IN-SILICO AND IN-VITRO ANALYSIS OF POTENTIAL PLANT EXTRACTS AGAINST ASPERGILLOSIS

IN-SILICO AND IN-VITRO ANALYSIS OF POTENTIAL PLANT EXTRACTS AGAINST ASPERGILLOSIS

V. Anantha Bhairavi and R. Sathish Kumar *

Department of Biotechnology, Kongunadu Arts and Science College, Coimbatore, Tamil Nadu, India.

ABSTRACT: Cutaneous aspergillosis is a rare locally invasive disease in immunocompromised patients caused primarily by Aspergillus species which induce primary infections through injured skin, burns, or locally infect via fungal contaminated adhesive dressings or intravenous catheter insertion, resulting in lesions to erythematous papules. About 80% of aspergillosis is caused by the ubiquitous and fastest-growing airborne fungi Aspergillus fumigatus leads to mortality in immunocompromised patients. Thioredoxin systems are one of the two main antioxidant systems in maintaining their cellular redox homeostasis essential for cell viability and occurring in all life forms. Thioredoxin reductase is one of the components of Thioredoxin system responsible for the normal growth of Aspergillus fumigatus. The present study deals with the in-silico analysis of binding efficiencies between phytochemicals from different medicinal plants with the target protein thioredoxin reductase of Aspergillus fumigatus using bioinformatics tools. The phytochemicals and target protein structure were retrieved from PubChem and PDB (ID:6BPY) databases, respectively. Qikprop module was used for ADME profiling of phytochemicals and the selected phytochemicals were subsequently docked with protein using Schrodinger software. Docking studies revealed that 3’Acetyl lycopsamine from Heliotropium indicum scored the significant glide score of -6.64 Kcal/mol. Based on the docking studies, antimicrobial screening was carried out for different extracts of plants with phytochemicals that scored best with the target protein. Among the extracts, Ethyl acetate extract of Heliotropium indicum showed better antifungal activity against Aspergillus fumigatus suggesting that Heliotropium indicum can be used as a potential drug molecule against Aspergillosis.

Keywords: Aspergillosis, Heliotropium indicum, Phytochemicals, Plant extract, Thioredoxin reductase

INTRODUCTION: Cutaneous aspergillosis is a rare locally invasive disease commonly caused by Aspergillus fumigatus and Aspergillus flavus that can occur commonly in immunocompromised patients ^{1, 2}.

The infection originates as primary cutaneous via injured skin, burns or as a local infection generally associated with adhesive dressings or intravenous catheter insertion contaminated with fungus by forming single or multiple lesions, which in extreme conditions leads to the appearance of erythematous papules followed by becoming pustular, necrotic central ulceration having elevated border with black eschar ^{3, 4, 5}.

However, predominantly these infections appear secondary to systemic or disseminated Aspergillosis of lungs ⁶.

The airborne fungi A. fumigatus are the major causative agent for about 80% of invasive aspergillosis and mortality in humans, especially in patients with compromised immune system. They are ubiquitous in nature, leads to dark eschars on reddish plaques or at the site of injuries ^{7, 8}. They are responsible for the fungal keratitis that are capable of inducing blindness ^{9, 10, 11} and are considered as one among the most common airborne pathogens as they develop stress-tolerant airborne spores as a part of their lifecycle ^{12, 13, 14}. Hydrophobic cell wall can withstand a wide range of pH, temperature and are thereby efficiently dispersed in the air. These fastest-growing fungal species promote the colonization of multiple niches with small hydrophobic spores facilitating penetration ^{15, 16}.

Generally, Fungi exhibit various mechanisms to withstand antifungal resistance by the host immune system. The sulfur-based cellular system is important as they possess unique redox properties ^{17, 9, 18}. Thioredoxin systems are one of the two main antioxidant systems having Trx, Trx reductase (TrxR), and NADPH as components in maintaining cellular redox homeostasis, which is essential for the viability of cells and occurs in all forms of life ^{19, 20, 21}. The low molecular weight form of these dimeric flavoenzymes exhibits specificity for Trx substrate in fungi and catalyze the electron transfer from NADPH to dithiol, which is a redox-active of Trx and is also responsible for the antioxidant defense and synthesizes DNA ^{22, 23, 24}.

Although various drug combinations and antifungal agents have been used against these infections, most lead to multidrug resistance and other side effects on prolonged or uncontrolled usage, suggesting the need for novel drugs ^{25, 26}. Since ancient times, medicinal plants have been extensively used for a wide spectrum of diseases and infections due to their rich phytochemicals and are considered pharmaceutical lead ²⁷. Ethnopharmacological studies have shown that a plant's phytochemicals can exhibit activities synergistically or by a single compound ^{28, 29}. Plants like Heliotropium indicum were used in folklore medicine with its leaf paste against various ailments, and they possess excellent wound healing activity ^{30, 31}.

Some plants like Grona triflora have anticonvulsant, antinociceptive, antibacterial, anti-inflammatory, and antioxidant activities ^{32, 33}. Although plant-based medicines are effective against diseases, they may sometimes lead to adverse health effects. These side effects are associated with poor drug target interactions or increased compound toxicity ²⁸. In-silico studies help to overcome these by predicting the protein target interactions, and prior optimization of the drug can be carried out at an early stage of development from the most suitable drug compound ³⁴. The present study focuses on the structure-based identification of plants whose phytocompounds can actively target the protein thioredoxin reductase of Aspergillus fumigatus followed by their in-vitro study.

MATERIALS AND METHODS:

Databases: Protein Data Bank (www.rcsb.org) database was utilized to retrieve the 3D structure of the target protein Thioredoxin reductase, and their active site residues were predicted via Ligsite online tool (http://projects.biotec.tu-dresden.de/pocket/) ^{35, 36}. Structure of phytocompounds from plants like Heliotropium indicum, Grona triflora, Evolvulus alsinoides, Ziziphus mauritiana, Commelina benghalensis, Aristolochia bracteolata, Pyrus communis and Coccinea grandis were retrieved from PubChem database (http://www.ncbi.nlm.nih.gov/pccompound) and scrutinized for docking studies. Their pharmacological profile was verified with PASS online tool ³⁷.

Prediction of Drug Likeliness: Initially, LigPrep module (Schrodinger, LLC, NY, USA, 2009) of Maestro 9.0.211 version of the Schrodinger suite was employed to optimize the ligands via converting the two-dimensional structure of the ligands into the three-dimensional structure by the addition of hydrogen atoms. The geometry of the ligands can be optimized with processes like correction, energy minimization, and ionization ^{38, 39, 40}. After preparing the ligands, the pharmacological properties of phytocompounds can be validated by ADME profiling with various parameters of QikProp module of Schrodinger ⁴¹. Parameters like donor hydrogen bond, acceptor hydrogen bond, molecular weight, skin permeability, blood brain barrier coefficient and Lipinski’s rule of three and five were analyzed. Lipinski’s rule of five helps discerning drug-like compounds from non-drug-like compounds ⁴². Phytocompounds exhibiting sensible results were considered for docking studies with target protein ⁴³.

Protein Preparation and Receptor Grid Generation: Protein Preparation Wizard of Schrodinger software performed the protein preparation that adds hydrogen atoms to rectify missing side chains and residues and stabilize the charges ⁴⁴.

The X-Ray crystallographic structure of proteins may be bound to water molecules that affect the docking process thus the water molecules were removed to increase the entropy of target molecules followed by optimization and minimization ³⁸.

The protein's active site was identified by generating a grid box where water molecules and hetero atoms are removed via the receptor grid generation module of Maestro. The area of interaction between the ligands and protein was defined with the help of the grid generated ^{45, 46}.

Molecular Docking: Based on the ADME profile, the compounds that passed the drug-like parameters were docked against the target protein via GLIDE module of Schrodinger suite. GLIDE scores provided will represent the binding free energy of the interactions ⁴⁷.

Plant Collection and Extract Preparation: Plants like Heliotropium indicum and Grona triflora were collected from Kanyakumari district and their identity was confirmed by The Botanical Survey of India, Coimbatore (BSI/SRC/5/23/2022/Tech/517and BSI/SRC/5/23/2022/Tech/516). Leaves of the collected plants were shade dried and powdered separately. About 25 g of each powdered sample was placed in the thimble, and sequential Soxhlet extraction was carried out with 250ml of solvents liken-Hexane, Ethylacetate and Methanol. The solvents were then evaporated and the concentrated extracts were taken for further study ⁴⁸.

Antifungal Activity: The fungicidal activity of the extracts was screened against Aspergillus fumigatus clinical isolates procured from Bioline Laboratory, Coimbatore. Agar well diffusion method was performed using Sabouraud dextrose agar medium where overnight cultures were prepared by inoculating the fungal strains in the SDA broth and incubated at 28°C.

The cultures were then swabbed on to the agar medium and 6mm wells were punched using sterile cork and borer. Each well was loaded with 50µl (2mg/ml) of each extract and incubated at 28°C. The diameter of zone of inhibition was measured in mm with standard Ketoconazole ^{49, 50}.

RESULTS AND DISCUSSION:

Protein Structure Retrieval: The 3D structure of protein Thioredoxin reductase was retrieved from PDB with PDB ID: 6BPY as shown in Fig. 1.

FIG. 1: 3D STRUCTURE OF THIOREDOXIN REDUCTASE FROM PDB

ADME Analysis: Phytocompounds structures were retrieved and analyzed for their ADME properties. The parameters of drug ability like rotatable bonds, acceptor and donor hydrogen atoms, molecular weight and Lipinski’s rule of five limits were fulfilled by these 18 compounds as shown in Table 1.

TABLE 1: ADME PROFILE OF PHYTOCOMPOUNDS USING QIKPROP MODULE

Molecular Docking: Docking studies revealed that from the 18 compounds taken only 17 compounds successfully docked as shown in Table 2 with the target in which 3̍Acetyllycopsamine showed the best G score of -6.64 Kcal/mol with the residues ALA124, ASP294, GLN45 and THR49 having bond lengths 1.9, 2.1, 2.5 and 2.0 Å respectively was visualized using PYMOL software as given in Fig. 2. This was followed by Indicine-N-Oxide and Quercetin with significant G score of -6.54 and -6.36 Kcal/mol. Compounds like 3̍Acetyl-lycopsamine, Indicine-N-Oxide, Caffeic acid, Syringic acid and Vanillic acidfrom Heliotropium indicum exhibited good GLIDE scoresof -6.64, -6.54, -5.30, -4.64 and -4.30Kcal/mol respectively. Followed by this, Compounds like Quercetin and Melilotic acid from Grona triflora showed promising interactions with G score -6.36 and -5.28 Kcal/mol. In addition to that, Arbutin from Pyrus communisalso exhibited a good interaction of -6.02 Kcal/mol. Clindamycin from the plant Ziziphus mauritiana exhibits a significant interaction of -5.33 Kcal/mol. From the plant Evolvulus alsinoides the compound Cytidine bounds to the target with a score of -5.20 Kcal/mol whereas Luteolin from the same plant exhibited good interactions by forming seven bonds with the target protein together with a G score of -3.47 Kcal/mol. Compounds like 3̍Acetyllycopsamine and Arbutin effectively bound to the active site pockets GLN45, SER11 and ASN 125 amino acid residues of target protein. Similarly, Indicine-N-Oxide and Caffeic acid bound to the active sites SER 11 where Indicine-N-Oxide also bound to the active site ASN 125 and Caffeic acid towards ASP 294 active sites. Apart from these compounds, Melilotic acid and syringic acid also effectively bound to the active site pockets of the target protein GLN 45, ASN 125, ASP 294 and SER 11. However, Ethisterone from Coccinea grandis alone showed no particular interactions with the target protein.

TABLE 2: INTERACTION OF PLANT COMPOUNDS WITH 6BPY

FIG 2: DIAGRAMMATIC REPRESENTATION OF INTERACTION BETWEEN 3’ACETYLLYCOPSAMINE WITH THIOREDOXIN REDUCTASE (6BPY). (a) represents the surface image of protein in which the pale green colour compound bounds to the active pocket of the target protein in deep olive colour. (b) represents the cartoon image of the interaction with amino acid residues with which the compound binds with bond lengths.

In-vitro Antifungal Studies: From the docking results, it was validated that the greatest number of phytocompounds from Heliotropium indicum and Grona triflora showed better ADME and docking results. Thus, the results of antifungal study conducted for different extracts of these plants are shown in Table 3.

TABLE 3: ANTIFUNGAL ACTIVITY OF EXTRACTS AGAINST ASPERGILLUS FUMIGATUS

Results revealed that Ethyl acetate leaf extract of Heliotropium indicum effectively inhibits the growth of Aspergillus fumigatus with a maximum of 34mm zone of inhibition when compared with Grona triflora extracts and standard drug as given in Fig. 3.

FIG 3: ANTIFUNGAL ACTIVITY OF PLANT EXTRACTS AGAINST ASPERGILLUS FUMIGATUS. (a) represents the antifungal activity of different extracts of Heliotropium indicum and hexane extract of grona triflora (d.t). b) represents the antifungal activity of ethyl acetate and methanol extracts of Grona triflora (d.t) and standard ketoconazole.

DISCUSSION: Aspergillus species are ubiquitous in nature that are capable to cause infections in human body like skin, lungs and even the central nervous system ⁵¹. Azole-based drugs are extensively used against these infections as they inhibit the ergosterol synthesis pathway, thereby affecting the fungal cell membrane. However, due to prolonged exposure to these drugs during treatment results in drug resistance that are responsible for the increase in mortality rate ^{52, 53}.

Thus, the current study is targeted against Aspergillus fumigatus to identify efficient drug molecules using several bioinformatic tools. Molecular docking is one of the most widely used strategies in the structure-based drug designing (SBDD) technique, which exploits the structural properties of targets for drug development and extensively studies the ligand-target interaction with their binding energies ^{54, 55}. Since, studies have revealed that targeting Thioredoxin reductase fungal proteins can reduce the growth of fungi, they were chosen as a target to evaluate their interactions with potential therapeutic candidates. They also showed limited similarities with human proteins and are shown to be substantially expressed during aspergillosis ^{56, 57}. They significantly impact thioredoxin's redox reaction, which is important for various physiological processes and pathological conditions in various organisms, leading to conditions like apoptosis and even autoimmune diseases ⁵⁸. As a potential methodology that may be used in the early phases of drug development, in-silico approach was used in this research as a preliminary step to determine the potent anti-fungicidal plants from the selected medicinal plants followed by in-vitro analysis ⁵⁹. Plants primarily produce bioactive components or secondary metabolites that possess various pharmacological properties ⁶⁰.

The presence of these metabolites provides medicinal properties like antimicrobial properties to plants where these compounds might have disrupted the cell wall or inhibited biofilm formation ⁶¹. The structures of phytocompounds collected from PubChem databases were subjected to ADME analyses 62 to balance the important features of a drug-like molecule. It was evident from the ADME profiling data that the majority of compounds from plants that passed the test were in the order of Heliotropium indicum, Grona triflora, Evolvulus alsinoides, Ziziphus mauritiana, Commelina benghalensis, Aristolochia bracteolata, Coccinea grandis and Pyrus communis. Due to the widespread belief that natural remedies are safe, the use of herbal medicines is rising dramatically nowadays. However, natural products can occasionally cause adverse effects by modifying conventional drugs' performance ²⁸.

Therefore, the compounds' biological activities have been understood and validated using the PASS online programme, which confirmed that all the compounds that passed the ADME exhibit drug-like properties. Molecular docking experiments have been performed to anticipate potential drug molecule binding orientations with the target protein ^{42, 63}. According to docking experiment results from Table 3, more phytocompounds from Heliotropium indicum and Grona triflora plants have substantial G scores for binding to the target protein's active residues. Studies also revealed that Heliotropium indicum and Grona triflora extracts have been extensively used against skin infections for years ^{63, 64}.

The compound 3’Acetyllycopsamine from Heliotropium indicum is the most effective compound when compared to other compounds suggesting that it could be an excellent lead against the organism Aspergillus fumigatus. From Table 2 it is evident that apart from 3’acetyllycopsamine, the compound Indicine-N-Oxide from Heliotropium indicum also possesses significant interactions towards the target whose anticancer activity was already reported ⁶⁵. Quercetin also exhibited a G score of -6.36 Kcal/mol with three bonds whose antifungal activity has already been reported ⁶⁶. However, in-silico interaction with Thioredoxin reductase of Aspergillus fumigatus was not reported early. Table 2 indicates that Arbutin from Pyrus communis, in addition to the aforementioned compounds, also exhibited high binding activity towards the target; nevertheless, plants with more ADME passed docked compounds were taken into consideration for the following investigations. In order to validate the results of docking, in-vitro antifungal screening was conducted with different extracts of plants that ranked first and second, with the greatest number of compounds having significant docking scores. Different parts of the plants were considered for the in-vitro screening as the phytochemical profile of a plant varies with different parts of the plant ⁶⁷.

Ethyl acetate leaf extracts of Heliotropium indicum and Grona triflora inhibited the fungal growth effectively when compared with other solvent extracts indicating the effect of sequential solvent extraction ⁴⁸. Hence, the results suggest that the leaf extracts of Heliotropium indicum can be an excellent source of antifungal agent as they possess several active phytocompounds that was observed to be a potential drug compound.

CONCLUSION: Primary cutaneous aspergillosis caused via skin injury is often connected with cutaneous invasive aspergillosis, which sometimes leads to mortality. Development of multidrug resistance and prolonged usage of drugs result in side effects disseminating the need for novel medicines. The present study suggests that Heliotropium indicum and Grona triflora possess activity against the growth of tested fungi. Further studies will be carried out to understand the antifungal profile of extracts from these plants, thereby analyzing their stability.

ACKNOWLEDGEMENT: The authors thank the administration of Kongunadu Arts and Science College, Coimbatore, for providing the software to carry out this work.

Author Contributions: Anantha Bhairavi V had done the ADME, Docking, antifungal studies and drafted the manuscript. Sathishkumar R had designed the study, edited the manuscript and guided to carry out this work. The final manuscript was read and approved by all authors.

Funding: No funding was received for conducting this study.

Declarations:

Ethical Statement: This article does not contain any studies involving animals and human participants by any of the authors.

CONFLICTS OF INTEREST: We declared no conflict of interest in the studies.

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    How to cite this article:

    Bhairavi VA and Sathish KR: In-silico and in-vitro analysis of potential plant extracts against aspergillosis. Int J Pharm Sci & Res 2023; 14(8): 4142-52. doi: 10.13040/IJPSR.0975-8232.14(8).4142-52.

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