Identification of effective plant extracts against candidiasis: an in silico and in vitro approach

Abstract

Background

Globally, millions of people suffer from repeated fungal infections affecting the skin, keratinized tissues, and mucosal membranes. Approximately 1.7 million cases result in death with an elevated incidence rate among immunocompromised people that may later result in severe infections. Among the causative agents, Candida albicans are the most prevalent fungi inducing subcutaneous to invasive candidiasis. Although they are commensals in human body alteration in gut or prolonged treatments results in candidiasis. Several virulence proteins of C. albicans are involved in infections and secreted aspartic proteases2 (SAP2) plays an important role among them by causing damage to the reconstituted human epithelium. In the present study, phytochemicals from Heliotropium indicum, Grona triflora, Ziziphus mauritiana, Atalantia racemosa, Coccinia grandis, Caryota urens, Aristolochia bracteolata, Evolvulus alsinoides, Pyrus communis and Commelina benghalensis were studied against SAP2 with the help of bioinformatic tools to understand their binding efficiency.

Results

The phytochemical structures were retrieved from PubChem database and the target protein structure was retrieved from PDB database with ID:3PVK. ADME profiling for phytochemicals was performed with Qikprop module, followed by docking with protein using Schrodinger software. Docking studies showed that Indicine-N-Oxide from H. indicum scored the significant glide score of − 5.54 kcal/mol. Finally, antifungal studies against C. albicans were conducted using several extracts of plants containing phytochemicals with considerable glide scores on docking studies. The Ethyl acetate leaf extract of H. indicum prominently inhibited the fungal growth when compared with the control.

Conclusion

Identification of effective therapeutic candidates for the treatment of fungal infection is facilitated by the potential of H. indicum to hinder fungal growth and the interaction of their phytocompounds with fungal targets paves a way for developing a novel drug.

Background

Mycoses or fungal skin infections are the leading cause of human skin infections caused by dermatophytes or yeast. Although these infections are non-fatal, they are prone to superficial life threatening conditions in immunocompromised patients. According to a global survey, around 150 million people suffer from severe fungal infections and 1.5 million death cases were reported in which approximately 700,000 cases were due to invasive candidiasis [1]. The broad term Candidiasis refers to cutaneous, mucosal or organ infections through fungi of Candida genus that results in white patches on skin [2,3,4]. Candida is used to describe a class of fungi having more than 150 species. Among various species of Candida, C. albicans is one of the most common causes of Cutaneous candidiasis as they can grow on moist areas of the body and cause superficial infections characterized by the formation of lesions to wide rashes with high frequency in diabetic and HIV patients [2]. They are dimorphic commensals in healthy individual skin, mucosa and gastrointestinal tract which when imbalanced results in overgrowth and induce candidiasis [5,6,7]. These organisms are able to form biofilms that helps them to develop antifungal resistance by two mechanisms. In the first step, biofilms reduce the drug ability to penetrate followed by the survival of organisms under the increased oxidative stress by the development of persister cells that uses various mechanisms [8, 9]. Risk factors associated with candidiasis include long term usage of various drugs, rupture of mucosal or cutaneous barriers or via immunosuppression [10]. These yeast-type fungus are opportunistic fungi responsible for candida intertrigo in all hair bearing skin causing white patches on the skin where the epithelial cells are damaged by the cytolytic peptide toxin called candidalysin [11,12,13,14]. For the physiology and development of fungi proteolytic enzymes plays an important role by promoting morphogenesis and metabolism of fungi [15, 16]. Proteases are a family of protein comprises of ten members in which secreted aspartic proteinase1 (SAP1), secreted aspartic proteinase2 (SAP2) and secreted aspartic proteinase3 (SAP3) are responsible for the damage in the reconstituted human epithelium (RHE) followed by adhesion and are secreted by the yeast form [17,18,19,20]. The optimal pH for the SAP activity ranges from 2.0 to 7.0 thus they may be considered as a virulence factor in human infections [20]. Candidapepsin2 are also called as secreted aspartic proteinase2 (SAP2) capable of digesting C3b, C4b, and C5 complement components of host and cause inhibition of terminal complement complex formation (TCC) are one among the SAP genes required for the disease development and tissue degradation [21, 22]. Although various treatments are available with azole drugs and polyenes, prolonged usage of which can leads to resistance and as the available drugs are expensive and some are nephrotoxic it is better to choose an alternative drug [23,24,25]. Medicinal plants have been used traditionally for various ailments due to the presence of myriads of phytocompounds. Some of them have proved to be effective antifungal agents as crude or as a pure compound [26,27,28,29]. Plants like Heliotropium indicum have been widely used as a folk medicine due to its antimicrobial and anti-inflammatory effect [30, 31]. Due to its medicinal properties, survey has shown that Grona triflora can also be employed to treat various ailments [32]. However, scientific evidence is required in order to understand how these plant components interact with in pathogenesis of candidiasis. The current study focuses on the analysis of various phytocompounds selected from 10 different medicinal plants possessing activity against skin infections based on literature survey through their binding efficiency with the protein candidapepsin2 of C. albicans, followed by in vitro antifungal studies.

Methods

Protein structure retrieval

The 3D structure of Candidapepsin2 (SAP2) protein of Candida albicans was retrieved from Protein Data Bank (www.rcsb.org) [33]. The active site of the protein was predicted using online tool Ligsite (http://projects.biotec.tu-dresden.de/pocket/) [34].

Ligand structure retrieval

Based on the literature survey, structure of phytocompounds from 10 different medicinal plants like H. indicum, G. triflora, Caryota urens, Evolvulus alsinoides, Ziziphus mauritiana, Atalantia racemosa, Commelina benghalensis, Aristolochia bracteolata, Pyrus communis and Coccinea grandis was retrieved from PubChem database (http://www.ncbi.nlm.nih.gov/pccompound) [35].

Preparation of proteins and ligands

Maestro 9.0 version of Schrodinger suite was used for Protein preparation where protein preparation wizard was used to add hydrogen atoms and remove the water molecules within het groups [36]. Using algorithms optimization of 3D and ligand geometry were carried out with minimization of energy and LigPrep module (Schrodinger, LLC, NY, USA, 2009) was used for ligand preparation where addition of hydrogen atoms can generate the 3D structure of ligands [37, 38].

ADME studies

The properties of absorption, distribution, metabolism, excretion and toxicity of the ligand are a vital step in drug designing which was determined with Qikprop module in the Schrodinger suite [39] where the selected phytocompounds were assessed to find their drug likeliness within the parameters like rotatable bonds, molecular weight, dipole moment, hydrogen bond acceptor, hydrogen bond donor, blood brain barrier coefficient, human oral absorption, skin permeability and Lipinski’s rule of three and five on QikProp module. Only those compounds that have significant ADME profile were considered for further docking studies and their pharmacological effects were validated using PASS online tool [40, 41].

Docking studies

Grid-based Ligand Docking with Energetics (GLIDE) module of Schrodinger software (http://www.schrodinger.com/) was used for to identify the ligand protein interaction and their significant binding efficiencies with the help of glide score using XP (Xtra Precision) visualizer in which the best hits can be identified [42, 43].

Preparation of plant extract

Plant collection

Whole plants of Heliotropium indicum and Grona triflora were collected from Kanyakumari District, Tamil Nadu between November 2021 and March 2022. The collected plant materials were authenticated (BSI/SRC/5/23/2022/Tech/517 and BSI/SRC/5/23/2022/Tech/516) from the Botanical Survey of India, Southern Regional Centre, Coimbatore.

Extract preparation

Leaves, stem and root of the plants were washed, shade dried and grinded with a blender. About 50 g of each powdered samples were taken for Soxhlet extraction with 500 ml analytical grade Hexane, Ethyl acetate and Methanol solvents based on the polarity. After the extraction the crude extracts were concentrated and dried [44].

Antifungal screening

Antifungal studies of different plant extracts were carried out using Agar well diffusion method [45]. Overnight cultures of Candida albicans (MTCC 183) were swabbed on the Sabouraud Dextrose agar plates, 6 mm wells were punched and loaded with 100µl of different extracts (1 mg/ml) and kept for overnight incubation at 37 °C. After incubation the diameter of the zone of inhibition was measured for each well where Fluconazole was used as positive control and the respective solvents were taken as negative control [46].

Results

Structure retrieval

Structure of phytocompounds from different plants was retrieved from PubChem database with their PubChem ID. Three-dimensional structure of target protein was retrieved from PDB with ID:3PVK (Fig. 1) with about 342 amino acids in length whose active site residues were found to be THR222, THR221, ASP86, ASP32, TYR84, TYR225, ASP218, GLY220 and GLU193.

Fig. 1

Three-dimensional structure of Secreted aspartic proteinases2 from PDB

ADME studies

Three-dimensional structure of ligands generated was taken for ADME studies where seven compounds (Indicine-N-Oxide, 3′ Acetyl lycopsamine, p-Coumaric acid, Ferulic acid, Syringic acid, Vanillic acid and Caffeic acid) from H. indicum and four compounds (Melilotic acid, Phloretic acid, Kaempferol and Quercetin) from G. triflora, two compounds (Cytidine and Luteolin) from E. alsinoides and one compound each from Z. mauritiana, C. grandis, A. bracteolate, P. communis and C. benghalensis, respectively, showed drug likeliness in ADME profiling. Their PubChem structures and ADME profile was shown in Table 1 and Table 2, respectively. Pharmacological effects of these compounds were validated with PASS prediction where no antifungal activity was found for Indicine-N-Oxide and only the antibacterial and antiviral activities are recorded (Table 1).

Table 1 Retrieved 2D structure of phytocompounds with PubChem ID and PASS online result

Table 2 ADME profile of phytocompounds using QikProp Module

Structure based docking studies

Docking studies provides an insight of how each phytocompounds binds to the active pockets of the target protein (PDB ID: 3PVK) based on their structure. It was found that among the phytocompounds Indicine-N-Oxide from Heliotropium indicum exhibited the better binding efficiency with target with a significant glide score of − 5.54 kcal/mol when compared with the standard Fluconazole possessing a G score of − 4.66 kcal/mol followed by phytocompounds like Clindamycin, Arbutin, 3′Acetyl lycopsamine and Quercetin with glide scores of − 5.20, − 5.18, − 5.06 and − 4.98 kcal/mol, respectively, with hydrogen bond formation (Table 3). The binding mode of Indicine-N-Oxide with target protein was visualized using PYMOL visualizing tool where their mode of interaction was found to be with the formation of hydrogen bond. Significant interactions were observed with active site residues TYR84 and ASP86 having bond lengths 1.9 and 2.0 Å, respectively, as shown in Fig. 2 where cyan green colour represents the ligand Indicine-N-Oxide and deep olive colour represents the active pocket of the target protein. The interaction of fluconazole with the active site residues of the target protein was also visualized using PYMOL software (Fig. 3). Most of the compounds from the plant Heliotropium indicum like Indicine-N-Oxide, Syringic acid, Ferulic acid and Vanillic acid bound to the active sites TYR84 of the target protein with the formation of H–O bond. Similarly, Quercetin, Kaempferol and Melilotic acid from the plant Grona triflora also bound to the active site residues ASP32, TYR84 and THR222 of the protein. Compounds like Cytidine, Luteolin from Evolvulus alsinoides and Clindamycin from Ziziphus mauritiana formed five bonds on interacting with the protein. These compounds had successfully bound to the active site residues ASP32, GLU193 and THR222 of the target. However, compounds like 21-Diazoprogesterone, Ethisterone and Phloretic acid showed no interactions with the target protein.

Table 3 Interaction of Phytocompounds with Candidiapepsin2 (SAP2)

Fig. 2

Interaction of Indicine-N-Oxide with Candidapepsin2 with least Glide Score − 5.54 kcal/mol

Fig. 3

Interaction of Fluconazole with Candidapepsin2 with least Glide Score − 4.66 kcal/mol

Antifungal screening

Based on the docking results two plants were selected as most of their phytocompounds exhibited better ADME profile and binding efficiencies compared to other plants. Among the different extracts of plant samples, Ethyl acetate leaf extract of Heliotropium indicum shows a maximum diameter of zone of inhibition of 18 mm when compared to all other extracts (Fig. 4). Both positive and negative controls exhibited no efficacy against the organism as depicted in Table 4.

Fig. 4

Antifungal activity of plant extracts against Candida albicans by Agar well diffusion method. a Represents the antifungal activity of methanolic extracts of root (R), stem (S) and leaf (L), b represents the antifungal activity of hexane extracts of root (R), stem (S) and leaf (L), c represents the antifungal activity of ethyl acetate extracts of root (R), stem (S) and leaf (L), d represents the control plate with methanol (M), ethyl acetate (EA), hexane (H)and distilled water (DW) as negative controls and a positive control Fluconazole (Flu)

Table 4 Antifungal activity of Plant Extracts against Candida albicans

Discussion

Skin is a protective barrier of the body that carries numerous microbes as commensals for a healthy body which includes bacteria and fungi species within that the fungi C. albicans plays an important role in human microbial biota [47]. Although these are beneficial, at times they may progress into infectious agent affecting the skin and mucosal membranes which is also a major concern. Studies have considered fungal proteases as virulent factors as they can interfere with many biological assays and thereby results in inflammation in the host at lower pH and interfere the immune system [15, 21, 48]. Reports have been revealing the importance of SAP2 virulence protein in the pathogenesis of candidiasis in diseased models [49, 50]. Thus, the current study focussed on SAP2 protein to identify novel safe drug against candidiasis. The target protein of this study obtained from PDB possess chain A with 342 amino acids sequence length and 36.92 k Da in weight. Generally, drug designing was carried out with clinical trials as an important step to understand the pharmacokinetic profile of the drug compound their side effects and metabolism. However, this stage often leads to adverse findings which results in the failure of the drug development as the compound studied may not be suitable for a particular disease [51]. Thus, employing computational approach of ADME profiling for compounds prior to in vitro and in vivo studies helps to reduce these issues [52]. In the present work, Computational approaches have been extensively used to identify potent phytocompounds containing medicinal plants to target C. albicans causing Candidiasis. All phytocompounds were selected from different plants based on their literature survey and subjected to ADME profile and found that only a few showed the drug likeliness within the given parameters of Lipinski’s rule of five suggesting that they possess excellent pharmacological properties to be utilized in drug development. Poor drug likeliness of the compounds in the ADME toxicity studies implies that those compounds have poor absorption and permeability in the human body which in turn affects the bioavailability of drugs and results in drug failure [53]. From Table 2 it is evident that most of the compounds that showed drug likeliness in ADME toxicity profile are phenols, flavonoids and alkaloids that possess properties like good absorption contributing to the drug bioavailability. According to studies, Molecular docking plays a vital role in drug discovery process with most promising candidates from ADME profile were considered [54, 55]. Docking helps us to understand the binding affinity and interactions of compounds with the active pockets of target [56]. Thus, we performed docking studies for compounds showing drug likeliness in ADME profiling and only those compounds with their structures have been discussed throughout the study. In silico docking studies of the compounds in the present study revealed that for each compound the interaction differs due to their differences in interacting moieties. However, most compounds that were subjected to docking successfully binds to at least one active site residues of the protein with the formation of hydrogen bond. The removal of water molecules and other residues from the protein structure improves the ability of drug target interactions. Based on the findings, it's obvious that the phytocompounds from H. indicum and G. triflora seems to be more drug-like and actively interacted with Candidapepsin2, suggesting that these plants could be exploited to establish effective Candidiasis therapies. Studies have showed that Indicine-N-Oxide is the active component responsible for the antitumour activity of H. indicum [57]. Present study revealed that Indicine-N-Oxide also possess better antifungal activity against Candidapepsin2 when compared with all other phytocompounds and standard drug taken for study. Therefore, presence of this active component may be responsible for the effectiveness of H. indicum as traditional folk medicine against infections followed for several years. Figure 3 represents how the standard drug fluconazole bound to the active site THR222 and TYR84 of the target protein where salmon colour represents the drug and deep olive green colour represents the active sites of the target protein. With the formation of O–H and H–O bonds having bond lengths 2.0 and 1.9 Å respectively the standard molecule also effectively binds to the target. From these docking results it is evident that the phytocompound present in Heliotropium indicum and Grona triflora exerts much efficiency than the standard drug against Candida albicans. However, further studies are also required to validate the effect of these compound against infections. Apart from Indicine-N-Oxide, the compound Quercetin also exhibited significant interactions (Additional file 1: Fig. S5) whose antifungal activity has been proved in previous studies [58]. Current study elucidates its efficiency in binding to the drug target SAP2 as shown in Table 3 with the formation of 3 bonds. As we have implicated in silico studies as a prior step in identification of effective medicinal plants to develop phytomedicine against fungal infection, it was observed that more phytocompounds from the plants H. indicum and G. triflora exhibited drug likeliness and successfully docked with the target protein in its active sites. Thus, those two plants were chosen for screening antifungal activity and from the results it is evident that ethyl acetate leaf extracts of both the plants showed antifungal activity whereas ethyl acetate leaf extracts of H. indicum possess potential anticandidal activity when compared to G. triflora. However, fluconazole does not exhibit antifungal potential on in vitro studies which may be due to the drug resistance developed by the strain used in the current study. The plant H. indicum was reported to possess various medicinal properties like anti-inflammatory and antitumour activity wherein its leaf paste was used for skin related ailments traditionally [59]. Current study also reveals that leaves of the plant possess potent antifungal activity when compared to other parts of the plant. Recently, studies have shown that the ethyl acetate leaf extracts of O. americanum exhibited antimicrobial activity against varied microbial species [46]. Similarly, in vitro antifungal screening unveils that ethyl acetate extract among other extracts possess better antifungal activity and the plant H. indicum rich in alkaloids and phenolic compounds are excellent drug candidates against fungi.

Conclusion

Candidiasis is a fungal infection caused Candida limited to skin and mucous membranes often leads to systemic infections. Although it is not life threatening, it may be one of the causes for other chronic lethal diseases. Prolonged usage of drugs available in the market results in resistance and side effects which paves a way for efficient drug discovery. In the present study, suitable drug candidates were screened from ADME results and subjected to docking against target unveiled that Indicine-N-Oxide from H. indicum have significant binding affinity towards TYR84 and ASP86 residues of target with glide score − 5.54 kcal/mol. In addition to that, antifungal screening showed the ability of inhibiting the growth of C. albicans by H. indicum leaf extracts. Further studies will be conducted to evaluate the stability of compounds and to understand the efficiency of H. indicum and G. triflora plant extracts against candidiasis.