Molecular docking analysis of estrogen receptor binding phytocomponents identified from the ethyl acetate extract of Salicornia herbacea (L)

It is of interest to evaluate the secondary metabolites using high performance thin layer chromatography (HPTLC) finger printing and Gas chromatography-Mass spectroscopy (GC-MS) in S. herbaceaextract. The powdered plant material extracted using different solvents were used for the qualitative analysis of alkaloids, flavonoids, terpenoids and saponins followed by HPTLC finger printing and GC-MS analysis. The components identified in the GC-MS were docked with estrogen receptor (ER) to identify the binding specificity of isolated compounds. The ethyl acetate extract of S. herbaceashowed the presence of high number of secondary metabolites when compared to other solvent system. The qualitative analysis of the plant material also showed the presence of carbohydrates, protein, amino acid, phenol, flavonoids, terpenoids, glycosides, saponins and steroids. The HPTLC finger printing analysis revealed the existence of alkaloid, flavonoid, terpenoid and saponin compounds and GC-MS. GC-MS was performed to identify the phytocomponents constituents in the extract. 8 phytocompounds were identified to analyse binding with ER. The binding affinity score (-6.8 kcal/mol) and interacting ER residues (28) the phyto compound di-n-octyl phthalate showed best docking score with ER α than the standard drugs lasofoxifene, and 4-hydroxytamoxifen. The binding affinity and number of interacting ER residues was -6.9 kcal/mol; 10 and -6.2; 11, respectively. The results identified the presence of ER antagonist in S. herbaceaand warrants further investigation to explore for treating ER regulated diseases.

HPTLC finger printing analysis of ethyl acetate extract of S. herbacea plant: 10 µl each of the plant samples was dissolved in 50 µl ethyl acetate and diluted. This diluted sample was centrifuged at 3000 rpm for 5 minutes. 2 µl of sample were loaded as 5 mm band rpm length in the 3x10 silica gel 60F254 TLC Plate using Hamilton syringe and CAMAG LINOMAT 5 Instrument. The sample loaded plate was kept in TLC twin through developing chamber with mobile phase, ethyl acetate-methanol water (5:3:1), n-Hexane -Ethyl acetate (7.2:2.9), Chloroform-Glacial acetic acid-Methanol-Water (6.4:3.2:1.2:0.8) Alkaloid, flavonoid, terpenoid, saponin respectively. The developed plates were dried and documented using CAMAG REPROSTAR 3 at visible light, 254 nm and 366 nm the plates were fixed in a scanner stage and scanned at 254 nm. The peak Table, display and densitogram were noted using win (ATS 1.3.4 version for derivation, respective spray reagents were used for detecting bands and the plates were documented in visible light and 366 nm [20].

Analysis of GC-MS:
The Clarus 680 GC become used inside the evaluation that's hired to a fused silica column, filled with Elite-5MS (5% biphenyl 95% dimethylpolysiloxane, 30 m × 0.25 mm identity × 250μm df) and the components had been separated using Helium as service fuel at a regular flow of 1 ml/min. at some stage in the chromatographic run the injector temperature changed into set at 260°C. The 1μl of extract pattern injected into the tool and the oven temperature become as follows: 60°C (2 min); observed by using 300°C on the charge of 10°C min−1; and 300°C, where it became held for six minutes. The mass detector situations including switch line temperature 240°C; ion source temperature 24°C; and ionization mode electron effect at 70 eV, an experiment time 0.2 sec and test c language of 0.1 sec. The fragments are from 40 to 600 Da. The spectrums of the components have been as compared with the database of the spectrum of the recognized additives saved in the GC-MS NIST (2008) library 27 [21].

Target preparation and Ligand Library:
The crystallographic structure of ER (PDB ID: 6VJD) alpha ligandbinding domain was retrieved from the protein data bank (PDB) with the ligand lasofoxifene. The downloaded ER structure was edited to remove the ligand lasofoxifene and water molecules using the Discovery Studio Visualizer v19.1.0.18287 (www.accelerys.com) and again saved in PDB format. The major phytoconstituents present in S. herbacea were retrieved in SDF format from the PubChem database. The obtained phytocompounds were then converted into PDB file format using OPEN BABEL software [22]. Also, the native ligand lasofoxifene and 4-hydroxytamoxifen were selected as standard drugs and docked with ER to compare the effect of S. herbacea phytocompounds.

Evaluation of Ligands Drug likeness and Toxicity:
The screened ligands were evaluated for the drugability, physicochemical properties, toxicity, toxicity classes, and lethal dose using Molinspiration server (www.molinspiration.com/cgibin/properties). The drugability properties were analyzed based on the Molar Weights (MW), Total polar surface area (TPSA), lipophilicity (log P), Hydrogen Bond Acceptor (HBA), Hydrogen Bond Donor (HBD) to identify Lipinski's rule of the drug-like compounds. In addition, the Simplified Molecular Input Line Entry System (SMILES) were downloaded from the PubChem Database to calculate ADMET properties with toxicity class. The ADMET properties were calculated by implementing ADME Tlab 2.0 [24].         Tables  3, 4 Table 8. The assessment of crude extract is an imperative a part of accurate identification. HPTLC is useful as a phytochemical marker and more effective in the field of plant taxonomy and also for the identification of plant secondary metabolites [27]. HPTLC finger printing is proved to be a linear, unique, and correct technique for herbal identification. Such finger printing is useful in the quality control of herbal products and checking for the adulterants. Therefore, it may be beneficial for the assessment of various advertised pharmaceutical preparations. HPTLC profiles also show the occurrence of secondary metabolites of medicinal importance which support the traditional therapeutic uses of the plant species [28]. The qualitative analysis of ethyl acetate extracts of S. herbacea through HPTLC confirmed the presence of many secondary metabolites like alkaloids, flavonoids, saponins, and terpenoids (Figure 1-8).
Four compounds with Rf values of 0.06, 0.84, 0.92, 0.94, are detected along with 3 unknown compounds ( Table 3). In S. herbacea ethyl acetate extract in chromatogram (Figure 1 and 2), Orange, brown colored zone at visible mode is observed in the tracks which after a derivatization of brownish violet at 366 nm confirms the presence of alkaloid compound in the samples. The Table demonstrates that alkaloid numbered as 3 found to be maximum in its concentration. Alkaloids constitute one of the major groups of plant constituents. The mobile phase of used was ethyl acetate: methanol: water (10: 1.35: 1) for the alkaloid profiling. They represent one of the largest and most diverse families of the natural compound [29]. The Table 5  The chromatographic finger printing for terpenoids is well resolved at 366 nm after derivatization Figure 7 and 8. The plates are sprayed with anisaldehyde sulphuric acid reagent followed by heating and visualized in day light which shows 10 prominent peaks in ethyl acetate extract. The 5,6,8,9,&10 peaks detect in the ethyl acetate extracts are identified as terpenoid and the best solvent system to scrutinize the above partition is n-hexane: ethyl acetate (7.2: 2.9). Most of the terpenoids are of plant origin; however, they are also synthesized by other organisms, such as bacteria and yeast as part of the primary or secondary metabolism. Terpenoids have been found to be useful in the prevention and therapy of several diseases, including oxidative stress, inflammation, diabetes, asthma, hepatitis, and cancer and gastro enteritis. A number of terpenoids exhibit cytotoxicity against an expansion of tumor cells and most cancers preventive in addition to anticancer efficacy in preclinical animal model [32].
In recent years, the complexity and risks of drug discovery and development procedures have grown significantly, resulting in greater expenditure on drug research [34,35]. The biopharmaceutical industry's productivity is declining due to poor ADMET (absorption, distribution, metabolism, excretion, and toxicity) qualities [36,37]. Oral administration is becoming the preferred method among patients due to its convenience and patient compliance [38,39]. In this heed, we have analyzed the ADMET properties for di-n-octyl phthalate and also for the selected standard drug lasofoxifene, and 4-hydroxytamoxifen to compare its efficacy ( Table 9). For a new oral drug, bioavailability is one of the most desirable attributes. In contrast, assessing oral bioavailability is extremely difficult since bioavailability is a combined effect of numerous biological and physicochemical variables [40,41]. Here, we analyzed the bioavailability for the selected phytocompounds and then compared with the standard drugs. The bioavailability for the lasofoxifene, di-n-octyl phthalate was determined as F = <20% with the probability of 0.382 and 0.402, respectively. The 4hydroxytamoxifen exhibits F = ≥20% with the probability of 0.589. As a result, the aforementioned event reminded us that human intestinal absorption (HIA) might serve as an alternate signal for oral bioavailability to some extent. As a result, it is also crucial in preclinical drug assessment [42][43][44]. The predicted HIA for the lasofoxifene, 4-hydroxytamoxifen and di-n-octyl phthalate is HIA = ≥ 30%, with the probability of 0.716, 0.689, and 0.672. The human colon epithelial cancer cell line, caco-2, is used to model human intestinal absorption of drugs. The optimal value for the papp (caco-2 permeability) is >-5.15 or -4.70 or -4.80 cm/s. The estimated papp (caco-2 permeability) for the lasofoxifene, 4hydroxytamoxifen, di-n-octyl phthalate is -5.141 cm/s, -5.002 cm/s, and -4.733 cm/s respectively. The predicted BBB (Blood-Brain Barrier) probability for lasofoxifene, di-n-octyl phthalate and 4-hydroxytamoxifen was 0.915, 0.7 and 0.995. It indicates all three drugs can penetrate the brain. Plasma protein binding (PPB) is a key criterion for a drug's effectiveness and safety to be explored during each drug-development program [45,46]. The plasma binding protein probability was identified as 89.32 %, 93.815 %, and 89.022 % for lasofoxifene, 4-hydroxytamoxifen, and di-n-octyl phthalate. The optimal value for the plasma binding protein is 90%. The higher the therapeutic index (TI), the safest the drug. If the TI is minimal (the difference in the two doses is extremely small), the medicine must be dosed cautiously [47,48].
The individual receiving the medicine should be continuously watched for any symptoms of drug toxicity. Therefore, the results suggested that all three drugs were safe for patients consuming. The CYP1A2 (Cytochromes P450) enzyme is responsible for the biotransformation of 8.9% of medicines that undergo hepatic metabolism.
CYP facilitates the metabolism of over half of all marketed medicines, making it the most significant enzyme in drug metabolism. The analyzed lasofoxifene, 4-hydroxytamoxifen, di-noctyl phthalate, and related CYP inhibition probability were predicted as 0.19, 0.088, and 0.051. It implies that the drug will be digested and eliminated, lowering the drug concentration in the blood and preventing toxicity. The half-life (t1/2) of a drug is the time necessary to reduce its concentration in the body by one-half via excretion and is important in deciding dose frequency. The predicted half-life for the lasofoxifene, 4-hydroxytamoxifen, and din-octyl phthalate is 2.073 h, 2.243 h and 1.649 h, respectively, which explains the necessity of frequent doses in treatment. However, the phytocompound di-n-octyl phthalate extracted from the S. herbacea might be consumed as a decoction. Clearance rate (CL) is a proportionality factor that relates the concentration of drug measured in the body to the elimination rate. The identified clearance rate for lasofoxifene, 4-hydroxytamoxifen, and di-n-octyl phthalate was 1.826, 1.704, and 1.394 mL/min/kg. It described that the clearance rate was low might sustain in plasma for a long time. The hepatotoxicity (from hepatic toxicity) implies chemical-driven liver damage. The predicted hepatotoxic probability for the selected standard drugs lasofoxifene, 4-hydroxytamoxifen, and di-n-octyl phthalate was 0.868, 0.96, and 0.2. It distinctly describes that the standard drug will lead to high liver injury, and the phytocompound di-n-octyl phthalate is a hepatic-friendly drug.

Conclusion:
Phyto constituents are identified by qualitative methods and the identified phyto constituents are ascertained using HPTLC. Ethyl acetate extract of S. herbacea plant is rich in terpenoids compounds with biological activities. The data based on the HPTLC finger print approach which can also be proposed as a quick and reliable analytic model for the pharmacognostic study of plant raw materials used in commercial products. Hence, the extracted phytocompounds from the plant S. herbacea using ethyl acetate was analyzed using molecular docking with ER. The phyto compound di-n-octyl phthalate extracted from the S. herbacea had the highest docking score towards ER,. Furthermore, the ADME/T characteristics of the di-n-octyl phthalate revealed that it might be deemed a potential drug-like chemical. The di-n-octyl phthalate is widely accessible, allowing for the earlier development of appropriate medications against estrogen driven breast cancer.