Chemical characterization and antifungal potential of Trigonella foenum-graecum L. followed by molecular docking studies

Abstract The present study involves the chemical characterization and antifungal potential of fenugreek Trigonella foenum-graecum L. (fenugreek) seeds/leaves essential oils and their major constituents along with computational molecular docking calculations. The fenugreek seeds and leaves essential oils were extracted by hydro-distillation and characterized by Gas chromatography-mass spectrometry and Gas chromatography-flame ionization detector. Benzyl acetate (51.70%) and γ-tocopherol (42.09%), the major compounds in fenugreek seeds and leaves essential oils respectively were isolated by column chromatography. The essential oils and their major compounds were investigated for their antifungal activity against Rhizoctonia solani and Drechslera oryzae by the Poison food technique. Among all the treatments, fenugreek leaves essential oil was the most effective with ED50 values of 850 and 1120 ppm against R. solani and D. oryzae respectively. All the treatments were found more effective against R. solani than D. oryzae. Molecular docking studies of the major compounds present in the essential oils against R. solani agglutinin (PDB ID 4G9N and 4G9M) suggested that 9,19-cyclolanost-24-en-3-ol present in abundance in fenugreek leaves essential oil than the seeds essential oil bound effectively with the targeted protein with the docking score of -6.14 kcal mol−1. Overall, our combined experimental and computational study provides scientific justification for the use of fenugreek leaves essential oil as a natural antifungal agent against R. solani. Moreover, fenugreek leaves essential oil being most effective against D. oryzae may also be used as a natural antifungal agent against it.


Introduction
Rice (Oryza sativa L.) is the main staple food crop all over the world, but it frequently suffers from various bacterial, viral and fungal diseases.The most damaging disease affecting rice is the sheath blight which is caused by Rhizoctonia solani and results in huge yield losses 1 .The fungus is soil borne that mainly remains confined to the leaf sheath and also attacks the aerial parts.The illness normally occurs at the height of the tillering stage and affects the entire plant above the waterline 2 .Additionally, the management of the fungal disease becomes challenging as the fungal sclerotia can lay latent in the soil for several years 3 .The fact that this fungus causes such a significant economic loss has drawn the attention of plant pathologists and rice farmers 4 .Another serious disease of rice is the brown leaf spot disease caused by Drechslera oryzae.It is transmitted through the seeds and results in severe yield loss globally 5 .The symptoms of the disease appear as the blight of seedlings and finally affects mature plants including spots on leaves.The disease results in 10-60% seed mortality and a drastic reduction in yield 6,7 .
Several fungicides are available in the market to combat these fungal diseases but ongoing use of these fungicides has seriously threatened the environment and human health along with creating fungal resistance to these synthetic fungicides 8 .Further, there is an increase in the level of toxic metabolites of synthetic fungicides when used at higher doses in the resistant strains.Moreover, they cannot be used on organic foods produced with "green" laws 9 .The European Parliament also signed the text regarding plant protection products regulations (Regulation 1107/2009/EC, 2011) 10,11 .Therefore, it is necessary to prioritize the search for alternatives to synthetic fungicides.
Numerous natural substances with plant origin have been used for a long time to control plant pathogenic fungi 12 .They are generally synthesized as secondary metabolites by plants to protect themselves from environmental stress and pathogens and have a huge potential to inspire the modern agrochemical industry 13,14 .These natural products are safe to use as they are biodegradable and have no or fewer side effects 15 .
Essential oils, which are natural products, could replace synthetic fungicides used to control plant pathogenic fungi through the implementation of a more effective integrated pest management strategy 16 .They are safe to use as they are nonphytotoxic in nature, have a low shelf life and are easily degradable 17 .Additionally, because the components of essential oils can work together as synergists, there is little chance of developing new resistant strains when using them as natural fungicides 18 .Moreover, the directive 2009/128/ CE has promoted the use of essential oils as biocontrol agents in Europe as a substitute for synthetic pesticides 19 .
Trigonella foenum-graecum L. (T.foenumgraecum L.) commonly called fenugreek/methi is a herbaceous aromatic crop and is a member of the family Fabaceae.Fenugreek seeds and leaves have been used as spices to enhance the flavor of food products.The seeds contain fixed as well as essential oil 20 .
Fenugreek seeds also contain free amino acids (arginine, lysine and histidine), sterols (cholesterol and sitosterol), vitamins (A, B and C), alkaloids (choline, trigonelline and gentianine) and flavonoids (apigenin, orientin, luteolin and vitex) 21 .The seeds and leaves of fenugreek exhibit hypoglycemic 22 , hypocholesterolemic 23 , immunomodulatory 24 , antimicrobial 25 , anticancer 26 , antioxidant 27 and neuroprotective 28 properties.More importantly, the antifungal potential of organic extracts prepared from different parts of fenugreek has been tested against various fungal pathogens 29-31 and yeasts 32 .There are very few reports on testing the antifungal potential of fenugreek essential oil against plant pathogenic fungi (R. solani and D. oryzae).
In continuation with our previous communication 33 , this study has been planned to determine the chemical composition and antifungal potential of fenugreek seeds and leaves essential oils and their major compounds against rice fungi i.e., R. solani and D. oryzae.These studies are complemented with computational molecular docking studies of the major compounds present in the fenugreek essential oils to evaluate their inhibitory potential towards the rice fungi.Molecular docking analysis is of profound importance in this study since it is an effective structure-based drug design technique.It helps in locating the site where a given compound can bind in a noncovalent fashion to a target macromolecule.It also helps in predicting the associated strength of binding 34 .Docking studies lead to the optimization of the shape and orientation of both the ligand and protein to reduce the free energy of the system 35 .

Plant materials and reagents
Fenugreek leaves and seeds (3 kg each) were procured from the Department of Agronomy, Punjab Agricultural University (PAU), Ludhiana (latitude 30.900965, longitude 75.857277GPS coordinates of 30°54'3.4740''N and 75°51 '26.1972''E).They were identified by an expert taxonomist, Dr. Namrata Gupta (Associate Professor) in the Department of Botany, PAU, Ludhiana.A voucher specimen (NO: PAU2022-5951) was deposited in the same department.FT-IR spectra of isolated pure compounds were taken on a Perkin Elmer, Model RX-1 FT-IR spectrophotometer (in nujol). 1 H NMR (400 MHz) spectra were recorded in CDCl 3 with Bruker AC using tetramethylsilane (TMS) as an internal reference.The FT-IR and 1 H NMR spectra were recorded at the Sophisticated Analytical Instrumentation Facility (SAIF), Central Instrumentation Laboratories (CIL), Punjab University, Chandigarh.The standard antifungal agent i.e., Nativo WP used in this study was purchased from Sisco Research Laboratories Pvt. Ltd., Mumbai.The pure cultures of tested fungi were procured from the Plant Bacteriology Laboratory of the Department of Plant Pathology, PAU, Ludhiana.

Extraction of essential oils
The essential oils from dried (shade dried for 48 hours) fenugreek seeds and leaves (500 g each in 1L of water) were extracted by hydrodistillation method using the Clevenger apparatus for 4 hours.The essential oils obtained were stored in amber colored bottles at 4°C.The yields of essential oils obtained were calculated on a dry weight basis by using the following formulae 36 :

Characterization of essential oils
Both the essential oils were characterized by using gas chromatography-mass spectrometry (GC-MS) and gas chromatography coupled with a flame ionization detector (GC-FID).
GC-MS analysis was carried out by using an Agilent 7890A GC having HP-5MS column (30 m × 0.25 mm × 0.25 µm) using nitrogen (99.99%) as a carrier gas (1 mL/min).The essential oils were diluted in n-hexane (1µL/mL) and 1.0 μL was injected into the column (1 μL/ minute) in a split mode (1:10).The injector was set at 250°C.The oven temperature was set at 50°C for the first 2 min and was raised to 120°C (4°C/min) for 5 min and then to 230°C (10°C/ min) for 10 min.The temperature was finally increased to 280°C and was then kept at constant temperature for 10 min.Various MS parameters (Agilent MSD5975C MS detector) used include detector voltage 1.5 V, mass range 45-600 amu and ionization voltage of 70 eV.
Relative proportions of essential oils components were also analyzed by Agilent 7890A-GC-FID (Agilent Technologies, Palo Alto, CA, USA) using the same conditions as used for GC-MS.The carrier gas used was nitrogen at a flow rate of 1 mL/min.The injector and detector temperatures were kept at 250 and 300°C respectively.Relative proportions were calculated by dividing individual peak area by the total area of all peaks, response factor was not taken into account.
The components were identified on the basis of their mass spectra comparison with the data reported in NIST and WILEY libraries.The identification of compounds was made by comparing the retention time with the authentic samples.The various components were also identified by comparing their retention indices (RI) with n-alkane (C 7 -C 40 ) series under the same chromatographic conditions and also by comparing their RI with the literature 37 .The GC peak area normalization of the three injections has been expressed as the mean percentage of each essential oil component.

Isolation of benzyl acetate and γ-tocopherol from fenugreek seeds and leaves essential oils
Isolation of benzyl acetate and γ-tocopherol from fenugreek seeds and leaves essential oils respectively was carried out by column chromatography.
For the isolation of benzyl acetate, fenugreek seeds essential oil (5 g) was chromatographed on activated silica gel (300 g) and elution was done with petroleum ether:ethyl acetate (80-100%) as a solvent system and the structure was confirmed by spectroscopic studies.
For the isolation of γ-tocopherol, the fenugreek leaves essential oil (5.0 g) was chromatographed on silica gel (300 g) and the elution was carried out using ethyl acetate:hexane (1-10%).Thin layer chromatography was used to keep track of every fraction (TLC).The ethyl acetate:hexane (5%) underwent a second round of silica gel column chromatography and was further eluted using ethyl acetate:n-hexane (1-10%).

Antifungal activity
Using the Poison food technique, fenugreek seeds and leaves essential oils and their major constituents were evaluated for in vitro antifungal efficacy against D. oryzae and R. solani 39 .The pure cultures of the tested fungi were identified by the third co-author, Dr. Mandeep Hunjan from the Department of Plant Pathology, PAU, Ludhiana and were grown on PDA medium.The sub-culturing was done once in a month and was stored at 20-25°C in an incubator.After putting the cotton plug on the flask, it was autoclaved for half an hour at 120°C temperature and 15 psi pressure.Then, the media was poured into 90 mm Petri plates in the laminar airflow amended with fenugreek seeds and leaves essential oils at different concentrations i.e., 25 to 3000 ppm prepared in dimethyl sulfoxide (DMSO; 10 µL).Unamended plates containing DMSO only served as a control 40 .Nativo was used as a standard.The circular bits (5 mm) of the actively growing cultures of the fungi were inoculated in each Petri plate under aseptic circumstances.The Petri plates were stored in the incubator (27°C) after being individually sealed with paraffin film.The pathogens growth was observed by measuring the diameter of mycelial growth using a scale after seven days.Five replicas were performed for each treatment.The percentage inhibition was estimated using the formulae: Inhibition of mycelial growth (%) = Mycelial growth in control -Mycelial growth in treatment / Mycelial growth in control x 100 The ED 50 value, i.e., the concentration which inhibits 50% pathogen mycelia growth for each treatment was calculated from its percentage inhibition of mycelial growth at different concentrations using the probit table analysis method 41 .

Molecular docking studies
The major constituents present in fenugreek leaves and seeds essential oils were subjected to computational docking studies against the agglutinin of R. solani to ascertain their potential binding modes and associated binding affinities.Conformations of the docked ligands were studied from their docking score (kcal mol -1 ) and the hydrogen bond formation between the compounds and their target protein.The R. solani agglutinin (PDB Code: 4G9N) is a homodimer and its crystal structure was downloaded from Protein Data Bank for docking studies 42 .The structures of the receptor and the five compounds-9,19-cyclolanost-24-en-3-ol, stigmast-5-en-3ol oleate, γ-sitosterol, γ-tocopherol and benzyl acetate, the components of the essential oils present in fenugreek seeds and leaves were prepared using the Autodock Tools 43 .For preparing the protein for docking, a grid box (86 Å × 106 Å × 80 Å) with a grid spacing of 0.400 Å was defined over one of the chains (chain A) of the homodimer of R. solani.For ligand preparation, the root of each ligand was defined, followed by adding the Gasteiger charges 44 .AutoDock-GPU4 v1.5, which harnesses the power of GPUs, was used for molecular docking 45 .Docking calculations employed 200 runs of the Lamarckian genetic algorithm 46 , and each run comprised 500 iterations of the ADADELT algorithm 47 .For better prediction of the binding site, our docking protocol used a population size of 300 and up to 25000000 energy evaluations, along with a 100% local search rate, although default values were used for all other parameters.ChimeraX and Discovery Studio were used to analyse the protein-ligand interactions to find the conformation that had the lowest binding energy 48,49 .

Statistical analysis
The results were expressed as means and standard deviations.Using SAS software, twoway ANOVA and Tukey's B test were used to analyze the antifungal potential data at a level of significance of p<0.01.

Extraction and characterization of fenugreek seeds and leaves essential oil
The yield of fenugreek seed essential oil was more (6.4% v/w) as compared to leaves essential oil (4.56% v/w).The results were in confirmation with Arivalagan et al. 50, where the yield of essential oil obtained from different genotypes of fenugreek seeds ranged from 3.25 to 6.88%.The yield of fenugreek seeds essential oil extracted by supercritical fluid extraction was found to be 8.95% 51 .
Our data on chemical composition differs from the previous literature reports as the chemical composition of essential oils depends upon various factors 53 .
γ-Tocopherol, the major component isolated from the fenugreek seeds essential oil, is a vital form of Vitamin E and is regarded as an efficient free radical scavenger 58 .It protects the plants against photo-inhibition and other forms of photo-oxidative stress.Additionally, it protects human beings from oxidative stress brought about by active oxygen and nitrogen species 59 .It prevents the oxidation of lipids and lipidcontaining foods during storage, thus increasing their stability and shelf life.Nuts, which are a great source of γ-tocopherol, have been shown to lower the risk of myocardial infarction and ischemic heart disease death when consumed regularly 60 .

Isolation and characterization of benzyl acetate and γ-tocopherol from fenugreek seeds and leaves essential oil
Isolation of benzyl acetate and γ-tocopherol from fenugreek seeds and leaves essential oils respectively was carried out by column chromatography and their structures were confirmed by spectroscopic techniques.
FT-IR spectrum of benzyl acetate gave bands at 3034 cm -1 because of =C-H stretching of the aromatic nucleus, 2954 and 2893 cm -1 due to -C-H stretching, 1736 cm -1 due to C=O stretching of the ester group, 1608, 1589, 1497 and 1453 cm -1 due to C=C stretching of the aromatic nucleus, 1222 cm -1 due to C-O stretching of the ester group, 965 and 739 cm -1 due to -C-H def. of the monosubstituted benzene ring.
This data was further confirmed using 1 H NMR (CDCl 3 , 500 MHz, δ in ppm) which showed a multiplet in the range of δ 7.23-7.36due to the presence of 5 aromatic protons (Ar-H), singlet at δ 5.09 due to the presence of benzylic protons (-CH 2 ) at C-7, a singlet at δ 2.08 due to the presence of methyl protons (-CH 3 ) of acetate group at C-10.The confirmed structure of benzyl acetate is shown in Fig. S3. 1 H NMR data of γ-tocopherol shows (CDCl 3 , 500 MHz, δ in ppm) a singlet at δ 6.36 due to an aromatic proton (Ar-H) at C-6, a singlet at δ 4.19 due to a proton of hydroxyl group, a quartet in range of δ 2.64-2.68(J=10 Hz) due to two protons at C-4, a singlet at δ 2.15 due to methyl (-CH 3 ) protons at C-25, a singlet at δ 2.10 due to methyl (-CH 3 ) protons at C-26, two multiplets in range of δ 1.74-1.80and 1.67-1.73due to two protons at C-3, a multiplet in range of δ 1.49-1.58due to six protons (methylene protons -CH 2 ) at C-12, C-16 and C-20, a multiplet in range of δ 1.33-1.43due to methyl (-CH 3 ) protons at C-24 and two protons at C-11, a multiplet in range of δ 1.22-1.32due to due to 10 protons at C-13, C-15, C-17, C-19 and C-21, a multiplet in range of δ 1.03-1.09due to the presence of three protons of C-14, C-18 and C-22 and a multiplet in the range 0.85-0.87 of due to nine protons of three methyl groups at C-27,C-28 and C-29 and a multiplet in the range of 0.83-0.84due to methyl group at C-23.The confirmed structure of γ-tocopherol is shown in Fig. S3.

Antifungal activity
Fenugreek leaves essential oil showed maximum antifungal activity with maximum inhibition of mycelial growth (86.5 and 77.0%) at 3000 ppm having ED 50 values of 850 and 1120 ppm against R. solani and D. oryzae respectively.The seeds essential oil showed 81.2 and 71.3% mycelial growth inhibition at 3000 ppm having ED 50 values of 1320 and 1880 ppm against R. solani and D. oryzae respectively.Hence, the fenugreek leaves essential oil exhibited greater antifungal potential than seeds essential oil against both the fungi.
Among the major compounds, benzyl acetate was more effective than γ-tocopherol at higher concentrations and less effective at lower concentrations against both the tested fungi.It showed 63.2 and 57.3% mycelial growth inhibition at 3000 ppm having ED 50 values of 2390 and 2850 ppm against R. solani and D. oryzae respectively whereas γ-tocopherol showed 61.3 and 51.2% mycelial growth inhibition at 3000 ppm having ED 50 values of 2160 and 2900 ppm against R. solani and D. oryzae respectively.Benzyl acetate was found ineffective at 100 ppm whereas γ-tocopherol showed 3.4% inhibition of mycelial growth at 100 ppm against R. solani.On the other hand, both compounds were not effective at 100 ppm against D. oryzae.The standard used i.e., Nativo showed 100% inhibition of mycelial growth at 50 ppm (Table 2 and 3).
Based on ED 50 values, the antifungal potential of various treatments against R. solani followed  Leaves essential oil > seeds essential oil > γ-tocopherol > benzyl acetate Similarly, based on ED 50 values, the antifungal potential of various treatments against D. oryzae followed the following order.

Leaves essential oil > seeds essential oil > benzyl acetate > γ-tocopherol
After comparing ED 50 values of fenugreek seeds/leaves essential oil and their major compounds against both the tested fungi, it was found that all the treatments were more effective 8 fig 1 against R. solani than D. oryzae (Fig. 1).
The antifungal potential of fenugreek seeds/ leaves essential oils, benzyl acetate and γ-tocopherol has been reported by various researchers against different fungi on fruits.The fenugreek seeds essential oil exhibited antifungal potential against Aspergillus niger and A. fumigatus having diameters of inhibition zones of 22 mm and 24 mm at 100% respectively 61 .
On the other hand, hexane leaves extract of Dittrichia viscosa L. containing tocopherols as the major compounds exhibited antifungal activity against Malassezia spp.and Candida krusei 62 .Peganum harmala seeds essential oil enriched with tocopherol isomers showed antifungal activity against various plant pathogens 63 .
Similarly, various benzoic acid derivatives isolated from methanolic leaves extract of Piper lanceaefolium revealed antifungal properties against Candida albicans with a minimal inhibitory concentration of 100 µg/ml 64 .The antifungal potential of cinnamic and benzoic acid esters has also been reported against various C. albicans strains 65 .
Antifungal potential of fenugreek seeds/leaves essential oils identified in our work may be ascribed to the presence of aromatic esters, sterols, their fatty acid esters and γ-tocopherol 63,65,66 .The lower amounts of these phytochemicals in fenugreek seeds essential oil might be responsible for their lesser antifungal potential than the leaves essential oil.Various reports in the literature reveal the antifungal potential of sterols, their fatty acid esters and γ-tocopherol.Choi et al. 67 revealed the antifungal potential of sterols isolated from roots of Dipsacus asper against various phytopathogenic fungi.A mixture of stigmasterol and β-sitosterol isolated from the pericarp of Areca catechu inhibited spore germination and germ tube elongation of C. gloeosporioides having ED 50 values of 86.9 and 50.0 μg/ml respectively 68 .
Different theories have been advanced to explain the antifungal nature of essential oils.One school of thought states that essential oils are lipophilic in nature and can easily pass through cell walls and membranes resulting in the loss of their integrity 69 .However, it is also believed that these oils can alter the cell membrane permeability to various cations such as H + thereby changing the pH of the cells and affecting their metabolism and intracellular osmotic pressure 70 .Changes in intracellular osmotic pressure by altered cell membrane permeability upset intracellular organelles, which in turn causes leakage of cytoplasmic contents and ultimately cell death 70 .Numerous researchers have also noted changes in the ultrastructure of fungal cells in response to essential oils.Rasooli et al. observed changes in A. niger cells after treatment with Thymus eriocalyx and Thymus x-porlock essential oils when studied under transmission electron microscopy 71 .Soylu et al. 72 noticed considerable morphological changes in the

Molecular docking studies
Since all the above experimental treatments revealed greater effectiveness against R. solani, molecular docking of the major compounds present in the essential oils of fenugreek seeds and leaves essential oils was carried out against R. solani agglutinin (PDB ID 4G9N and 4G9M), a homodimer lectin-type protein with specificity towards N-acetyl galactosamine and galactose 42 .
Overall, 9,19-cyclolanost-24-en-3-ol which is present both in the essential oils of fenugreek seeds (11.89%) and leaves (15.26%) interacted most effectively with the targeted protein than stigmast-5-en-3-ol, oleate and γ-sitosterol, which are present in greater abundance in fenugreek leaves essential oil than the seeds essential oil.This correlates with the higher antifungal potential of fenugreek leaves essential oil as compared to seeds essential oil against R. solani.In contrast, the lower predicted binding affinities calculated for γ-tocopherol and benzyl acetate with R. solani agglutinin protein suggested their relatively lower antifungal potential, compared to their respective essential oil against R. solani.
Hence, the docking study provided an elaborate method for molecular modulation of R. solani agglutinin protein and the biologically active compounds present in fenugreek seeds and leaves essential oils.

Conclusion
In conclusion, our study provides experimental and computational justification for the utilization of fenugreek leaves essential oils as a natural antifungal agent against R. solani.Further, fenugreek leaves essential oil being the most effective in our experimental studies, may also be used as a natural antifungal agent against D. oryzae.However, in vivo studies of essential oils along with their bioactive components are still needed to be explored to test their bioefficacy in the fields accompanied by their mode of action and phytotoxicity studies.

Figure 1 .
Figure 1.Comparison of ED 50 values of fenugreek seeds/leaves essential oil and their major compounds against R. solani and D. oryzae

Table 1 .
GC-MS analysis of fenugreek seeds and leaves essential oil

Table 2 .
In vitro efficacy of fenugreek seeds/leaves essential oils and their major compounds against R. solani Mean values shown in the table above followed with different superscripts are significantly different (p<0.01)using twoway ANOVA followed by Tukey's B-test

Table 3 .
In vitro efficacy of fenugreek seeds/leaves essential oils and their major compounds against D. oryzae