INTRODUCTION:

The process of lactation emphasizes its role in infant health, particularly in developing countries where breast milk is a major necessity. It contrasts this with the often underappreciated importance of breastfeeding in developed nations. The passage outlines the numerous benefits of breastfeeding, including the reduction of risks for various infant diseases such as diarrhea, respiratory illnesses, acute otitis media, necrotizing enterocolitis, atopic eczema, leukemia, and both types of diabetes.

Additionally, breastfeeding is linked to improved blood pressure and cholesterol levels, lower cancer risk, and reduced obesity rates¹. The World Health Organization (WHO) and UNICEF recommend initiating breastfeeding within the first hour after birth and continuing exclusively for the first six months of a baby's life. This early and exclusive breastfeeding has long-term benefits, including higher IQs, better cognitive development, improved academic performance, and a lower likelihood of obesity and diabetes in later life. Breastfeeding is a significant public health measure that should be actively supported and promoted^2,3.

Various factors can lead to lactation failure, resulting in malnourished babies. These factors include psychological issues such as stress and anxiety, which can negatively impact milk production^4,5. Obesity is another factor that can reduce lactation due to hormonal changes that may interfere with milk production and breastfeeding success^6,7. Effective weight and lactation management are therefore crucial for some mothers. When counseling fails to improve lactation, many women turn to medications, including herbal and pharmaceutical galactagogues, to support milk production⁸. The use of herbal medicines, particularly among women with chronic conditions has been increased. The importance of plants in human survival, noting that a small number of plant species provide the essential nutrients humans need. Medicinal plants, used for therapeutic purposes, are particularly valuable for supporting health⁹. The importance of addressing lactation challenges to ensure the well-being of both mothers and infants, and it advocates for greater support and solutions to promote successful breastfeeding^10,11^,21.

Prolactin, a polypeptide compound, it binds to prolactin receptors, which are structurally similar to growth hormone receptors. The cytoplasmic domain is activated when the hormone binds to the prolactin receptor, thereby activating cytoplasmic tyrosine kinases. As a result, the JAK-STAT signaling pathway activates transcription factors that trigger DNA and RNA synthesis. As an outcome, the formation of mRNA, this promotes specific protein synthesis increases in ribosome. Casein, lipids, and lactose are formed through this process. As a result, milk production is boosted¹².

Different ethnic groups have long utilized Oxalis corniculata Linn, also known as creeping wood sorrel, as a raw vegetable and in folk medicine to treat a variety of human diseases. Nevertheless, there aren’t many studies on the pharmacological justification for its widespread use¹³. O. corniculata is a weed worldwide but this is consumed as a green leafy vegetable by the local residents of Nilgiris, Tamil Nadu, India. This plant belongs to the Oxalidaceae family. The entire plant has high vitamin C content and is edible. The plant contains phytochemical like fatty acids, glycosides, tannins, phytosterols, phenol, flavonoids, and galacto-glycerolipid in addition to volatile oils. Anxiolytic, antifungal, anticonvulsant, antiulcer, antinociceptive, anticancer, antidiabetic, hepatoprotective, hypolipidemic, abortifacient, antibacterial, and wound healing qualities are among the many benefits of this medicinal plant that have been the subject of several research¹⁴. The plant provides credence to the traditional usage of O. corniculata as a natural cure for many illnesses. The present was conducted to identify the potential of isoflavones as a bioactive compound for inducing activation through the substitution of Prolactin using an in silico approach.

a b

c d

Figure 1: a –Flower; b – Leaves; c – Fruit; d – Whole Plant

Interaction Studies Between Docking and Binding In silico:

Software Used¹⁵

· RCSB

· PubChem Database

· SwissADME

· Way2Drug

· Openbabel

· Auto Dock Vina

· Pymol

· BIOVIA Discovery Studio

In silico docking study on isoflavones using Auto Dock Vina.

STEP 1: Identifying protein of interest:

Prolactin receptor (3NPZ) was identified and downloaded from the PDB (Protein Data Bank) of the RCSB (Research Co-laboratory for Structural Bioinformatics).

The procedures are:

· Open PDB database.

· Find the prolactin receptor.

· The receptor 3NPZ was identified as prolactin and downloaded in PDB format.

· The protein was visualized using BIOVIA Discovery Studio.

· Check for missing residues.

STEP 2: Obtaining the identified ligands:

PubChem is a largest chemical information database which provides us with chemical, physical, and structural features of the desired ligand.

The procedures are:

· Open PubChem database.

· Search for the desired ligand.

· Download the ligand in SDF (Structural Data File) format.

STEP 3: Determine the ADMET properties:

SwissADME is a software tool that computes the physiochemistry and pharmacokinetics, drug likeness, lipophilicity, water solubility and medicinal chemistry of small molecules^16,17.

The procedures are:

· Open PubChem.

· Search for suitable ligand.

· Copy the Canonical Smiles.

· Open SwissADME.

· Paste the Canonical Smiles in the input dialogue box.

· Click “run”.

STEP 4: Analysis of biological activity:

Way2drug is a pass server (Prediction of Activity Spectra for substances) is a software product designed as a tool for evaluating the general biological potential of an organic drug like molecule.

The procedures are:

· Open PubChem.

· Search for a suitable ligand.

· Copy the Canonical Smiles.

· Open Way2Drug.

· Create a user account.

· Login into the Way2Drug.

· Paste the Canonical Smiles in the input dialogue box.

· Click gate predict.

STEP 5: Changing the format of the ligand from PDB to PDBQT:

Openbabel is a tool used to convert the chemical file format.

The procedures are:

· Open the Openbabel.

· Choose the prescribed input file format (SDF).

· Select the ligand of ligand of choice as input.

· Choose the prescribed output file format (PDBQT).

· Choose the output file destination.

· Click convert.

· Convert desirable ligands into PDBQT format using Openbabel.

· Check whether the file is saved in the chosen destination.

STEP 6: Preparation of protein:

mgltools are a software used to prepare the protein. mgltools are used for the deletion of water, addition of kollman charges, addition of polar hydrogen atom, merge non -polar hydrogen atom.

The procedures are:

· Copy the protein to the bin folder of mgltools.

· Open AutoDockTools.

· Open file and click read molecule.

· Open the desired protein file.

· Click > edit > select delete water to delete water molecules.

· Click >edit > select charges and then select add kollman chargers.

· Click > edit > select hydrogen >add > select polar method only.

· Click> edit> select hydrogen > merge non-polar.

· Select Grid > Grid box.

· Choose the appropriate coordinates.

· Note the XYZ coordinates.

· Grid > macromolecule > choose protein > select molecule >save the protein in PDBQT format.

STEP 7: Molecular docking using AutoDock Vina:

Using the prepared protein and desirable ligands in PDBQT format, docking calculation where run in AutoDockVina. AutoDockVina is a free/ open -source molecular docking software¹⁸.

· Create a new folder.

· Paste the prepared ligand and protein in that folder.

· Plays the AutoDockVina software file inside the same folder.

· Run Vina screen.

· The output and lock file will be saved in the name of the ligand in the destination folder.

· In the log file we can analyse the docking score.

STEP 8: Pymol:

Pymol is a 3D structure visualizing software which is used to view the PDB structure of a protein.

The procedures are:

· Place the ligand and protein file inside the output file.

· Open the ligand and protein file using pymol to create a complex file.

STEP 9: BIOVIA Discovery Studio:

BIOVIA Discovery Studio is 3D structure visualizing software which is used to view the PDB structure of a protein. It is used to identify the hydrogen bond interaction between the protein and the ligand.

The procedures are:

· Open the complex file using BIOVIA Discovery Studio.

· View the protein ligand binding in a 3D structure.

· By clicking show 2D diagram we can able to find hydrogen bond interaction between the protein and the ligand in a 2D structure.

RESULTS AND DISCUSSION:

Identifying protein of interest:

The 3D structure of 3NPZ is identified and downloaded from PDB data base

Figure 2 : Prolactin Receptor (PRLR) Complexed with the Natural Hormone (PLR)

Obtaining the identified ligands:

The 3D structure of Isovitexin and vitexin were identified and downloaded from PubChem. (isovitexin and vitexin were plant compounds).

Figure 3: Isovitexin

Figure 4:Vitexin

Determination of ADMET properties:

ADMET properties of a Isovitexin and vitexin were analysed using Swiss ADME^16,17.

The phytochemical properties provided by the ADME software follow:

Weight in molecules (MW):

Isovitexin: 431.38 g/mol on average

Vitamin X: On average, 432.39 g/mol

LogP for lipophilicity:

Isovitexin: On average, 0.71 LogP

Vitexin: On average, 0.39 LogP

The polarity, or PSA (Polar Surface Area):

Isovitexin: PSA on average equal to 95

Their low lipophilicity also will cause them to have widespread diffusion likely metabolism.

Isovitexin and vitexin are two flavonoids that are extensively metabolized, mainly in the liver, through conjugation reactions in the form of glucuronidation and sulfation

Analysis of biological activity:

Get on the PASS Server:

· Go to the Way2Drug website or platform of the PASS server.

· Verify that the ligands are being used with the right canonical smiles.

· State the ligand's chemical structure that you want to examine (in this case, vitexin or isovitexin). Generally, the structure can be directly drawn using one of the molecular editor tools on the website, or it can be submitted as a file in a compatible format (such as SDF, MOL, or SMILES).

· Forward the structure for review. The PASS server will process the input structure and generate predictions using its database and algorithms.

· The database values are then processed to return a bioactivity, property or characteristic that might be associated with the input ligand and the server will generate a report containing all the potential bioactivities if any

· This type of mass-tailored biological activities like Receptor binding, enzyme inhibition and many more which have been predicted.

Changing the format of the ligand from PDB to PDBQT:

The Openbabel software provided the pdbqt file from the sdt file format of the files. The main process of the software is to convert the sdt format files into pdbqt file formats. This conversion is done for the further process (i.e) auto dock vina.

This is done because the autodock vina software accepts only the pdbqt format files for the docking process.

Preparation of protein:

Copy the protein code directly to the mgltoolsovu bin folder.

· Launch AutoDockTools.

· Click "read molecule" after going to the folder.

· Find the desired protein file that was generated or downloaded using one of the methods outlined.

· To signal that eliminating water molecules will result in their deletion (click Edit > Delete Water).

· Add charges, click edit, then choose the charges, then choose "add," followed by Kolllman chargers.

· To bring up the options, click the word, choose it, and then click the "add" button. From the list of alternatives, you can only select the polar approach. To alter something, select the option Hydrogen. Click the chosen file and select ‘edit merger’, then ‘non-polar’.

· Click your right mouse button and then click on ‘Selected Grid’ on the menu to open the Grid box.

· It is possible to find an infinite number of objects with the right coordinates.

· These coordinates are likely to be the specific location and the XYZ axis location of a particular place or item.

· Grid: This will save the protein in either PDBQT format or macromolecule/protein/molecule.

Molecular docking using auto dock vina:

Using the prepared protein and desirable ligands in PDBQT format, docking calculation where run in AutoDockVina.

The output that was given by the autodock vina software shows that isovetixin and vetixin plays a promising role in enhance the lactation compared to the standard drugs.

The galactagogue activity of isoflavone compounds belonging to the Oxalidaceae family was evaluated by the utilization of the Auto Dock vina software. The Auto Dock software was utilized to acquire values for several parameters, such as bond energy, length, and number of interactions. Prolactin is used as a reference compound to compare all measured values.

Four conformations were generated with AutoDock Vina. In AutoDock, vina conformations are arranged based on binding energy. More negative values denote higher binding affinity, and lower binding energy indicates that the ligand and receptor are more compatible. When two variants of the same chemical or ligand are docked to the same protein structure, the rmsd (Root Mean Square Derivation) of the lower and upper bound indicates that no known atomic levels were assumed between the two files.

Isovitexin:

Isovitexin has a binding energy of -8.4 (kcal/mol).

Figure 5: Binding energy of Isovitexin

Vitexin:

Vitexin has a binding energy of -8.3 (Kcal/mol).

Figure 6: Binding energy of Vitexin

Discovery studio:

The discovery studio is a tool used to view the 3D structures. The complexed file of a protein (3NPZ) and ligands^{19, 20}

Figure 7: Prolactin – Prolactin receptor

Complex file of 3NPZ and Prolactin:

The White region in the picture indicates the Prolactin Receptor, and the red region indicates the protein Prolactin. If prolactin binds to the prolactin-receptor through the initiation of the JAK-STAT signaling pathway, breast milk is stimulated.

Complex file of 3NPZ and Isovitexin:

The White region indicates the Prolactin Receptor, and the red region shows the compound Isovitexin. The ligand isovitexin has an equal and better affinity to the protein prolactin, which may induce milk supply.

Figure 8: Prolactin – Isovitexin

Figure 9: Prolactin- Vitexin

The White region indicates the Prolactin Receptor, and the red region shows the compound vitexin. The ligand vitexin has an equal and better affinity to the protein prolactin, which may induce milk supply.

CONCLUSION:

The study examines the galactagogue action of isoflavone, a chemical known for its many characteristics. Isoflavone molecules have anticancer, anti-inflammatory, and anti-neurodegenerative effects. Using Autodock Software, isoflavones from O.corniculata were assessed for their galactagogue activity. Isovitexin and vitexin reported in this plant was taken for further study. Isovitexin has binding affinity is -8.4 and Vitexin -8.3 towards prolactin receptor. Compared to vitexin, isovitexin have higher binding affinity. When compared to other isoflavonoids, isovitexin and vitexin was found to have bond energy and bond lengths that were significant to the reference compound Prolactin. The binding energy of vitexin and isovitexin is -8.3 and -8.4 (kcal/mol) respectively.

Isovitexin and vitexin exhibited a positive interaction with prolactin receptors, according to molecular docking analysis. The reason for this was the low binding energy, strong bond length, and 6 to 9 contacts with the prolactin receptor's active region. Previous work on galactagogue activity has been established using quercetin from Spergula arvensis, a plant belonging to the Caryophyllaceae family contain compounds of isoflavones, a type of phytoestrogen which stimulate milk production. The quercetin, an isoflavone has the binding affinity of -8.8 towards prolactin receptor^10,21. In addition, O. corniculata shows slightly lower binding affinity when compared to quercetin. The study suggests that isoflavones can substitute prolactin²¹, and increase lactation during poor milk production. Isovitexin and vitexin may also be used as a framework for future research and development to build more effective therapeutic agents.

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Received on 17.09.2024 Revised on 09.01.2025

Accepted on 17.03.2025 Published on 08.11.2025

Available online from November 13, 2025

Research J. Pharmacy and Technology. 2025;18(11):5487-5492.

DOI: 10.52711/0974-360X.2025.00791

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.