Percentage of yield
The extraction of Hardwickia binata leaf in organic solvent showed good yield compared to others like ethyl acetate and hexane. Results showed the significant difference in yield using different solvents. Among solvents tested, methanol showed higher extraction of Yield (3.1%) followed by ethyl acetate (2.7%) and Hexane (2.4%), indicating the extraction efficiency favors the high polar solvents and methanol extracts showed high polyphenol and terpenoid content [15]. Similar results were finding in Knoxia sumetrensis methanolic extract of yield 3.1% [16] and methanol extract of Citrullus colocynthis yield 3.20% [17]. It is possible that the high yield in aqueous media is owing to the fact that most phytochemicals have electronegative functional groups, making the compound hydrophilic in nature [18].
Qualitative analysis of H.binata
The phytochemical screening of Hardwickia binata was analysed in methanol leaf extract. (Supplementary file, Table 1) Results shows the presence and absence of secondary metabolites. Alkaloids, phenol, Tannin, Flavonoids, Steroids and glycosides, Carbohydrate and terpenoids were present in methanolic leaf extract of H. binata and proteins are absent.
Quantitative analysis
Estimation of total phenolic content
The total phenolic content was measured using different concentration of Gallic acid equivalents in milligram per gram (mg GAE/g) of dry weight (DW). Our results demonstrated total phenolic content of methanol extract of Hardwickia binata (Y= 0.004x + 0.018, R2 = 0.9995). Similar results were finding in methanol extract of Ficus carica (R2= 0.953) and Olea europaea (R2= 0.973) [19]. Phenol and flavonoids are correlated to antioxidants with the ability to break chains [20]. Phenolic compounds exhibit good antioxidant activity, because the plant extract contains the hydroxyl group responsible for facilitating free radical scavenging [21]. Phenolics can serve as antioxidants in a range of methods. Hydroxyl groups in phenolics are good hydrogen donors: hydrogen-donating antioxidants may interact with reactive oxygen and nitrogen species in a termination reaction, breaking the cycle of radical formation [22].
Total flavonoid content
Total flavonoid content of H. binata differs according to different concentration of sample. The results are expressed as mg/g of rutin equivalent. Total content of flavonoid was expressed in methanol extract of Hardwickia binata leaf (Y= 0.059x + 0.019) R2= 0.996. Equivalent in previously reported the flavonoid content of Ficus carica (R2= 0.922) and Olea europaea (R2= 0.934) [19]. Flavonoids have strong free radical scavenging of free radicals, chelation of metal ions and oxidizing molecules to implicate various diseases [23].
Functional group identification of Methanol leaf extract Hardwickia binata
The FTIR spectrum of Hardwickia binata methanol leaf extract and ethyl acetate extracts were analysed from 500 cm-1 to 4000 cm-1 (fig.1) and the intensity of peak, group and character (table 1). The dominant stretching aliphatic primary amine (N-H) group's intense peak 3370.50 cm-1. Stretching alkene group (C-H) is responsible for the 2916.70 cm-1 peak and strong stretching group of carbon dioxide (O=C=O) is represented by the band at 2362.94 cm-1. The peak at 667.70 cm-1 report (C=C) strong alkene group. Strong primary alcohol group indicate the intense peak at 1053.53 cm-1. Similar results are reported in methanolic leaf extract of Coriandrum sativum have functional group such as amines (N-H), alkene (C=C) and primary alcohol (C-O) was observed [24]. In other studies, functional group such as N-H (amine), C-H (alkene), C=O (Primary alcohol) were observed from Schefflera vinnosa leaf extract [25].
Phytocompounds identification of MeoH - LE Hardwickia binata
Phytochemicals constitutions of methanol leaf extracts were identified by using GC-MS. Methanol extract show 30 compounds in chromatogram, which were identified based on retention time between 3 to 28 min (fig. 2). The name, molecular structure, molecular formula and biological activities of the identified compounds are given in Table 2 (supplementary file). Among these 30 compounds; six biomolecules were identified as Hydrazinecarbothiomide, Undecane, Mome Inositol, N-Hexadecanoic Acid, 1-Hexadecanol, Acetate, 1,3-Benzenedicarboxylic Acid, Bis (2-Ethylhexyl) Ester. These reported phytomolecules have anticancer [26] anti-allergic, anti-inflammatory [27], anti-alopecic, anti-cirrhotic, anti-neuropathic, cholesterolytic [28], antioxidant, nematicide, anti-androgenic, Haemolytic, Pesticide [29]. In similar results were finding in Paracoccus pantotrophus contains tetratriacontane, heneicosane, hexadecanoic acid, methyl ester, eicosane, tetracosane, 1-heptacosanal [30]. The bioactive compound of ethanol leaf extract of Pergulaia daemia was shown tetracosamethyl-cyclododecasiloxae, 1,3 benzenedicarboxylic acid, bis (2-ethylhexyl) ester [31]. Other results were finding in endangered plant Ceropegia bulbosa chloroform extract of whole plant shown Eicosane, hexatriacontane, octadecanoic acid [32].
Biological applications
Antibacterial activity
The H. binata of Methanol leaf extracts of antibacterial activity was carried out in disc diffusion method using gram-positive and gram negative bacteria (fig. 3). In higher zone of inhibition was observed in gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus) compared to negative bacteria (Escherichia coli and Pseudomonas aureginosa). In comparison of negative bacteria, positive bacteria have higher zone inhibition (Enterococcus faecalis (14 ± 0.54 mm) and staphylococcus aureus (12 ± 0.63 mm) (Table 2). In comparison of positive bacteria, negative bacterial strains have less susceptible action because the presence of thin layer of peptidoglycan. In positive bacterial cell wall rich in peptidoglycan and polysaccharides. This structural difference may cause the low antibacterial activity in negative bacteria (Escherichia coli and Pseudomonas aureginosa) and higher activity in positive bacterial strains (Enterococcus faecalis and staphylococcus aureus) [33]. In similar results were finding in ethanolic extract of Psidium quayaquilense antibacterial activity against staphylococcus aureus (14 mm) [34] and Enterococcus faecalis (17 mm) using methanolic callus extract of Saraca asoca [35]. Other results were finding in antibacterial activity against Pseudomonas auroginosa (11 mm) in Pongamia pinnata [36] and cold water extract of Jasminum officinale antibacterial activity against Escherichia coli (7 mm) [37].
Antioxidant activity
The DPPH technique works on the basis of reducing DPPH in the presence of a hydrogen donating antioxidant [38]. The ability of extracts to donate hydrogen reduces the colour of DPPH. DPPH is one of the chemicals that contains a proton free radical with a distinct absorption that is greatly reduced when exposed to proton radical scavengers [39]. Scavenging activity of H. binata was carried out in DPPH assays. The free radical inhibition were increasing concentration (50, 100, 150, 200, 250 µg/ml) in a dose dependent manner of MeOH leaf extracts and standard were shown in fig 4. The highest free radical inhibition percentage was observed in maximum concentration (250 µg/ml) in 80% of inhibition. The studies clearly reveal that natural antioxidants obtained from natural have high radical scavenging activities, which are crucial to preventing the harmful effects of free radicals in diabetes [40]. Antioxidant activity using DPPH of H. binata IC50 (306.85 ± 3.35 µg/mL). In similar results were finding DPPH activity in Chaptalia nutans methanolic leaf extract (345.41 ± 5.35 µg/mL) [41]. Another report were finding in Alstonia angustifolia (IC50 = 384.77 µg/mL) ethanolic leaf extract [42].
ADMET
Many potential therapeutic drugs fail to progress to clinical trials due to negative ADMET (absorption, distribution, metabolism, elimination, and toxicity) parameters. This research includes quantitative measurements of drug-like properties, such as pKa, absorption, solubility, bioavailability, BBB penetration and so on [43]. The Lipinski, Veber, and Igan rules are based on a review of human medication ADME properties. These principles are incredibly beneficial for generating drugs based on the 2D structure of tiny molecules as well as the bioavailability of those molecules. All of the isolated compounds match Lipinski's rule of five with zero violations, according to ADME's computations [44]. We also evaluate other factor like, number of rotatable bonds (n-ROTB), topological polar surface area (TPSA), octanol-water coefficient (logP), HBD and HBA [45]. All of the compounds' Kp values are between –4.26 and –5.47 cm/s indicating limited skin permeability. They exhibit ideal lipophilicity, according to the predicted logP values (ranging from 2.43 to 4.37). Among 27 compounds, 13 compounds exhibit high gastrointestinal (GI) absorption and 5 compounds exhibit blood–brain barrier (BBB) permeability showed in table 3, there are Quinoline, 1,2-dihydro-2,2,4-trimethyl, Hexadecanoic acid, methyl ester, n-Hexadecanoic acid, 4,4'-((p-Phenylene) diisopropylidene) diphenol, 5-Phenyl-2,4-pyrimidinediamine, 2TMS derivative, that according ADME prediction parameters. All of the drugs are CYP1A2, CYP2C19, and CYP2C9 inhibitors, as per the CYP interaction results (table 4). One of most essential enzymes involved in the drug metabolism is CYP2D6 [46]. Compounds 3, 4, 23 and 26 were identified as probable CYP2D6 inhibitors, while other compounds were identified as non-inhibitors.
Molecular docking
The molecular docking is used to explore the molecular interaction of ligand and target. A careful examination of molecular docking interactions could aid in the development of ligands with the properties required for efficient binding to the target receptor, resulting in the accomplishment of organizational goals pharmacological and therapeutic Agents. Table.5 displays the binding energy, number of hydrogen bond contacts, bond length and the amino acids involved in the interactions. The result showed that most of the ligands (1P7Kand 5OTE) established amino acid interaction frequently with AQE: 501, ALA: 86 and PRO: 44, TYR: 36, PHE: 98.All hydrogen bond interaction showed hydrogen bond length below 2.6A°.The best docking (binding free energy) scores for all active bio compound were found in the range with 1P7K (Quinoline, 1,2-dihydro-2,2,4-trimethyl -4.6 / amino acid residues PRO:44 (4.11),TYR:36 (4.56)TYR:87 (4.37, 4.87) PHE:98 (4.76, 3.81), Hexadecanoic acid, methyl ester -6.2 kcal/mol/ residues of amino acids interaction LEU:46 (2.18),TYR:36 (4.44, 4.32) PRO:44 (5.02, 5.12),PHE:98 (3.63, 3.89, 4.30, 4.44),TYR:87 (3.99), and 5-Phenyl-2,4-pyrimidinediamine, 2TMS derivative = -7.50/ amino acid residues PHE:98 (2.20, 5.29, 4.88, 4.16)TYR:87(4.16),TYR:36 (4.90)PRO:44 (4.89)) (fig. 5-7)and 5OTE (Quinoline, 1,2-dihydro-2,2,4-trimethyl-= -4.2/ residues of amino acid interaction LYS:109 (4.82) VAL:35 (3.89) ASP:32 (2.21) CYS:36 (5.14), Hexadecanoic acid, methyl ester = -6.6,/ interaction of amino acid residues SER:221 (2.82), THR:118 (3.70),VAL:90 (5.11) AQE:501 (4.74), PHE:87 (5.15), ALA:86 (4.71), LYS:105 (4.63) and 5-Phenyl-2,4-pyrimidinediamine, 2TMS derivative = -8.11/ amino acid interaction THR:239 (2.26) LYS:202 (2.29), ASP:200 (3.66) ASP:218 (4.85) ALA:86 (4.82), AQE:501 (3.91, 2.10) ASP:201 (2.00)).(fig. 8-10).
Network Analysis of Protein–Protein Interactions (PPI)
Thirty-one primary hub nodes were selected and inserted into the STRING database, creating a link between two different nodes proteins/genes). The PPI network (44 edges and 21 nodes) was constructed (Figure 11) and may be important in the pharmaceutical effect phase of oncology. GTF31, RASL12, FCHO2 and CMIP were the only three targets that did not connect with one another. A rating of 0.883 or above indicates a high level of trust. UBXN2B, UBXN7, AMN and TRAF were two independent nodes with high degrees of trust.
Network Pharmacology
Molecular Analysis
At the statistical level, the p-value was 3.92e-05 <0.65. AT was mostly associated with the MF 34 and led to biological quality control, prostate gland growth, control of prostate epithelial cell proliferation found by transmission, and gene-set enrichment analysis knowledge along with a gene analysis set improvement for these 21 genes. GTF31, RASL12, FCHO2 and CMIP proteins have common biological functions as well molecular functions (specifically the characteristics of protein homodimerization). The analysis of drug aims is among the most recognized techniques for systematically discovering medicines based on ligands. Our bio-active products were found to be exposed to The best docking (binding free energy) scores for all active bio compound were found in the range with 1P7K (Quinoline, 1,2-dihydro-2,2,4-trimethyl -4.6 / amino acid residues PRO:44 (4.11),TYR:36 (4.56)TYR:87 (4.37, 4.87) PHE:98 (4.76, 3.81), Hexadecanoic acid, methyl ester -6.2 kcal/mol/ residues of amino acids interaction LEU:46 (2.18),TYR:36 (4.44, 4.32) PRO:44 (5.02, 5.12), PHE:98 (3.63, 3.89, 4.30, 4.44), TYR:87 (3.99), and 5-Phenyl-2,4-pyrimidinediamine, 2TMS derivative = -7.50/ amino acid residues PHE:98 (2.20, 5.29, 4.88, 4.16) TYR:87 (4.16), TYR:36 (4.90) PRO:44 (4.89)) and 5OTE (Quinoline, 1,2-dihydro-2,2,4-trimethyl-= -4.2/ residues of amino acid interaction LYS:109 (4.82) VAL:35 (3.89) ASP:32 (2.21) CYS:36 (5.14), Hexadecanoic acid, methyl ester = -6.6,/ interaction of amino acid residues SER:221 (2.82), THR:118 (3.70), VAL:90 (5.11) AQE:501 (4.74), PHE:87 (5.15), ALA:86 (4.71), LYS:105 (4.63) and 5-Phenyl-2,4-pyrimidinediamine, 2TMS derivative = -8.11/ amino acid interaction THR:239 (2.26) LYS:202 (2.29), ASP:200 (3.66) ASP:218 (4.85) ALA:86 (4.82), AQE:501 (3.91, 2.10) ASP:201 (2.00)). The dock complex exchanges for each ligand were analyzed and inspected with efficient remains.
KEGG and REATOME Pathways Enrichment Analysis
A total of 21 KEGG pathways (p-value < 0.05) have been mapped into active compound objectives. The results showed that EREG, EGF, TGFA, GRB2, and HRAS were substantially enhanced throughout the pathway. The number of target mapping pathways was higher than Leukocyte transendothelial migration (hsa04670 number = 5), Focal adhesion (hsa04510, number = 5), Salmonella infection (hsa05132 number = 5), Regulation of actin cytoskeleton (hsa04810 number=5) and Shigellosis (hsa05131, number = 5). The objectives of active compounds were mapped to twenty-one respite pathways (p-value < 0.05). The findings showed a substantial rise in EREG, EGF, TGFA, and GRB2 in paths. The mapping target paths were greater than the signaling paths are Smooth Muscle Contraction (HSA-445355, number= 3).