fungus in the genus mucorales (Butts and Krysan 2012).
Currently, there is a limited availability of the targets, lack of target specificity, and singularity limit the effectiveness of the existing antifungal medications (de andrade monteiro and Ribeiro alves dos Santos 2020).Emerging resistance, toxicity, and limited availability of systemic drugs limit the utility of the currently available regimen of antifungal drugs; hence, developing a bioactive composite of antimycotics, may yield beneficial outcomes (Gray et al. 2012).the potential use of plant extracts and their primary active phytochemicals present promising therapeutic antibacterial and antifungal effects (nazzaro et al. 2017;Jin 2019).Currently, the multitarget approach is a major research highlight, since it shows synergistic results for the desired antifungal activity.Hence, a multitarget bioactive combination for the antimycotic drug development can enhance the pharmacological action and reduce toxicity, besides reducing the chances of resistance (pan et al. 2018).
in the present work, the bioactive features of antifungal based on a bioactive synthesized lead were paired with their phytofragments in the form of a microemulsion.as it is a dispersion of water, oil, and surfactant(s) with a dispersed domain diameter that ranges from 1 to 100 nm, often 10 to 50 nm form as and superior synergistic properties of antifungal moiety (lone et al. 2020).

Synthesis and spectral characterization
Virtual screening was performed on five designed compounds a-E.We employed a synthetic scheme shown in Figures S1-S3. the list of synthesized derivatives are shown in table S1. the iR spectra of compounds showed the n-H stretching in the range of 3385-3415 cm −1 , C = o stretching between 1640 and 1734 cm −1 .the physical, chromatographic, iR spectra and nmR spectral characterization information of a1 and B5 is shown in table S2 and Figures S4-S7.

Docking studies
docking results revealed five molecules to possess lowest docking scores and thus a higher binding affinity with the selected four targets (Figure S8 and table S3). the phytochemicals and synthesized compounds presented a decent binding affinity towards the targets (table S3).From all designed compounds using scheme a (Figure S9a), compound a1 showed the highest docking score among all other compounds showed docking of −5.4,−6.9, −6.1, and 8.6 for the targets 1aap, 1Ea1, 4n5d, and 3o9a, respectively.the docking scores of all the designed compounds are shown in Supplementary table S4. the interactions of compounds with amino acid residues of binding cavities of each target are shown in table S5. the major phytoconstituents and their log p values were studied for composite formulation (table S6).docking studies revealed that the phytoconstituents and the designed compounds exhibited a high binding affinity towards the chosen targets.

Antifungal activity of synthesized compounds
table S7 shows the antifungal potential of the synthesized compounds and phytochemical extracts of Moringa oliefera on the Rhizopus arrhizus fungal strain.an increase in the area of the zone of inhibition was observed with the increase in concentration.the comparative of zones of inhibition of compounds with a standard against Rhizopus arrhizus.out of all synthesized compounds, compounds a1, d5, and B5 showed a higher zone of inhibition compared to terbinafine (table S7) indicating a modest antifungal profile.

Bioactivity of the developed product
Extraction of the targeted active constituents from Moringa oleifera was done by using four different solvents including, water extract, acid extract, alkaline extract and chloroform extract, and the biological evaluation of these extracts was carried out against the Rhizopus arrhizus strain.it was observed that the Moringa oleifera extract prepared in naoH showed a good antifungal activity than those prepared in other solvents (table S7).
the bioactive composite emulsion consisted of the aqueous phase containing 1 m naoH extract of Moringa oleifera extract, the oil phase containing cinnamon oil or clove oil, and a combination of surfactants including tween-80 and pEG-400 (table S8). the composition of the bioactive emulsion is shown in Supplementary table S8.Based on the bioactivity of the developed composite emulsion (Figure S10), it can be observed from table 1 that the bioactive composite emulsion batch B exhibited an excellent antifungal activity when compared with the standard drugs fluconazole (3 mm) and terbinafine (1 mm).

Conclusion
in the present study, 25 new glycosylated heterocycles that exhibit good socking scores and desired interactions with the selected targets were designed and 3mm (1 mg/ml) terbinafine 1.5 mm (1 mg/ml), 6 mm (2.5 mg/ml) synthesized, including piperazine and quinoxaline-n-oxide fragments.an antifungal study of designed molecules revealed that all of the synthesized piperazine and quinoxaline-n-oxide derivatives had the potential to be antifungal in the following order: d5> a1> B5.Compounds a1 and d5 were selected for further product development based on a variety of physicochemical considerations after being characterized using physicochemical methods, iR, and nmR spectroscopy on all synthesized piperazine and quinoxaline-n-oxide derivatives.Considering the phytochemical profile and antifungal effect of M. olifera in different solvents, the alkaline aqueous extract from Moringa oleifera leaves was chosen for product development.Finally, the essential oils including clove oil and cinnamon oil were chosen to emulsify the lead compounds a1 and d5 with the alkaline extract of M. olifera using a combination of tween-80 and pEG-400 to prepare four batches of bioactive composite emulsion.it was found that the bioactivity of the composite emulsion toward fungi was comparable to that of terbinafine and fluconazole for Rhizopus arrhizus.

Table 1 .
antifungal activity of prepared bioactive emulsion.