GC‐MS, alpha‐amylase, and alpha‐glucosidase inhibition and molecular docking analysis of selected phytoconstituents of small wild date palm fruit (Phoenix pusilla)

Abstract Phoenix pusilla (Arecaceae), commonly known as “small wild date palm”, is regarded as one of the underutilized fruit crops in South India. Methanol extract of P. pusilla ripened fruits (PPRF) was analyzed for in vitro porcine pancreatic alpha‐amylase (PPAA) and rat small intestine alpha‐glucosidase (RIAG) inhibition activities, and through gas chromatography–mass spectrometry (GC‐MS) analysis. The GC‐MS analysis showed the presence of 25 phytoconstituents from PPRF which was further assessed on the docking behavior of five targeted enzymes diabetes mellitus (DM) namely (i) human aldose reductase, (ii) protein tyrosine phosphatase 1B, (iii) pancreatic alpha‐amylase, (iv) peroxisome proliferator‐activated receptor gamma, and (v) dipeptidyl peptidase IV by using the AutoDock Vina method. In addition to this physicochemical, bioactivity score, absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis was performed using the Molinspiration and pkCSM free online servers. Methanolic extract of PPRF showed 50% inhibition concentration (IC50) at 69.86 and 72.60 μg/mL levels against PPAA and RIAG enzymes activities, respectively. Interestingly in the present study, GC‐MS analysis showed the presence of 25 phytoconstituents from PPRF. Physicochemical analysis of PPRF has exhibited that 13 ligands have complied well with Lipinski's Rule of Five (RoF). With regard to ADMET analysis, one ligand (9,12‐octadecadienoic acid [Z,Z]) has predicated to possess both the hepatotoxicity (HT) and skin sensitization (SS) effect. The docking studies showed that 1‐formyl‐2,5‐dimethoxy‐6,9,10‐trimethyl‐anthracene exhibited the maximum atomic contact energy (ACE) for all the five target enzymes of DM. Thus, the current study suggested that the methanolic extract of PPRF and its phytoconstituents could be considered as potent antidiabetic agents.


| INTRODUC TI ON
Plants have been recognized as excellent sources for both diet consumption and medicinal uses in humans.In India, since time immemorial, many plants have been used as a source of medicine (Balamurugan et al., 2019).Several plant parts namely leaves, bark, stem, flowers, roots, and fruits have been known to possess medicinal properties.Herbal drugs have advantages: (i) easily available, (ii) safe, (iii) with minimum side effects, and (iv) affordable when compared with synthetic drugs (Yadav & Singh, 2011).The medicinal values of few plants have been reported to possess chemically active substances that affect the physiological actions of the human body (Pradeep et al., 2014).Phoenix belongs to the Arecaceae family, which has approximately 22 species worldwide that include Phoenix abyssinica, P. acaulis, P. andamanensis, P. atlantica, P. caespitose, P. canariensis, P. canariensis x reclinata, P. dactylifera, P. farinifera, P. leonensis, P. loureiroi, P. paludosa, P. pumila, P. pusilla, P. reclinata, P. reclinata var.leonensis, P. roebelenii, P. rupicola, P. spinose, P. sylvestris, P. theophrasti, and P. zeylanica.Most of Phoenix (palm) species are utilized as ornamental plants.Nearly 80% of fruits (dates) from Phoenix (palm) species are edible and are regularly consumed in many countries in Asia, Africa, and Europe (Amorós et al., 2014).Leaves of P. loureiroi have been used as a natural broom by the tribal communities living in the Nilgiri biosphere reserve (NBR), Western Ghats, India (Rasingam & Jeeva, 2013).Similarly, the fruits (dates) of P. loureiroi have been used as folk astringent in intestinal problems (Murugan et al., 2017).
The GC-MS analysis showed the presence of 25 phytoconstituents from PPRF was further assessed on the docking behavior of five targeted enzymes of DM namely (i) human aldose reductase (HAR), (ii) protein tyrosine phosphatase 1B (PTP1B), (iii) human pancreatic alpha-amylase (HPAA), (iv) peroxisome proliferator-activated receptor gamma (HPPARG), and (v) dipeptidyl peptidase IV (HDPP-IV) by using the AutoDock Vina method.In addition to these physicochemical properties and bioactivity score, absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis was performed using the Molinspiration and pkCSM free online servers.

| Plant collection and authentication
The PPRF were collected from the Nemili village, near Arakkonam Tamilnadu, India during May and June 2022.Dr. K.N.Sunil Kumar, Research Officer and Head of Pharmacognosy Department, Siddha Central Research Institute (SCRI), Arumbakkam, Chennai has identified and authenticated the plant and fruit.The voucher specimen was prepared and deposited (reference number: P12072202P) in the SCRI herbarium for future reference.

| Extraction
The collected PPRF were washed thoroughly in distilled water 3 times, then subjected to sunshade drying after removing seeds, and followed by pulverized into fine powder using a electrical grinder.About 100 g of dry powdered fruits sap (PPRF) were extracted with 150 mL of 70% methanol using the Borosil Soxhlet apparatus for 6-8 h.The selection of methanol for the solvent extraction was considered to deliver many benefits compared to other organic solvents, as it is relatively safer protic solvent with a polarity index value of 5.1 and a dielectric constant of 33.70 at 25°C (Hikmawanti et al., 2021).
The 70% ethanol solvent in this research was capable to extract vital phytocompounds from the dry powder of PPRF.
The PPRF extract was then concentrated to dryness in a rotavapor.Half of the portion of the extract was stored in a deep freezer and another half portion of the extract was stored in a desiccator for future applications.

| In vitro PPAA inhibition assay
PPAA inhibition assay was carried out by adopting a slightly modified protocol of Shaikh et al. (2017).The soluble starch (0.15%) was used as substrate in 50 mM potassium phosphate buffer (pH 7.0), incubated with 50 μL of the enzyme (PPAA) at 37°C for 30 min.(In the case of test sample tubes, the buffer and sample [PPRF] volume were adjusted in order to maintain the final reaction volume.)Then the biochemical reaction was terminated by the addition of 1 mL of dinitro salicylic acid (DNS) reagent (1% 3,5-dinitro salicylic acid, 12% sodium potassium tartrate, 0.4 M sodium hydroxide).The test tubes were kept in boiling water for 15 min and then cooled under running tap water.Finally, the absorbance was determined at 540 nm using Epoch 2 (Bio Tek) microplate reader.Acarbose was used as a positive control.All the experiments were performed in triplicate.The data were fitted on a polynomial (regression) model, whereas vertical bars indicate standard error (±SEM), and Microcal Software (Sigma plot 11) was used to plot the graph.

| In vitro RIAG inhibition assay
RIAG preparation and as well inhibition assay was carried out by adopting Sakayanathan et al. (2018) protocol.The maltose (1 mM) was used as substrate in 100 mM potassium phosphate buffer (pH 7.0), incubated with an aliquot of the enzyme (RIAG) at 37°C for 30 min.(In the case of test sample tubes, the buffer and sample [RIAG] volume were adjusted in order to maintain the final reaction volume.)After incubation, the concentration of glucose was measured by using a glucose estimation kit as per manufacturer's instruction (Coral Clinical Systems).Finally, the absorbance was determined at 505 nm using Epoch 2 (Bio Tek) microplate reader.Acarbose was used as a positive control.All the experiments were performed in triplicate.The data were fitted on a polynomial (regression) model, whereas vertical bars indicate standard error (±SEM) and Microcal Software (Sigma plot 11) was used to plot the graph.

| GC-MS analysis of PPRF
One microliter of PPRF sample was taken for GC-MS (Agilent Technology, 7890B Model equipped with 5977A Mass Detector) analysis to determine the chemical constituents present in the PPRF sample.GC-MS was performed using the following conditions (i) column: C18 Silica (normal phase), (ii) oven temperature: 60°C, (iii) injection temperature: 250°C, and (iv) helium was used as carrier gas at the split ratio of (1:1).Finally, the obtained mass spectrum was interpreted and compared with the National Institute of Standards and Technology (NIST) library database (Srikalyani & Ilango, 2020).
Furthermore, the prepared ligands were converted into the pdbqt file format using the Open Babel free software.

| Physicochemical property and bioactive score analysis
The Molinspiration free online server was used to analyze physicochemical property and bioactive score of 25 selected ligands of P. pusilla (Mohan et al., 2022).

| ADMET analysis
The pkCSM online server was used to analyze the ADMET properties of 25 selected ligands of PPRF (Pires et al., 2015).

| Target protein identification and preparation
The three-dimensional (3D) structure of the target proteins: (i) human aldose reductase (PDB ID: IUS0 with resolution of 0.66 A°), (ii) protein tyrosine phosphatase 1B (PDB ID: 2QBS with a resolution of 2.10 A°), (iii) human pancreatic alpha-amylase (PDB ID: 2QV4 with a resolution of 1.97 A°), (iv) human peroxisome proliferator-activated receptor gamma (PDB ID: 3CS8 with a resolution of 2.30 A°), and (v) human dipeptidyl peptidase IV (PDB ID: 4A5S with a resolution of 1.62 A°) were obtained from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (www.rcsb.org).
A chain of these five proteins was preprocessed by removing other chains (B and C) and ligands, in addition to the crystallographically observed water particles (water without hydrogen bonds).UCSF Chimera software (www.cgi.ucsf.edu/chimera)was utilized to prepare the abovementioned protein (Radhakrishnan, 2017).Then each prepared protein was opened in Autodock 1.5.6 (MGL) tool, then polar hydrogen atoms and Kollman charges were added to each protein, and finally, protein (macromolecule) was saved the pdbqt file format for further docking analysis.

| Active site prediction
The active site of each protein was determined using the active site prediction web server (http://www.scfbio-iitd.res.in/dock/ActiveSite.jsp).The pre-registered email id was provided; the server will

| Molecular docking studies
The molecular docking studies were carryout by using the AutoDock Vina in Linux command mode (Vázquez-Jiménez et al., 2023).
Initially, a grid box was set according to the active site of the crystal structure of each protein mentioned in the above active site prediction section.The first-ranked pose for each ligand was considered for the binding energy calculation and similarly, binding sites were analyzed by using the protein-ligand interaction profiler (PLIP) free web server.

| RE SULTS AND D ISCUSS I ON
Phoenix pusilla (Arecaceae) is commonly known as "Parusakah" (in Sanskrit) and "Siruinju" (in Tamil).P. pusilla has been listed as one of the top 250 ayurvedic medicinal plants in India (Saravanan et al., 2021).The different parts of P. pusilla such as leaves, fruits, barks, and roots have been well documented (Charaka and Sushruta) and prescribed by Ayurvedic practitioners.Moreover, "sharbat-ephaalsaa" (Unani squash), a summer refreshing drink/juice prepared using P. pusilla fruits, has been used as an appetizer and cardiac tonic in India (Roop, 2018).Furthermore, P. pusilla unripened fruits have been known to possess biological activities such as antidiabetic and antioxidant (Sankar & Shoba, 2017).Thus, the abovementioned background engaged us to carry out the present study using the PPRF (underutilized fruit) of South India.The organoleptic feature of PPRF has been reported earlier and displays the following characteristics: (i) orange-red color, (ii) dates flavor odor, (iii) sweet taste, and (iv) oval shape (Farhanaz et al., 2017).About 10.01 ± 0.60 g of methanolic extract was obtained from 100 g of PPRF dry powdered extraction, which approximately accounts for 10% yield.In the current in vitro study, the inhibition potential of the methanolic extract of PPRF was determined against PPAA.Methanolic extract of PPRF showed a dose-dependent manner inhibition activity against PPAA (Figure 1).The 50% inhibition concentration (IC 50 ) of methanolic extract of PPRF was found to be 72.60 ± 0.7 μg/mL, whereas for the control (acarbose) it was found to be 26.31 ± 0.3 μg/mL, respectively.
To the best of our knowledge, we are the first to demonstrate the PPAA inhibition activity as well as GC-MS analysis of PPRF, though the unripened fruit inhibition activity has been reported previously by Sankar and Shoba (2017).
In the current study, physicochemical analysis of PPRF has exhibited that 13 ligands have complied well with Lipinski's Rule of Five (RoF), whereas 12 ligands have exhibited one violation (Table 3).
With regard to the bioactivity score analysis of PPRF, only 9,12-octadecadienoic acid (Z,Z) (one ligand) has shown an active (>0) bioactivity score against all the descriptors except kinase inhibitor (Table 4).
The ADMET analysis of PPRF, where one ligand [9,12-octadecadienoic acid (Z,Z)] has predicated to possess both the hepatotoxicity (HT) and skin sensitization (SS) effect (Table 5).The  physicochemical properties, bioactivity, and ADMET have been reported as pre-required properties to know before performing the docking studies (Mohan et al., 2022).
The present finding was in good agreement with the previous report, where Gln 63 has been shown as one of the binding site residues of HPAA (Akshatha et al., 2021;Omar et al., 2022).On the other hand, nine ligands (namely ligands 4, 7, 8, 11, 14, 15, 17, 19, and 23) do not exhibit any hydrogen bond interaction with HPAA.

| CON CLUS ION
In this study, the methanolic extract of PPRF showed the IC 50 as 72.60 and 69.86 μg/mL against PPAA and RIAG enzymes activities, respectively.Interestingly GC-MS characterization showed the presence of 25 phytoconstituents from PPRF.Furthermore, all the 25 selected ligands of PPRF showed the potential to dock with all the five targeted enzymes namely (i) human aldose reductase

F
The bar diagram of methanolic extract of Phoenix pusilla ripened fruit (PPRF) against porcine pancreatic alphaamylase (PPAA) and alpha-glucosidase inhibition.The data were fitted on a polynomial (regression) model, whereas vertical bars indicate standard error (±SEM) and Microcal Software (Sigma plot 11) was used to plot the graph.F I G U R E 2The GC-MS chromatogram of the methanolic extract of Phoenix pusilla ripened fruit (PPRF).Note: xaxis: time (minutes); y-axis: abundance (arbitrary units).TA B L E 1The GC-MS profiling of the methanolic extract of Phoenix pusilla ripened fruit (PPRF).
The phytoconstitutents identified from the methanolic extract of Phoenix pusilla ripened fruit (PPRF) by GC-MS analysis.
Abbreviation: RT, retention time.TA B L E 2