GC–MS analysis of phytoconstituents from Amomum nilgiricum and molecular docking interactions of bioactive serverogenin acetate with target proteins

Amomum nilgiricum is one of the plant species reported from Western Ghats of India, belonging to the family Zingiberaceae, with ethno-botanical values, and is well-known for their ethno medicinal applications. In the present investigation, ethyl acetate and methanol extracts of A. nilgiricum were analyzed by Fourier transform infrared spectrometer (FTIR) and gas chromatography-mass spectrometry (GC–MS) to identify the important functional groups and phytochemical constituents. The FTIR spectra revealed the occurrence of functional characteristic peaks of aromatic amines, carboxylic acids, ketones, phenols and alkyl halides group from leaf and rhizome extracts. The GC–MS analysis of ethyl acetate and methanol extracts from leaves, and methanol extract from rhizomes of A. nilgiricum detected the presence of 25 phytochemical compounds. Further, the leaf and rhizome extracts of A. nilgiricum showed remarkable antibacterial and antifungal activities at 100 mg/mL. The results of DPPH and ferric reducing antioxidant power assay recorded maximum antioxidant activity in A. nilgiricum methanolic leaf extract. While, ethyl acetate leaf extract exhibited maximum α-amylase inhibition activity, followed by methanolic leaf extract exhibiting aldose reductase inhibition. Subsequently, these 25 identified compounds were analyzed for their bioactivity through in silico molecular docking studies. Results revealed that among the phytochemical compounds identified, serverogenin acetate might have maximum antibacterial, antifungal, antiviral, antioxidant and antidiabetic properties followed by 2,4-dimethyl-1,3-dioxane and (1,3-13C2)propanedioic acid. To our best knowledge, this is the first description on the phytochemical constituents of the leaves and rhizomes of A. nilgiricum, which show pharmacological significance, as there has been no literature available yet on GC–MS and phytochemical studies of this plant species. The in silico molecular docking of serverogenin acetate was also performed to confirm its broad spectrum activities based on the binding interactions with the antibacterial, antifungal, antiviral, antioxidant and antidiabetic target proteins. The results of the present study will create a way for the invention of herbal medicines for several ailments by using A. nilgiricum plants, which may lead to the development of novel drugs.

Gas chromatography-mass spectrometry (GC-MS) analysis. The GC-MS chromatogram of methanol and ethyl acetate leaf extracts of A. nilgiricum recorded a total of 15 peaks corresponding to the bioactive compounds that were recognized by relating their peak retention time, peak area (%), height (%) and mass spectral fragmentation patterns to that of the known compounds described by the National Institute of Standards and Technology (NIST) library. Results revealed that 9 and 6 compounds were identified in methanol and ethyl acetate A. nilgiricum leaf extracts, respectively ( Table 2 & 3). The GC-MS chromatogram of the methanol extract from A. nilgiricum rhizomes recorded 10 peaks (Table 4). Overall, the structure of 25 phytocompounds identified in the ethyl acetate and methanol extracts from leaves, and from the methanol extract from rhizomes of A. nilgiricum are presented in Table 5 along with their retention time.
Antibacterial activity. Antibacterial activities of leaf and rhizome extracts of A. nilgiricum were tested against different bacterial pathogens at different concentrations (25,50 and 100 mg/mL). Maximum zone of inhibition of 21.43 mm was recorded with ethyl acetate leaf extract against Pseudomonas aeruginosa and highest zone of inhibition was observed for methanolic rhizome extract against Ralstonia solanacearum with 20.72 mm at 100 mg/ mL as compared to other tested concentrations (Fig. 5).  In silico studies. The GC-MS analysis revealed that A. nilgiricum leaf and rhizome extracts contained 25 bioactive compounds (Table 5). These phytocompounds were analyzed for activities against bacterial, fungal, viral, antioxidant and diabetic target proteins. The docking studies were carried out for phytoligands using the iGEMDOCK program to elucidate the binding affinities to the target proteins. The binding interaction and conformation of each phytocompound with each target protein were predicted and ranked based on the iGEM-DOCK-computed lowest energy and total binding energy, respectively. The best binding conformation for each phytocompound with each target protein was determined, if it showed the lowest total binding energy among its different conformations (Table 6).  (Table 6). Our findings are supported by the results of a previous study that showed that the lower the binding energy score was found the better the protein-ligand binding stability was identified 24 .
The serverogenin acetate formed the most excellent ligand-protein complexes (e.g. with six of seven tested target proteins) compared with other compounds. Next, docking analysis was then carried out for serverogenin acetate with the target proteins through AutoDock program. According to the binding energies, the docking

Discussion
In the present study, the investigation of ethyl acetate and methanol extracts from leaves and methanol extract from rhizomes of Amomum nilgiricum revealed the presence of various phytoconstituents, including flavonoids, carbohydrates, anthocyanin, cardiac glycosides, tannins, phenols, amino acids, alkaloids, anthroquinone, steroids, proteins, terpenoids, leucoanthocyanin, phytosterols, saponins and diterpenes. These bioactive phytoconstituents could be responsible for the therapeutic ability of various extracts of A. nilgiricum. The analysis was carried out by gas chromatography-mass spectrometry (  www.nature.com/scientificreports/ anti-inflammatory activities 27 . The trimethyl-(2-trimethylsilylphenyl)silane is an aromatic hydrocarbon, and has been reported in aqueous extract of Centella asiatica displayed anticancer, antioxidant and antimicrobial activities 28 . The cyclic compounds are unsaturated; and hence, play a key role in the antioxidant defense. 3,5-bis(trimethylsilyl)cyclohepta-2,4,6-trien-1-one is a ketone compound with antioxidant activity 29 , which has been isolated from methanol leaf extract of Psidium guajava and methanol leaf extract of Syzygium alternifolim 30 . The phytoconstituent 3,5-bis(trimethylsilyl)cyclohepta-2,4,6-trien-1-one which was identified from n-hexane seeds extract of Garcinia kola 31 [34][35][36] . Similarly, 1,1,1,3,5,5,5-heptamethyltrisiloxane is a siloxane compound which has been isolated from methanol leaf extract of C. italica 37 , aqueous bark extracts of Khaya grandifoliola and Enantia chlorantha 38 as well as from ethanol rhizome extract of Dryopteris cochleata 39 exhibited anti-inflammatory and antimicrobial properties 38,39 . While, octadec-1-yne, an alkene compound has been reported from chloroform extract of Spermadictyon suaveolens flowers showed antibacterial activity 40 . On contrary, octadec-1-yne extracted from petroleum ether and ethyl acetate leaves extracts of Leucaena leucocephala from Malaysia 41 did not possess any phytopharmaceutical properties. In an independent study, the monoterpeniod compound, 3,4-heptadien-2-one, 3-cyclopentyl-6-methyl-reported from hexane flower extract of Acmella uliginosa 42   www.nature.com/scientificreports/ Serverogenin acetate is a bioactive compound known for its application in pharmacology, this bioactive compound isolated from methanol leaf extracts of Trichilia connaroides demonstrated broad spectrum of biological properties such as antioxidant, anti-insect, antimicrobial, anticancer and antiulcerogenic activities 44 . The trimethylsilyl derivative compound trimethyl-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]silane identified from n-hexane seed extract of G. kola 31 , chloroform and ethanol leaf extracts of Acacia karroo and Ziziphus mauritiana 45 produced significant antibacterial activity against clinical bacteria and also recorded anti-inflammatory, anticancer, analgesic and diuretic activities. Ethyl 2-oxopropanoate is an androstane natured compound reported from methanol leaves extract of A. nilotica 46 and Kamettia caryophyllata 47 showed potent bioactivities of acidifier, acidulant and inhibits production of uric acid 47 . (1,3-13 C 2 )propanedioic acid has been isolated from methanol extract of Alysicarpus monilifer whole plant 48 however its biological activity is yet to confirm. The flavonoid compound 2,4-dimethyl-1,3-dioxane known to possess anti-inflammatory, analgesic, antibacterial and antifungal activities was isolated from Arjunarishta, a modified ayurvedic medicine 49 . 5-amino-6-nitroso-1H-pyrimidine-2,4-dione from methanol leaf extract of Stachytarpheta jamaicensis capable of producing high multi therapeutic properties such as antioxidant, anti-arthritic, anti-inflammatory and bactericidal potentials 50 . (2S,4S,5R,6R)-3,8,9-trioxatricyclo[4.2.1.0 2,4 ]nonan-5-ol is a sugar moiety natured compound identified from ethanol extract of aerial parts of Crotalaria longipes 51 and ethanol extracts of whole plant of Sarcostemma secamone 52 reported to be used as a preservative. In this study, the leaf and rhizome extracts of A. nilgiricum showed remarkable in vitro antibacterial and antifungal activities by suppressing their colony and growth rate. In support of this study, earlier our group reported the potential antimicrobial activities of rhizome extracts of A. niligiricum 53 . In addition we have also noticed remarkable antioxidant and antidiabetic activities from the leaf and rhizome extracts of A. nilgiricum which is the first kind of report from this study. Previously, the antibacterial and antioxidant activities using the essential oil extracted from A. subulatum was documented Shrestha 54 . Similarly, Sharma et al. 55 reported the antioxidant activity by seed extract of A. subulatum.
From the above evidences, it can be elucidated that A. nilgiricum plant consists of enormous potential of pharmacological constituents, therapeutic phytocompounds responsible for various pharmacological actions like antimicrobial, antioxidant anti-inflammatory, antidiabetic, analgesic, antiaging, anticancer, hepatoprotective, hypercholesterolemic, antihistaminic, antiandrogenic, antifibrinolytic, diuretic, antiasthma activities, preservative etc. These major chemical compounds identified from different crude extracts are considered to be a part of plants' defense systems and they may be grouped as protective compounds found in this plant referring to them as 'phytoanticipins' and 'phytoprotectants' 56 . Thus, the identification of a various phytochemical compounds from ethyl acetate and methanol extracts from leaves and rhizomes of A. nilgiricum display significant medicinal properties of the plant A. nilgiricum. Further studies like bio-prospecting are essential to support its biological properties and biological importance of these innovative bio-molecules will be interesting to be studied. To the best of our knowledge, this is the first report on GC and MS investigation of A. nilgiricum leaf and rhizome extracts.
Bioinformatics tools provide a great support to pharmaceutical companies in the process of drug discovery in a short duration of time with less cost using all primary information obtained from in vivo and in vitro analysis. In silico molecular docking in one of the greatest methods to determine new ligand for proteins of identified www.nature.com/scientificreports/ structure and thus play an important role in structure based drug discovery. Investigators worldwide use computer docking programs to discover and investigate the binding affinity for compounds that fit a binding site on the protein. The structure of the protein and ligand should be three dimensional 57 . The study and documentation of structural compounds from different medicinal plant species are gaining interest and importance. In the structure based drug design, molecular docking is generally used to predict and inter molecular complex between the drug compounds with its target protein 58 . In the present study, a total of 25 bioactive compounds were identified from A. nilgiricum leaf and rhizome extracts by GC-MS analysis and used for molecular docking studies. Further, the compounds were analyzed for bioactivity against the target proteins. Serverogenin acetate is the lead compound and exhibited antibacterial, antifungal, antiviral, antioxidant and antidiabetic activities.
Computational simulation studies revealed that serverogenin acetate compound recorded better affinity with low binding energy in comparison with other compounds. The docking study results also showed that various energy sources are consistent and contribute to the overall strength to the binding interactions of serverogenin acetate for each target proteins.

conclusions
The present investigation was focused on identification of various bioactive compounds from the leaf and the rhizome extracts of Amomum nilgiricum for the first time by GC-MS analysis. These compounds are responsible for the different therapeutic and pharmacological properties. We have also provided the evidence of leaves and rhizome extracts of A. nilgiricum for its antimicrobial (phytopathogens), antioxidant and antidiabetic activities.     www.nature.com/scientificreports/ methanol solvents (500 mL) in a Soxhlet apparatus for 8 h respectively at a temperature not beyond the boiling point of the solvents. The ethyl acetate and methanol extracts were filtered using Whatman No. 1 filter paper, and the filtrates were concentrated under reduced pressure with a rotary evaporator, freeze-dried in a lyophilizer, and then kept at 4 °C. Similarly, the freshly collected rhizomes were thoroughly washed in running tap water and rinsed in distilled water. Subsequently, the samples were cut into small pieces, shade-dried at room temperature for 10 days, powered and extracted with methanol in a Soxhlet unit for 8 h. The extracted samples were filtered by Whatman filter paper, and the filtrates were concentrated at reduced pressure with a rotary evaporator, dried using a lyophilizer and kept at 4 °C until further use.
Preliminary phytochemical screening. The qualitative phytochemical prescreening of leaf and rhizome extracts was performed following the previously published protocols 59 . The standard solution was prepared from 100 mg leaf or rhizome extract by dissolving the extract in 10 mL ethyl acetate or methanol. These solutions were then screened for the presence of different phytochemicals; namely, phenols, carbohydrates, flavonoids, anthocyanin, cardiac glycosides, saponins, tannins, alkaloids, steroids, terpenoids, amino acids, leucoanthocyanin, anthroquinone, proteins, phytosterols and diterpenes.    62 and AutoDock program 63 . Initially, all the target proteins and compounds were prepared by assigning hydrogen bonds, bond orders, charges and flexible torsions. Screening of these compounds for protein ligand interactions was carried out using the iGEMDOCK program with the preset parameters: population size of 150 was set with 60 generations and two solutions were selected for customized docking parameter. From the customized docking study, the probable binding conformations of phytocompounds were determined based on the iGEMDOCK total energy.
The best binding conformation of phytocompounds against target proteins were determined based on the lowest total binding energy among the different conformations generated. The identified phytocompounds were imported into the iGEMDOCK graphical user interface and were sorted by the post-docking analysis based on their binding energies and compound fitness score measured by the iGEMDOCK docking algorithm 64 . To determine the relative strengths of the binding interactions of the best identified phytocompound, screening for its best binding pose and the series of energy values, such as binding energy, ligand efficiency, inhibition constant and Van der Waals (VDW) + hydrogen bonding (Hbond) + desolvation energy of each target protein, were analyzed using the AutoDOCK program. Furthermore, the detailed interactions between the best phytocompound identified, and its binding sites with bacterial, fungal, viral and diabetic target proteins were visualized in PyMol 3D visualization 65 . Antioxidant activity. The 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) is a generally used method to evaluate the free radical scavenging ability of natural compounds. The free radical scavenging activity of the leaf and rhizome extracts of A. nilgiricum were measured against DPPH 66 . The Ferric reducing antioxidant power assay (FRAP) was used to measure the total antioxidant power from the leaf and rhizome extracts 67 .
Antidiabetic activities. The α-amylase inhibition assay from A. nilgiricum leaf and rhizome extracts was determined using the 3,5-dinitrosalicylic acid method 68 . The α-glucosidase inhibition activity of A. nilgiricum leaf and rhizome extracts was measured using the method of Eom et al. 69 . Acarbose was used as positive control. The aldose reductase (AR) inhibition assay from A. nilgiricum leaf and rhizome extracts was determined according to Suryanarayana et al. 70 using Quercetin as positive control. The inhibitory action was stated as the half maximal inhibitory concentration (IC 50 ), which is a measure of the effectiveness of the extract in inhibiting enzymes activities.