Saudi Journal of Biological Sciences 29 (2022) 3899–3910

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Saudi Journal of Biological Sciences

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Original article

Platyphylloside, a potential inhibitor from epicarp of B. aegyptiaca

against CYP450 protein in T. rubrum – In vitro and in silico approaches
Mohamed Hussain Syed Abuthakir a, Munirah Abdullah Al-Dosary b, Ashraf Atef Hatamleh b,
Hissah Abdulrahman Alodaini b, P. Perumal c, Muthusamy Jeyam a,⇑
a
Department of Bioinformatics, Bharathiar University, Coimbatore, Tamil Nadu, India
b
Department of Botany and Microbiology, College of Science, King Saud University, P.O.Box 2455, Riyadh 11451, Saudi Arabia
c
Laboratoire Information Genomique et Structurale (IGS), Marseille, France

a r t i c l e i n f o a b s t r a c t

Article history: Trichophyton rubrum is one of the major disease causing pathogens in human; mainly it causes tinea
Received 13 December 2021 pedis, tinea cruris and tinea corporis. Cytochrome P450 which considered to be an important protein that
Revised 3 March 2022 can impact ergosterol biosynthesis pathway. B. aegyptiaca is rich source of secondary metabolites with
Accepted 8 March 2022
tremendous medicinal values and it has sweet pulp, leaves with spine, strong seed and oily kernel. The
Available online 12 March 2022
epicarp of the fruit was taken for this study to inhibit T. rubrum using in vitro and in silico techniques.
The epicarp portion was extracted using various solvents and water. The anti-dermatophytic activity
Keywords:
on T. rubrum of these extracts was assessed utilizing poison plate technique with 5 individual concentra-
Balanites aegyptiaca
Trichophyton rubrum
tions. The fractioned chloroform extract of epicarp had fully inhibited the growth of T. rubrum at 3 mg/ml.
CYP450 Further, the chloroform extract was subjected to LC-MS analysis, in total, 40 compounds were elucidated.
Platyphylloside Then, the derived compounds were included for predicting ADMETox properties using Qikprop module.
MD simulation From the analysis 40 compounds were identified to be eligible for docking process. Then the desirable
compounds, drug Ketoconazole were subjected to docking analysis using Glide module of Schrödinger.
It shows that Platyphylloside has better docking result than other compounds and drug Ketoconazole.
Further, MD simulation was carried out for Ketoconazole-Cyp450 and Platyphylloside-CYP450 complexes
using Desmond, Schrödinger. MD simulation study also confirmed that the Platyphylloside-CYP450 com-
plex more stable. This study suggests that Platyphylloside may act as potential inhibitor and it could be
further subjected to experimental analysis to inhibit the T. rubrum growth.
Ó 2022 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Dermatophytoses or Tinea are a type of skin infections in

human and other living organisms caused by group of filamentous
Abbreviations: B.aegyptiaca, Balanites aegyptiaca; T.rubrum, Tricgophyton pathogenic fungi called Dermatophytes (Behzadi et al., 2014; Kaul
rubrum; NCBI, National Center for Biotechnology Information; BLAST, Basic Local et al., 2017). Microsporum, Trichophyton, Epidermophyton cause der-
Alignment Search Tool; ADME-Tox, Absorption, Distribution, Metabolism, Excretion matophytoses by digesting the keratin of the host by producing
and Toxicity.
⇑ Corresponding author. keratinases and it grows in keratinized tissues like skin, hair, nail,
E-mail addresses: [email protected] (M.H.S. Abuthakir), [email protected].
feathers, hooves and horns (Burmester et al., 2011; Navone and
sa (M.A. Al-Dosary), [email protected] (A.A. Hatamleh), [email protected] Speight, 2018).
(H.A. Alodaini), [email protected] (P. Perumal), [email protected] Trichophyton rubrum is anthropophilic in nature. It is the pre-
(M. Jeyam). dominant disease causing agent accounting for 69.5% followed by
Peer review under responsibility of King Saud University. other species of Trichophyton (Bristow and Spruce, 2009). Most
commonly T. rubrum causes the infections in the area of feet, groin,
hands and scalp throughout the world (Blutfield et al., 2015). Pro-
tein Lanosterol 14a-demethylase encrypted by the gene CYP51, is
Production and hosting by Elsevier the most conserved protein in the CYP (CYP450) superfamily and

https://doi.org/10.1016/j.sjbs.2022.03.017
1319-562X/Ó 2022 The Author(s). Published by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Mohamed Hussain Syed Abuthakir, Munirah Abdullah Al-Dosary, Ashraf Atef Hatamleh et al. Saudi Journal of Biological Sciences 29 (2022) 3899–3910

it has more specificity. CYP450s have concerned in various biolog- development of mycelia of T. rubrum was analysed every day. After
ical processes, together with production of basic and secondary 21 days of incubation the pathogen inhibitory percentage (I) was
products in the pathogen (Lamb et al., 2007). measured using the below formula stated below,.
All azole drugs have the same function of inhibiting the protein
I ¼ fðDcontrol  DtreatedÞ=Dcontrolgx100
Cytochrome P450 14a-demethylase. The azoles like Ketoconazole,
Itraconazole, Fluconazole have the great capacity to inhibit the Experiments were carried out in triplicates. The results were
growth of dermatophytes especially T. rubrum (Ghannoum, expressed as the mean ± standard error (SE). Data were analyzed
2016). Major steroid of fungal cell membrane exists in ergosterol statistically using SPSS software version 20.
that regulates fungal natural functions such as fluidness and integ-
rity (Martinez-Rossi et al., 2008). Blocking of ergosterol biosynthe- 2.4. Minimum Inhibitory Concentration (MIC)
sis and accumulation of other sterols on cell membrane lead to
increased membrane rigidity and permeability, interrupt Two fold broth dilution methods were followed in line with tips
membrane-bound enzymes, pathogen growth inhibition and of NCCLS for filamentous fungi (M38-A2) for examining the Mini-
finally cell death (Zhang et al., 2007; Parker et al., 2014). mum Inhibitory Concentration (MIC). 21 days old culture of T.
According to WHO, 75% of complete population happen treating rubrum was used for preparing the inoculum and spore suspension
their ailment accompanying herbs and other ancient beneficial with 0.1 to 1.5  106 CFU/mL was designed for MIC (Nascente et al.,
methods (Balakumar et al., 2011). Medicinal plants exist a fashion- 2009).
able supply of secondary metabolites and are terribly helpful in The concentrations of 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39,
treating varied diseases that ends up in new drug researches 0.19 and 0.1 mg/ml were prepared, the MIC90 was noticed to reach
(Shakya, 2016). B. aegyptiaca bears potential medicative values 90% of inhibition during seven days of incubation at 30 °C.
and is employed as herbaceous medicine. The fruit has 4 layers,
(i) Epicarp or outer layer, ripened fruit has crispy outer layer, (ii) 2.5. Phytochemical analysis
Mesocarp or fleshy, sticky fruit pulp which is also known as desert
date, (iii) Endocarp or woody shell, it is stone like very strong, (iv) Phytochemical screening process was carried out using frac-
inner seed or kernel (Al-Thobaiti and Zeid, 2018). It is used as tioned chloroform extract of fruit epicarp using different exper-
purgative and to treat jaundice, diabetes (El-Nagerbi et al., 2013), iments aimed at analyzing the existence of phytochemicals such
whooping cough, dysentery, leukoderma, constipation, skin and as carbohydrates, proteins, amino acids, tannins, saponins, phe-
liver diseases (Pandit et al., 1996; Kamal, 1998), it has potent anti- nols, flavonoids, terpenoids, coumarins, glycosides and emodins
helmintic, fish poison and antifungal activities (Archibald, 1933; (Harborne, 1998; Edoga et al., 2005; Kokate et al., 2006).
Abdallah et al., 2012).
This study focuses on the antidermatophytic activity of epicarp 2.6. Compound analysis
of B. aegyptiaca by inhibiting the progress of Trichophyton rubrum
using experimental studies, distinguishing compounds from the Liquid Chromatography-Mass Spectrometry (LC-MS) was used
extract with terrible vital activity through LC-MS, docking and to detect the macromolecules and secondary metabolites
MD simulation analysis. which were extent in the fractionated chloroform extract of fruit
epicarp. LC-MS analysis was carried out using UPLC-QToF-MS
system (Taamalli et al., 2015). Elucidated mass of compounds
2. Materials and methods
from LC-MS were equated with GNPS (Global Natural
Products Social Molecular Networking) database (Wang et al.,
2.1. Plant collection
2016).
The plant was subjected to diagnosis and edible part of vegeta-
2.7. Protein – Homology modeling, refinement and confirmation
tive growth developed after flowering was collected from the Nil-
ambur area which is located 15 km distance from the Coimbatore
The crystal structure of CYP450 of T. rubrum was not obtainable
city, Tamil Nadu, India. Then, the plant was authenticated with the
in PDB. Hence, BLAST search carried out for finding suitable tem-
aid of Botanical survey of India, Coimbatore (BSI/SRC/5/23/2018/T
plates for modeling using NCBI-BLAST. Templates identified for
ech.1582). Further, the plant was washed well and the epicarp por-
CYP450 of T. rubrum with more than 40% similarities (5FRB), hence
tion was separated from fruit pulp region, then shade drying,
protein was modeled by homology modeling using Schrödinger.
grinding and sequential extraction were done using the various
The loops of designed protein were refined and energy was
solvents like Petroleum ether (PE), Hexane (HEX), Chloroform
reduced by ModRefiner for which algorithm is employed for
(CHL), Ethyl acetate (EA), Methanol fraction (MET), Aqueous frac-
atomic level, high resolution protein structure refinement (Xu
tion (WAT), Methanol (DMET) and Water (DWAT).
and Zhang, 2011). Finally, modeled protein quality was confirmed
utilizing the Ramachandran plot study by PROCHECK server
2.2. Fungal culture (Laskowski et al., 1993).

The fungal culture MTCC.8477, Trichophyton rubrum was 2.8. Protein active site prediction
received from the MTCC (Microbial Type Culture Collection and
GENE Bank), Chandigarh for this work. Active site identified by compared with existing crystal struc-
tures of identical protein from Candida species utilizing diversified
2.3. Antifungal assay sequence lining up method by Clustal Omega server (Sievers and
Higgins, 2017).
Poison food assay (Grover and Moore, 1962) was used to assess
the antidermatophytic action of various extracts of epicarp on T. 2.9. Ligand preparation
rubrum. Various concentrations (1–5 mg/ml) of epicarp extracts
were prepared and mixed with SDA medium, control plate without The crystal structure of drug Ketoconazole and compounds
extract and positive control with Ketoconazole were prepared. The which are elucidated from the fruit epicarp were downloaded in
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Mohamed Hussain Syed Abuthakir, Munirah Abdullah Al-Dosary, Ashraf Atef Hatamleh et al. Saudi Journal of Biological Sciences 29 (2022) 3899–3910

Fig. 1. Antidermatophytic activity of various extracts of fruit epicarp on T. rubrum.

Table 1
Effect of fruit epicarp of B. aegyptiaca on radial growth of Trichophyton rubrum.

Conc. mg/ml PE HEX CHL EA MET WAT DMET DWAT

1 28.33 ± 0.88 25.33 ± 0.66 18 ± 0.00 24 ± 0.00 30 ± 0.00 10.66 ± 0.66 10.66 ± 0.66 20.00 ± 0.00
2 22.00 ± 1.15 17.00 ± 0.57 12.33 ± 0.33 21 ± 0.57 21 ± 0.57 8.33 ± 0.33 8.33 ± 0.33 17.33 ± 0.66
3 16.00 ± 0.00 10.33 ± 0.33 6.00 ± 0.00 13.33 ± 0.66 18 ± 0.00 6.00 ± 0.00 4.00 ± 0.00 11.66 ± 0.33
4 13.33 ± 0.66 6.00 ± 0.00 – 10.33 ± 0.88 11.66 ± 0.33 4.00 ± 0.00 – 10.00 ± 0.00
5 10.66 ± 0.33 2.00 ± 0.00 – 6.66 ± 0.66 7.33 ± 0.66 – – 8.00 ± 0.00

Radial growth expressed as Mean ± S.E.

Values are calculated using Bonferroni test and they are significant (P < 0.05).

Table 2
Phytochemical analysis of fractioned chloroform extract of B. aegypptiaca fruit
epicarp.

S. no Groups Name of the test Presence/

absence*
1. Proteins and Amino acids 1.Biuret Test +
2.Ninhydrin Test +
2. Carbohydrates 1.Benedict’s Test +
2.Fehling’s Test +
3. Flavonoids Alkaline reagent test +
4. Phenols Ferric chloride Test +
5. Tannins Ferric chloride test –
6. Alkaloids Mayer’s Test +
7. Terpenoids Salkowiski’s test +
8. Steroids Liebermann Burchard test +
9. Saponins Froth Test +
10. Anthraquinones Glycosides Borntrager’s test –
11. Coumarins Coumarin Test –
Fig. 2. Anti-dermatophytic activity of fractioned chloroform extract of B. aegyptiaca 12. Emodins Emodin test –
epicarp on T. rubrum C – control, D – DMSO, K – Ketoconazole, 1 – 1 mg/ml, 2 –
(+) – Presence () – Absence.
2 mg/ml, 3 – 3 mg/ml, 4 – 4 mg/ml, 5 – 5 mg/ml.

Fig. 3. MIC of fractioned Chloroform extract on T. rubrum.

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Pubchem. Further, these compounds were alloted for ADME-Tox 2.11. Molecular dynamics simulation
examination utilzing QikProp component (Zhang et al., 2021).
The various attributes of absorption, distribution, metabolism, The Molecular Dynamics (MD) simulation process was per-
excretion and toxicity were analysed for each compound (Sabitha formed for an extent of time of 10 ns with Desmond module of
and Vijayalakshmi, 2012). Schrödinger for checking the stability of the docked complex struc-
tures of Ketoconazole-CYP450 and Platyphylloside -CYP450.
The simulation process was started with Protein preparation
2.10. Molecular docking wizard for optimizing and minimizing the complex structure using
OPLS3e force field. Further, the minimized structure was built by
GLIDE module of Schrödinger was used to perform protein–li- system builder using TIP3P solvent model, orthorhombic box shape
gand docking (Castro-Alvarez et al, 2017). The molecular docking with minimized volume and permitting 10 Å of buffer region. Then
was executed through the procedures of protein generation, recep- ions were added and the system was neutralized by adding neces-
tor grid formation, ligand generation and ligand docking. sary positive (Na+) and negative (Cl) ions and 0.15 M salt was
Protein preparation phase was initiated with protein prepro- added. The system was minimized for 2000 iterations on a conver-
cessing by increasing hydrogen atoms, improvement of amino acid gence threshold of 1 kcal/mol/Å and pre-equilibrated by using the
orientation of hydroxyl groups, amide groups, creating disulfide inbuilt relaxation protocol (Aier et al., 2016).
bonds. Energy minimization process was performed with OPLS3e Further modeled system was loaded and the Molecular Dynam-
force field, non-hydrogen atoms were decreased till the common ics simulation run was administered up to 10 ns with recording
Root Mean Square Deviation (RMSD) reached standard value of interval of every 100 ps for total energy. For system equilibration
0.3 Å. the NPT ensembles were utilized and temperature maintained at
Grid was surrounded for the residues of Cytochrome P450 300 K (Gaonkar et al., 2020).
active site region. The drug and ligand molecules were prepared Further result was analyzed by using the parameters like
by optimization process using the OPLS3e force field. Protein-Ligand RMSD, protein RMSF, ligand RMSF, Protein-ligand
Finally, the ligand docking was implemented using XP (extra contacts which includes various types of interactions like hydrogen
precision) process (Chen et al., 2016) by adding the derived output bond interactions were the important to analyze for obtaining the
files of ligand preparation and grid generation. stability of the complex.

Fig. 4. Chromatogram of LC-MS evaluation of fractioned chloroform extract of fruit epicarp with both positive (above) and negative (below) mode.

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3. Results 3.3. Phytochemical screening

3.1. Antifungal assay From the result of phytochemical analysis, the chloroform
extract of fruit epicarp contains carbohydrates, proteins, flavonoids,
The various extracts of epicarp of Balanites aegyptiaca was com- phenols, alkaloids, terpenoids, steroids and saponins (Table 2).
posed and their antidermatophytic activity (Fig. 1) was assessed
(Table 1). 3.4. LC-MS study
Upon reviewing the top of results, it had been detected that
the mycelial growth of T. rubrum was completely suppressed LC-MS evaluation of both positive and negative mode with dis-
by fractionated CHL extract from 4 mg/ml (Fig. 2), aqueous tinct retention time (Fig. 4) showed that totally 41 compounds
fraction extract completely inhibited by 5 mg/ml and methanol have been elucidated from fractioned chloroform extract of fruit
(DMET) extract was 4 mg/ml. As, CHL fraction of fruit epicarp epicarp (Table 3). For identifying the compound, the mass value
was found to possess higher activity, additional study was pri- of LC-MS derived compounds were compared with GNPS (Global
marily centered on the CHL fraction of fruit epicarp. Natural Products Social Molecular Networking) database.

3.2. Minimum Inhibitory Concentration (MIC) 3.5. ADME-Tox analysis

After incubation period, the MIC90 was ascertained because 90% 3D structures of 40 compounds were downloaded from the
of inhibition was firm wherever no visible growth was detected. Pubchem database. The ADME-Tox (Absorption, Distribution,
The MIC90 for CHL extract of fruit epicarp on T. rubrum was Metabolism, Excretion and Toxicity) result showed that all the
3.12 mg/ml (Fig. 3). compounds expected inside the range of all the limits, thus these

Table 3
LC-MS derived compounds from fractioned chloroform extract of epicarp of Balanites aegyptiaca.

S. Compound name Mass from Original mass Chemical Ion

no LC-MS (GNPS) formula mode
1. Theobromine 180.08 180.0647 C7H8N4O2 +
2. N-Acetyl Phenylalanine 207.08 207.0895 C11H13NO3 +
3. Aerugine 209.11 209.0510 C9H15N5O +
4. N-Acetyl-D-Galactosamine 221.11 221.0899 C8H15NO6 +
5. Forchlorfenurone 247.07 247.0512 C12H10ClN3O +
6. 3-[(E)-2-(3-Hydroxyphenyl)vinyl]-5-methoxyphenol 242.16 242.0943 C15H14O3 +
7. p-Coumaric acid 164.19 164.0473 C9H8O3 +
8. 7-methoxy-9,10-dihydrophenanthrene-2,5-diol 242.10 242.0943 C15H14O3 +
9. N  5  -Carbamoyl-N  2-(phenylacetyl)ornithine 293.08 293.1376 C14H19N3O4 +
10. Glucosaminate 195.10 195.0743 C6H13NO6 +
11. Caffeate 180.05 180.0423 C9H8O4 +
12. Phenylalanine 164.09 165.0790 C9H11NO2 +
13. Lycorine 287.09 287.1158 C16H17NO4 +
14. Paraxanthine 180.09 180.0647 C7H8N4O2 +
15. Adenosine 267.10 267.0968 C10H13N5O4 +
16. N-methylaurotetanine 341.13 341.1627 C20H23NO4 +
17. 1,3,6-trihydroxy-5-methoxy-2-[(2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]xanthen-9-one 437.11 436.1006 C20H20O11 +
18. N-[2-(4-sec-Butyl-phenoxy)-4,5-dihydroxy-6-hydroxymethyl-tetrahydro-pyran-3-yl]-acetamide 353.20 353.1838 C18H27NO6 +
19. Allocryptopine 369.45 369.1576 C21H23NO5 +
20. Pentaleno[1,6a-c]pyran-9-carboxylic acid, 1,3,4,5,6,7,7a,9a-octahydro-4,6,6-trimethyl-3-oxo-, (4S,4aR,7aS,9aR)- 264.14 264.1362 C15H20O4 +
21. 2-[5-[2-[2-[5-(2-oxopropyl)oxolan-2-yl]propanoyloxy]butyl]oxolan-2-yl]propanoic acid 398.26 398.2305 C21H34O7 +
22. Deoxycytidine 227.11 227.0906 C9H13N3O4 –
23. Pulvinic acid 308.12 308.0410 C18H12O5 –
24. 2-hydroxy-4-methoxy-3-(3-methylbut-2-enyl)-6-(2-phenylethyl)benzoic acid 341.13 340.1675 C21H24O4 –
25. Divaricatinic acid 211.08 210.0892 C11H14O4 –
26. Cortisol 362.20 362.2093 C21H30O5 –
27. Norepanorin 421.15 421.1525 C24H23NO6 –
28. 5-Chlorodivaricatinic acid 245.12 244.0502 C11H13ClO4 –
29. Citreorosein 285.11 286.0477 C15H10O6 –
30. 5-hydroxy-7-[4-hydroxy-2-methoxy-3-(3-methylbut-2-enyl)phenyl]-2,2-dimethyl-7,8-dihydropyrano[3,2-g] 437.11 436.1886 C26H28O6 –
chromen-6-one
31. 2-{[6-O-(beta-D-Glucopyranosyl)-beta-D-glucopyranosyl]oxy}-2-phenylacetamide 475.29 475.1690 C20H29NO12 –
32. Tetradecanoic acid 228.11 228.2089 C14H28O2 –
33. Hexadecanoic acid 256.17 256.2402 C16H32O2 –
34. Platyphylloside 476.19 476.2050 C25H32O9 –
35. (3E)-7-Hydroxy-3,7-dimethyl-3-octen-1-yl 6-O-(6-deoxy-alpha-L-mannopyranosyl)-beta-D-glucopyranoside 481.22 480.2571 C22H40O11 –
36. N-Acetyl Neuraminate 309.12 309.1060 C11H19NO9 –
37. 11-eicosenoic acid 311.16 310.2872 C20H38O2 –
38. 4-[(2-{[(2-Ethyl-2,3-dihydroxybutanoyl)oxy]methyl}phenyl)amino]-4-oxobutanoic acid 353.20 353.1475 C17H23NO7 –
39. Thelephoric acid 351.19 352.0219 C18H8O8 –
40. (3S,5S,8R,9S,10S,13R,14S,17R)-3-[(2R,3R,4S,5R,6S)-3,4-dihydroxy-6-methyl-5-[(2S,3R,4S,5S,6R)-3,4,5- 724.46 724.3305 C36H52O15 –
trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-5,14-dihydroxy-13-methyl-17-(6-oxopyran-3-yl)-
2,3,4,6,7,8,9,11,12,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthrene-10-carbaldehyde

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compounds were incorporated for molecular docking studies

(Supp. Table 1).

3.6. Modeling and validation

Protein 3D-structure of Cytochrome P450 of T. rubrum was

modeled using the templates 5FRB with 62% similarity by homol-
ogy modeling method. The modeled protein (Fig. 5) was further
validated and the result showed that 88.1% residues were present
in most favored region (Fig. 6).

3.7. Active site prediction

The sequence of Cytochrome P450 protein of T.rubrum was

aligned with the experimentally solved fungal target of Candida
species and predicted active site residues were highlighted in
Fig. 7. From this analysis, Tyr 106, Tyr 120, Lys 131, His 365 and
Arg 369 were perceived as the active site residues of CYP450 pro-
tein in T. rubrum.

3.8. Molecular docking

The 40 compounds and the drug Ketoconazole were docked

with the Cytochrome P450 protein of T. rubrum using XP (Extra
Precision) docking. Analysis of docked complex structures revealed
the compounds Platyphylloside, (3E)-7-Hydroxy-3,7-dimethyl-3-
octen-1-yl-6-O-(6-deoxy-alpha-L-mannopyranosyl)-beta-D-gluco
pyranoside and 1,3,6-trihydroxy-5-methoxy-2-[(2S,3R,4R,5S,6R)-3
,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]xanthen-9-one had
top ranked compounds with interactions to the active site residues
and 20 compounds had good dock score than Ketoconazole
(Table 4). Fig. 6. Ramachandran plot analysis of modeled Cytochrome P450 protein.
The drug Ketoconazole had dock score 7.6 Kcal/mol and inter-
actions with the active site residues Tyr 120, His 369 and struc-
beta-D-glucopyranoside had interactions with active site residues
turally conserved residue Tyr 52 (Fig. 8a). LC-MS derived plant
Tyr 106, Tyr 120, His 365 and structurally conserved residue Ser
compound Platyphylloside had dock score 13.2 kcal/mol and
366 and Tyr 492 with dock score 12.6 kcal/mol. The compound
interactions with active site residues Tyr 106, Lys 131, His 453, with
1,3,6-trihydroxy-5-methoxy-2-[(2S,3R,4R,5S,6R)-3,4,5-trihydroxy-
structurally conserved residues Pro 447, Phe 448
6-(hydroxymethyl)oxan-2-yl]xanthen-9-one had 11.7 Kcal/mol
and Phe 496 (Fig. 8b). The compound (3E)-7-Hydroxy-3,7-
dock score and interactions with active site residue Tyr 106, Tyr
dimethyl-3-octen-1-yl-6-O-(6-deoxy-alpha-L-mannopyranosyl)-
120, Lys 131 and His 453. The above compounds which were eluci-
dated from the epicarp of B. aegyptiaca had better anti-
dermatophytic activity against the protein Cytochrome P450.

3.9. Molecular dynamics simulation

Molecular Dynamics (MD) simulation is an important study to

understand the stability of binding affinity of the ligand accompa-
nying the protein during the simulation period. It was carried out
using Desmond, Schrödinger for observing the stability of complex
structures of Cyp450 with Platyphylloside and Cyp450 with
Ketoconazole.

3.10. CYP450 with Platyphylloside

From the analysis of RMSD (Root Mean Square Deviation) plot,

the complex structure of protein CYP450 and ligand Platyphyl-
loside had showed that the fluctuations of both Ca atoms and
ligand heavy atoms were within the acceptable range of 1–3.0 Å
up to end of the simulation period (Fig. 9a) hence this complex
was more stable.
RMSF (Root Mean Square Fluctuation) plot shows that the
fluctuation of individual residue of CYP450 protein. From the
analysis, the residues from 280 to 320 and 380 to 410 had fluc-
Fig. 5. 3D structure of Modeled Cytochrome P450 protein. tuated more than other residues but they were not out of the
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Fig. 7. Predicting active site residues of CYP450 protein using multiple sequence alignment.

range (Fig. 9b). The active site residues and other important Pro 447 had maintained their interactions with maximum time per-
residues were situated within the range. Hence protein was iod of simulation (Fig. 10d). The 2D image shows that the drug Keto-
stable. conazole had hydrogen bond interactions with Tyr 52, Ser 363, Pro
From the analysis of protein–ligand contacts, compound had 447 and Pi-Pi interaction with active site residue Tyr 106 (Fig. 10e).
hydrogen bond interactions with active site residues Tyr 120, Lys From the study of various factors of MD simulation, the docked
131, His 365, Arg 369, His 453 with structurally and functionally complex structure of CYP450-Platyphylloside was more stable
conserved residues Leu 127, Ser 363, Phe 448, Ala 450, Arg 454, than CYP450-Ketoconazole complex, also Platyphylloside had
Cys 455, Leu 495 and Hydrophobic interactions with Tyr 106, Val more number of interactions with active site residues. This study
119, Ile 364, Phe 496 (Fig. 9c). Among these interactions the com- finds that the compound Platyphylloside has good antidermato-
pound Platyphylloside had maintained its interaction with active phytic activity against the CYP450 protein in T. rubrum.
site residues Tyr 106, Tyr 120, His 365, Arg 369, and His 453 from
start to end of the simulation period (Fig. 9d). The Fig. 9e shows
that the compound platyphylloside had hydrogen bond interac- 4. Discussion
tions with Tyr 120, His 365, Arg 369, Ala 450, His 453, Leu 495 at
the 10th ns of simulation period. Among the human diseases, dermatophytoses are the fourth
most prevalent affecting nearly quarter of the world’s population
(Hay et al., 2014). T. rubrum is the predominant disease causing
3.11. Cyp450 with Ketoconazole agent accounting for 69.5% followed by other species of Trichophy-
ton (Bristow and Spruce, 2009). Providing safer and cost-effective
RMSD plot derived that the protein Ca atoms and heavy atoms treatment is a need of the hour.
of Ketoconazole were continuously fluctuated up to the end of the The water and methanol extract of B.aegyptiaca fruit had appar-
simulation period but the fluctuation is within the acceptable ent antifungal activity on Candida and Aspergillus species (Abdallah
range of 3.0 Å (Fig. 10a). From the RMSF plot analysis, the residues et al., 2012). The bark of this plant is also used for wounds and skin
from 280 to 320 were fluctuated more and other residues were diseases (Anani et al., 2015). According to Hussain et al. (2019), the
within acceptable range (Fig. 10b). fractioned methanol extract of sweet mesocarp had potential
The protein–ligand contact plot shows that the hydrogen bond against M. gypseum and T. rubrum.
interactions were observed with the structurally conserved residues Saponins are very rich bioactive properties; especially they had
Tyr 52, Ser 363, Pro 447 and hydrophobic interactions with active antifungal activity especially anticandidal activity (Saha et al.,
site residue Tyr 106 and other structurally conserved residues Leu 2010; Nwachukwu, 2017). The rhizomes of C. cuspidatus had sapo-
109, Phe 114, Ile 364, Met 368, Cys 455, Leu 495 and Phe 496 by drug nins which showed anticandidal and antidermatophytic activity
Ketoconazole (Fig. 10c). Among these, Tyr 52 and Tyr 106 Ile 364 and especially against T. rubrum (Cota et al., 2020). Further, flavonoids,
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Table 4
Docking result of drug Ketoconazole and LC-MS compounds from epicarp of B. aegyptiaca with Cytochrome P450 protein.

S. Compound name Dock score Interacting residues Bond length (Å)

no (kcal/mol)
Ketoconazole 7.6 Tyr 52, Tyr 120, His 369 1.96, 1.80, 2.16
1. Platyphylloside 13.2 Tyr 106, Lys 131, Pro 447, Phe 5.39, 1.82, 1.78, 2.24,
448, His 453(2), Phe 496 1.84, 2.17, 5.48
2. (3E)-7-Hydroxy-3,7-dimethyl-3-octen-1-yl 6-O-(6-deoxy-alpha-L- 12.6 Tyr 106, Tyr 120, His 365, Ser 366, 2.03, 2.36, 1.95, 1.63,
mannopyranosyl)-beta-D-glucopyranoside Tyr 492 1.714
3. 1,3,6-trihydroxy-5-methoxy-2-[(2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6- 11.7 Tyr 106, Tyr 120, Lys 131, His 453 4.28, 1.79, 2.16, 1.88
(hydroxymethyl)oxan-2-yl]xanthen-9-one
4. 2-{[6-O-(beta-D-Glucopyranosyl)-beta-D-glucopyranosyl]oxy}-2-phenylacetamide 11.2 Pro 447(2), Phe 448, His 453, Ile 2.05,2.31, 1.94, 2.07,
464 2.03
5. 5-hydroxy-7-[4-hydroxy-2-methoxy-3-(3-methylbut-2-enyl)phenyl]-2,2- 10.5 – –
dimethyl-7,8-dihydropyrano[3,2-g]chromen-6-one
6. 4-[(2-{[(2-Ethyl-2,3-dihydroxybutanoyl)oxy]methyl}phenyl)amino]-4- 10.1 Tyr 106, Tyr 120, Ser 366(2), Arg 2.10, 2.15, 1.63, 2.44,
oxobutanoic acid 389, Phe 496 2.34, 5.37
7. N-[2-(4-sec-Butyl-phenoxy)-4,5-dihydroxy-6-hydroxymethyl-tetrahydro-pyran-3- 10.0 Tyr 106, Tyr 120, His 453(2) 5.02, 2.29, 1.71, 1.75
yl]-acetamide
8. Cortisol 9.9 Tyr 106, Ser 366 2.49, 2.13
9. 2-[5-[2-[2-[5-(2-oxopropyl)oxolan-2-yl]propanoyloxy]butyl]oxolan-2-yl] 9.2 Tyr 106, Arg 369 2.17, 2.78
propanoic acid
10. Thelephoric acid 9.0 Leu 495 1.94
11. Citreorosein 8.8 Tyr 106, Ser 366 2.09, 1.72
12. 7-methoxy-9,10-dihydrophenanthrene-2,5-diol 8.4 Tyr 106, Ser 366 4.09, 1.84
13. 2-hydroxy-4-methoxy-3-(3-methylbut-2-enyl)-6-(2-phenylethyl)benzoic acid 8.2 Tyr 120, His 453(2) 1.98, 1.75, 2.72
14. Lycorine 8.2 Tyr 106, Ser 366, Leu 495 4.28, 2.00, 2.42
15. 3-[(E)-2-(3-Hydroxyphenyl)vinyl]-5-methoxyphenol 8.2 Tyr 120, Leu 495 1.94, 1.74
16. 11-eicosenoic acid 8.1 Tyr 120, His 453 1.81, 1.94
17. Norepanorin 8.1 Tyr 106, Tyr 120 4.03, 2.09
18. Deoxycytidine 7.9 Tyr 106(2), Ser 366 2.10, 5.37, 1.69
19. Pentaleno[1,6a-c]pyran-9-carboxylic acid, 1,3,4,5,6,7,7a,9a-octahydro-4,6,6- 7.7 Arg 369 4.57
trimethyl-3-oxo-, (4S,4aR,7aS,9aR)
20. N-Acetylneuraminate 7.7 Ser 366, Leu 495 1.75, 1.89
21. N-methylaurotetanine 7.5 His 453, Phe 496 2.03, 5.36
22. Forchlorfenuron 7.2 Tyr 106, Tyr 120(2) 5.33, 2.16, 2.20
23. N-Acetyl Phenylalanine 7.1 Tyr 106 1.83
24. Allocryptopine 7.0 – –
25. Caffeate 6.9 Ser 366(2) 1.99, 2.03
26. Pulvinic acid 6.8 – –
27. Adenosine 6.8 Tyr 106, Tyr 120, Ser 366 2.34, 2.61, 1.85
28. N-Acetyl-D-Galactosamine 6.7 Tyr 106, Ser 366 2.10, 1.68
29. N  5  -Carbamoyl-N  2-(phenylacetyl)ornithine 6.7 Tyr 106, His 453 4.06, 1.77
30. Divaricatinic acid 6.6 Leu 495 2.16
31. Glucosaminate 6.2 Tyr 120, Lys 131, His 453(2) 1.69, 1.94, 2.04, 2.16
32. 5-Chlorodivaricatinic acid 6.1 – –
33. Aerugine 5.9 Tyr 120 2.07
34. p-Coumaric acid 5.9 Ser 366 1.92
35. Paraxanthine 5.9 Tyr 106, Leu 495 2.48, 1.81
36. Phenylalanine 5.4 Tyr 106, Phe 496 2.19, 6.19
37. Theobromine 5.1 Tyr 106(2), Phe 496 2.17, 4.80, 5.08
38. Hexadecanoic acid 5.0 Tyr 120, His 453 2.08, 1.81
39. Tetradecanoic acid 4.3 Tyr 120, His 453 2.03, 1.81

steroids and phenolic compounds had potent antifungal activity pyranosyl)-beta-D-glucopyranoside and 1,3,6-trihydroxy-5-meth
(Hassan et al., 2006). In the present study, the chloroform extract oxy-2-[(2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-
of epicarp displays saponins, steroids, flavonoids and phenols can 2-yl]xanthen-9-one are organic compounds under the class of
be showed the inhibitory activity against T. rubrum. diarylheptanoids, fatty acyl glycosides and benzopyrans com-
According to Wu et al. (2018), the CYP51 is an important for pounds, respectively. Diarylheptanoids have good antifungal activ-
mycelial growth and elongation. The drug VT1161 showed more ity against F. equiseti, F. tricinctum, C. albicans, S. cerevisiae and A.
than 20 interactions including H-bonds and other types of interac- flavus, A. parasiticus (Ganapathy et al., 2019). According to Akpinar
tions in the region of helix, strand and loops in Lanosterol 14- et al. (2018), the benzopyrans have good antimicrobial activity
alpha demethylase (CYP450) of C. albicans in the crystal structure against gram positive (S. aureus) and gram negative (E. coli) bacteria.
5TZ1 (Hargrove et al., 2017). In the present docking study, the resi- Platyphylloside found to interact with influential residues of
dues Tyr 106, Lys 131, Ser 366, His 453 interacted in the docked CYP450 protein and the stability of the complex is good because
complex structure of Platyphylloside, (3E)-7-Hydroxy-3,7- RMSD and RMSF values were within the acceptable area. Also,
dimethyl-3-octen-1-yl-6-O-(6-deoxy-alpha-L-mannopyranosyl)- Platyphylloside never lost the interaction from start to end with
beta-D-glucopyranoside and 1,3,6-trihydroxy-5-methoxy-2- Tyr 106, Tyr 120, His 365, Arg 369, His 453, Ile 456 which were
[(2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl) oxan-2-yl] an important residue in Helix B region of CYP450 protein for ergos-
xanthen-9-one with CYP450 in T. rubrum which were the equivalent terol biosynthesis in dermatophytes (Zhang et al., 2019). Platyphyl-
residues in the C. albicans. The compounds Platyphylloside, (3E)-7- loside is an important compound in class diarylheptenoids from
Hydroxy-3,7-dimethyl-3-octen-1-yl-6-O-(6-deoxy-alpha-L-manno black alder and this compound has good antimicrobial activity

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Fig. 8. Docking result of CYP450 with (a) drug Ketoconazole, (b) Platyphylloside.

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Mohamed Hussain Syed Abuthakir, Munirah Abdullah Al-Dosary, Ashraf Atef Hatamleh et al. Saudi Journal of Biological Sciences 29 (2022) 3899–3910

Fig. 9. MD simulation result of complex structure of CYP450 with Platyphylloside (a) Protein-Ligand RMSD, (b) Protein RMSF, (c) Protein-Ligand interactions, (d) Protein-
Ligand contacts, (e) 2D diagram of interactions at 10th ns.

especially against C. albicans and S. cerevisiae (Novakovic’ et al., 5. Conclusion

2015). Hence, this study strongly recommends Platyphylloside
had better antidermatophytic activity than the drug ketoconazole The plant B. aegyptiaca has potent medicinal properties; this
hence further this compound will be evaluated for finding its effi- study determined that through the fractioned chloroform extract
ciency against T. rubrum. of epicarp against tinea causing pathogen T. rubrum. Further, in sil-

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Fig. 10. MD simulation result of complex structure of CYP450 with Ketoconazole (a) Protein-Ligand RMSD, (b) Protein RMSF, (c) Protein-Ligand interactions, (d) Protein-
Ligand contacts, (e) 2D diagram of interactions at 10th ns.

ico study confirmed that the compounds Platyphylloside, (3E)-7- acted with the influential residues of CYP450 protein of T. rubrum.
Hydroxy-3,7-dimethyl-3-octen-1-yl-6-O-(6-deoxy-alpha-L-manno This study exhibited that the compound Platyphylloside is highly
pyranosyl)-beta-D-glucopyranoside and 1,3,6-trihydroxy-5-meth potential against dermatophytes. Platyphylloside will be subjected
oxy-2-[(2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan- to further studies in order to determine their efficacy through
2-yl]xanthen-9-one that were elucidated from the epicarp inter- in vitro and in vivo experiments against T. rubrum.

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Authors Contribution Ganapathy, G., Preethi, R., Moses, J.A., Anandharamakrishnan, C., 2019. Diarylheptanoids
as nutraceutical: a review. Biocatal. Agric. Biotechnol. 19 (101109), 1–14.
Gaonkar, S.L., Hakkimane, S.S., Bharath, B.R., Shenoy, V.P., Vignesh, U.N., Guru, B.R.,
MJ – Planned and monitored, SAMH - Execute and written all the 2020. Stable isoniazid derivatives: in silico studies, synthesis and biological
works, MAA – Supporting lab facilities and funding, AAH – Con- assessment against mycobacterium tuberculosis in liquid culture. RASAYAN J.
Chem. 13 (3), 1853–1870.
tributed in manuscript preparation and funding, HAA – Contributed
Ghannoum, M., 2016. Azole resistance in dermatophytes: Prevalance of mechanism
in data analysis and financial support, P – Data analysis and writing. of action. J. Am. Pediat. Med. Assoc. 106, 79–86.
Grover, R.K., Moore, J.D., 1962. Toxicometric studies of fungicides against brown rot
organisms Sclerotinia fructicola and S. laxa. Phytopathology 52, 876–880.
Declaration of Competing Interest Harborne, J.B., 1998. Phytochemical Methods: A Guide to Modern Technique of
Plant Analysis. Chapman and Hall, London.
The authors declare that they have no known competing finan- Hargrove, T.Y., Friggeri, L., Wawrzak, Z., Qi, A., Hoekstra, W.J., Schotzinger, R.J., York,
J.D., Guengerich, F.P., Lepesheva, G.I., 2017. Structural analyses of Candida
cial interests or personal relationships that could have appeared to albicans sterol 14_-demethylase complexed with azole drugs address the
influence the work reported in this paper. molecular basis of azole-mediated inhibition of fungal sterol biosynthesis. J.
Biol. Chem. 292 (16), 6728–6743.
Hassan, S.W., Bilbis, F.L., Ladan, M.J., Umar, R.A., Dangoggo, S.M., Saidu, Y., Abubakar,
Acknowledgement S.M., Faruk, U.Z., 2006. Evaluation of antifungal activity and phytochemical
analysis of leaves, roots and stem barks extracts of Calotropis procera
The authors extend their appreciation to the Researchers Sup- (Asclepiadaceae). Pakistan J. Biol. Sci. 9 (14), 2624–2629.
Hay, R., Johns, N., Williams, H., Bolliger, I., Dellavalle, R., Margolis, D., Marks, R.,
porting Project number (RSP-2021/316), King Saud University, Naldi, L., Weinstock, M., Wulf, S., et al., 2014. The global burden of skin disease
Riyadh, Saudi Arabia. in 2010. An analysis of the prevalence and impact of skin conditions. J. Invest.
Dermatol. 134, 1527–1534.
Hussain, M.S.A., Velusamy, S., Muthusamy, J., 2019. Balanites aegyptiaca (L.) Del. for
Ethical statement dermatophytose: ascertaining the efficacy and mode of action through
experimental and computational approaches. Inform. Med. Unlocked 15, 1–11.
Kamal, M.S., 1998. A furosyanol saponin from fruits of Balanites aegyptiaca.
This study has not used the human and animal samples.
Phytochemistry 48, 755–757.
Kaul, S., Yadav, S., Dorga, S., 2017. Treatment of dermatophytosis in elderly, children
Appendix A. Supplementary data and pregnant women. Ind. Dermatol. Online 8 (5), 310–318.
Kokate, C.K., Purohit, A.P., Gokhale, S.B., 2006. Pharmacogognosy, Edn 35, Nirali
Prakashan, Pune, pp. 98–114.
Supplementary data associated with this article can be found, in Lamb, D.C., Waterman, M.R., Kelly, S.L., Guengerich, F.P., 2007. Cytochrome P450
the online version, at http://dx.doi.org/10.1016/j.sjbs.2022.03.017. and drug discovery. Curr. Opin. Biotechnol. 18, 504–512.
Laskowski, R.A., MacArthur, M.W., Moss, D.S., Thornton, J.M., 1993. PROCHECK - a
program to check the stereochemical quality of protein structures. J. Appl.
References Crystallogr. 26 (2), 283–291.
Martinez-Rossi, N.M., Peres, N.T.A., Rossi, A., 2008. Antifungal resistance
Abdallah, E.M., Hsouna, A.B., Khalifa, K.S.A., 2012. Antimicrobial, antioxidant and mechanisms in dermatophytes. Mycopathologia 166, 369383.
phytochemical investigation of Balanites aegyptiaca (L.) Del. edible fruit from Nascente, P.d.S., Meinerz, A.R.M., Faria, R.O.d., Schuch, L.F.D., Meireles, M.C.A., Mello,
Sudan. African J. Biotechnol. 11 (52), 11535–11542. J.R.B.d., 2009. CLSI broth microdilution method for testing susceptibility of
Aier, I., Varadwaj, P.K., Raj, U., 2016. Structural insights into conformational stability Malassezia pachydermatis to thiabendazole. Braz. J. Microbiol. 40 (2), 222–226.
of both wild-type and mutant EZH2 receptor. Sci. Rep. 6 (34984), 1–10. Navone, L., Speight, R., 2018. Understanding the dynamics of keratin weakening and
Akpınar, D.E., M. Ozgur, H.Aslam, O.Alagoz, A.Oktemer, H.Dal, T.Holelek, E.Logoglu. hydrolysis by proteases. PLOS ONE 13 (8), e0202608.
2018. Synthesis, characterization, and investigations of antimicrobial activity of Novakovic’, M., Novaković, I., Cvetković, M., Sladić, D., Tesević, V., 2015.
benzopyrans, benzofurans and spiro [4.5]decanes. An International Journal for Antimicrobial activity of the diarylheptanoids from the black and green alder.
Rapid Communication of Synthetic Organic Chemistry. 48(19): 2510-2521. Braz. J. Bot. 38 (3), 441–446.
Al-Thobaiti, S.A., Zeid, I.A., 2018. Medicinal properties of desert plants (Balanites Nwachukwu, I.N., 2017. Antifungal activities and phytochemical constituents of
aegyptiaca) – an overview. Global J. Pharmacol. 12 (1), 01–12. nicotiana tabacum leaf extracts on selected dermatophytes. Nijerial J. Microbiol.
Anani, K., Adjrah, Y., Ameyapoh, Y., Karou, S.D., Agbonon, A., de Souza, C., Gbeassor, 31 (2), 3871–3875.
M., 2015. Effects of hydroethanolic extracts of Balanites aegyptiaca (L.) Delile Pandit, B.R., Kotiwar, O.S., Oza, R.A., Kumar, R.M., 1996. Ethno-medicinal plant lore
(Balanitaceae) on some resistant pathogens bacteria isolated from wounds. J. from Gir forest, Gujarat. Adv. Plant Sci. 9, 81–84.
Ethnophamacol. 164, 16–21. Parker, J.E., Warrilow, A.G.S., Price, C.L., Mullins, J.G.L., Kelly, D.E., Kelly, S.L., 2014.
Archibald, R.G., 1933. The use of the fruit of tree Balanites aegyptiaca in the control Resistance to antifungals that target CYP51. J. Chem. Biol. 7 (4), 143–161.
of Schistosomiasis in the Sudan. Transactions of the Royal Society of Tropical Sabitha, K., Vijayalakshmi, R., 2012. Finding new inhibitors for EML4-ALK
Hygiene 27, 207. fusion protein: a computational approach. Int. Res. J. Pharm. 3 (3), 171–176.
Balakumar, S., Rajan, S., Thirunalasundari, T., Jeeva, S., 2011. Antifungal activity of Saha.S, S. Walia, J. Kumar and B. Parmar., 2010. Structure-biological activity
Ocimum sanctum Linn. (Lamiaceae) on clinically isolated dermatophytic fungi. relationships in triterpenic saponins: the relative activity of protobassic and its
Asian Pacific J. Trop. Med. 4 (8), 654–657. derivatives against plant pathogenic fungi. Pest Manage. Sci. 66, 825–831.
Behzadi, P., Behzadi, E., Ranjibar, R., 2014. Dermatophytic funfi: infections, Shakya, A.K., 2016. Medicinal plants: future source of new drugs. Int. J. Herbal Med.
diagnosis and treatment. SMU Med. J. 1 (2), 50–62. 4 (4), 59–64.
Blutfield, M.S., Lohre, J.M., Pawich, D.A., Vlahovic, T.C., 2015. The immunologic Sievers, F., Higgins, D.G., 2017. Clustal Omega for making accurate alignments of
response to Trichophyton rubrum in lower extremity fungal infections. J. Fungi 1 many protein sequences. Prot. Sci. 27 (1), 135–145.
(2), 130–137. Taamalli, A., Arraez-Roman, D., Abaza, L., Iswaldi, I., Fernadez-Gutierrez, A., Zarrouk,
Bristow, I.R., Spruce, M.C., 2009. Fungal foot infection, cellulitis and diabetes: a M., Segura-Carretero, A., 2015. LC-MS_based metabolite profiling of methanolic
review. Diab. Med. 26, 548–551. extracts from the medicinal and aromatic species Mentha pulegium and
Burmester, A., Shelest, E., Glöckner, G., Heddergott, C., Schindler, S., Staib, P., A., Origanum majorana. Phytochem Anal. 26 (5), 320–330.
et al., 2011. Comparative and functional genomics provide insights into the Wang, M, Carver, J.J., Phelan, V.V., Sanchez, L.M., N.Garg, Y.Peng, et al., 2016. Sharing
pathogenicity of dermatophytic fungi. Genome Biol. 12, R7. and community curation of mass spectrometry data with Global Natural
Castro-Alvarez, A., Costa, A.M., Vilarrasa, J., 2017. The performance of several Products Social Molecular Networking. Nature biotechnology 34 (8), 828.
docking programs at reproducing protein–macrolide-like crystal structures. Wu, Y., Wu, M., Wang, Y., Chen, Y., Gao, J., Ying, C., 2018. ERG11 couples oxidative
Molecules. 22 (136), 1–14. stress adaptation, hyphal elongation, and virulence in Candida albicans. FEMS
Chen, D., Oezguen, N., Urvil, P., Ferguson, C., Dann, S.M., Savidge, T.C., 2016. Yeast Res. 18 (foy057).
Regulation of protein-ligand binding affinity by hydrogen bond pairing. J. Xu, D., Zhang, Y., 2011. Improving the physical realism and structural accuracy of
Comput. Chem. 2 (3), 1–16. protein models by a two-step atomic-level energy minimization. Biophys. J.
Cota, B.B., Oliveira, D.B., Borges, T.C., Catto, A.C., Serafim, C.V., Rodrigues, A.R.A., 101, 2525–2534.
et al., 2020. Antifungal activity of extracts and purified saponins from the Zhang, A.Y., Camp, W.L., Elewski, B.E., 2007. Advances in topical and systemic
rhizomes of C.cuspidatus against Candida and Trichophyton species. J. Appl. antifungals. Dermatol. Clin. 25, 165–167.
Microbiol. 130, 61–75. Zhang, Q., Khetan, A., Er, S., 2021. A quantitative evaluation of computational
Edoga, H.O., Okwu, D.E., Mbaebie, B.O., 2005. Phytochemicals constituents of some methods to accelerate the study of alloxazine-derived electroactive compounds
Nigerian medicinal plants. African J. Biotechnol. 4 (7), 685–688. for energy storage. Sci. Rep. 11, 4089.
El-Nagerbi, A.F.S., Abdulkadir, E., Elamin, M.R., 2013. In vitro activity of balanites Zhang, J., Li, L., Quanzhen, L.V., Yan, L., Wang, Y., Jiang, Y., 2019. The fungal CYP51s:
aegyptiaca and tamarindus indica fruit extracts on growth and Aflatoxigenicity their functions, structures, related drug resistance and inhibitors. Front.
of Aspergillus flavus and A. parasiticus. J. Food Res. 2 (4), 68–80. Microbiol. 10 (691), 1–17.

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