1. INTRODUCTION:

Diabetic foot ulcers (DFUs) are a significant challenge for both patients and the global healthcare department. It contributes to a high rate of morbidity and mortality. It also suffers limited effective treatment with substantial global economic impact^1-2.The severity of the DFUs is due to uncontrolled blood sugar levels, which affect and damage organs in the body, as well as contributing to impaired wound healing, neuropathy, vascular insufficiency, and other complications^3-5.Approximately 25% of diabetics develop DFUs, and their risk of death is similar to that of cancer patients^6-9.

Natural products have been identified as promising therapeutic agents for various diseases and disorders, and numerous studies have validated that the bioactive compounds have efficacy against DFUs. We investigated the potential activities of two plants in the present study, Neem and Giloy, as potential treatments for DFUs.

As we all know, the United Nations has called Azadirachta indica (Neem) the “tree of the 21st century." Meliaceae is the botanical family to which this plant belongs. Its primary distribution is found in Asia, Africa, and various tropical regions worldwide¹⁰.It has a broad range of therapeutic activities against inflammation, bacteria, fungi, viruses, infertility, maternal health, cancer, and diabetes. Its chemical constituents include alkaloids, flavonoids, glycosides, triterpenoids, limomoids, fatty acids, and steroids^11-14.

In the annals of ancient Ayurvedic literature, the names “Amrita,” “Heavenly Elixir,” and “Nectar of Immortality” are widely recognized as synonyms for the botanical compound known as giloy¹⁵.Tinospora cordifolia (Guduchi or Giloy) is a climbing shrub. This plant is endemic to tropical parts of India and is a member of the Menispermaceae family^16-17.It contains alkaloids, steroids, glycosides, and polysaccharides. It has activity against diabetic, cancer, and hepatotoxic conditions, as well as immunomodulatory and antioxidant properties^18-23.

Further, in diagnosis, it clearly identified that, hyperglycemia in diabetic patients with foot ulcers can lead to critical complications due to irregular glycemic control, nerve damage, and poor blood flow to the ulcer sites. These complications can alter gene expression, resulting in decreased levels of SIRT1 and NOS3, and increased levels of MAPK14, F2, and TGFBR1. These gene alterations contribute to an increased risk of infection, chronic inflammation, and ulcer progression²⁴.Figure 1A illustrates the complications of diabetes with foot ulcers, highlighting the critical role of hyperglycemia in disease progression.

Figure 1: (A) Complications of diabetes with foot ulcers, highlighting the critical role of hyperglycemia in disease progression and (B) Altered gene expression associated with diabetic foot ulcers, Indicating the direct link to hyperglycemia in disease pathogenesis.

Natural products have the ability to influence gene expression²⁵, which in turn affects various biological processes. Higher levels of SIRT1 are linked to apoptosis, DNA repair, stress response, insulin secretion, and energy metabolism. On the other hand, lower levels of MAPK14 may lead to better blood flow, reduced chronic inflammation, and enhanced overall biological function. Increased NOS3 levels are associated with improved blood flow, greater NO bioavailability, and less inflammation. Reduced F2 levels could lower the risk of thrombosis, atherosclerosis, and excessive platelet activity, thereby promoting better blood flow and immune regulation. Furthermore, decreased TGFBR1 levels might help reduce fibrin clot formation, endothelial damage, and low permeability, potentially lowering the risk of infection, ulceration, and nerve injury. Together, these genetic changes could enhance tissue health, speed up wound healing, and lower the complications and risk in diabetic patients, ultimately aiding in the healing of foot ulcers^26-30.Figure 1B illustrates the altered gene expression associated with diabetic foot ulcers, indicating the direct link to hyperglycemia in disease pathogenesis.

The therapeutic efficacy of natural compounds lies in their ability to target crucial genes involved in the pathogenesis of DFUs. By focusing on the underlying molecular mechanisms, these agents could provide a valuable approach to enhancing healing from wounds and minimizing the complications and risk for diabetic patients. Both plants included in this study have substantial literature evidence supporting their bioactive molecules for wound and ulcer treatment. Additionally, these plants have been suggested as potential anti-diabetic agents^31-32.Based on their therapeutic potential, they may be effective in treating wounds in diabetic patients. In this research, we conducted molecular docking studies between targets and molecules from Neem and Giloy against DFUs. The marketed drug named Moxifloxacin was used as a reference molecule to compare binding affinities with the molecules from neem and giloy. We also conducted network pharmacology studies for the identified key targets and predicted the significant signaling pathways in the management of DFUs. Pharmacokinetic, physiochemical properties, and toxicity predictions for the screened molecules from neem and giloy were also supported by our study to fulfill the objective.

2. MATERIALS AND METHODS:

2.1 Plant and bioactive compound selection:

Azadirachta indica (leaf and fruit extracts) and Tinospora cordifolia (leaf and stem extracts) have been scientifically proven to possess antioxidant, anti-inflammatory, wound healing-enhancing, and tissue repair capabilities^33-37. These therapeutic properties make them promising candidates for DFUs. Thus, in the present in silico study of both plants’ molecules has been chosen in the present study. The bioactive molecules from both plants have been characterized and validated using liquid chromatography-mass spectrometry in earlier studies. Therefore, on the basis of an extensive review, we selected thirty-one molecules for further investigation, including Azadirachtin, Allivicin, Aurachin D, Bruceantin, Caulerpin, Copalliferol B, Deacetylnimbin, Deacetylsalannin, Gentisic Acid, Lactopiperanol C, Nimbidiol, Nimbidic Acid, Nimbin, Quercetin, Salannin, 20-Hydroxyecdysone, Berberine, Calopiptin, Moupinamide, Magnoflorine, Palmitine, Pantothenic Acid, Phloretin, Palmatoside-C, Piperanine, Syringin, Tembetarine, Tetrahydropalmatine, Tinocordioside, Tinosponone, and Yuanhunine. Fifteen compounds from neem and seventeen compounds from giloy, where quercetin is present in both medicinal plants^38-43.

2.2 Selection of reference molecule:

Moxifloxacin is selected as a reference compound due to its promising efficacy against DFUs. It is a broad-spectrum antibiotic belonging to the fluoroquinolone class of medications. Previous studies have demonstrated favorable safety and efficacy profiles in patients with DFUs. Oral or intravenous administration of moxifloxacin, either alone or in combination therapy, has shown comparable efficacy to conventional antibiotic combinations, such as piperacillin-tazobactam and amoxicillin-clavulanate, in the treatment of moderate to severe DFUs. Furthermore, an intravenous or oral moxifloxacin regimen has been clinically and bacteriologically indistinguishable from a TDS intravenous regimen of piperacillin-tazobactam followed by a BD oral regimen of amoxicillin-clavulanate in remission. These findings suggest that moxifloxacin may be a valuable alternative antibiotic therapy for DFUs, offering simpler dosing regimens and the potential for enhanced patient outcomes^44-46.

2.3 In silico and network pharmacology-based studies:

2.3.1. Compound’s drug likeness score and toxicity predictions:

The canonical SMILES representations of 30 compounds were generated utilizing the online web server PubChem by utilizing a specific credential (http://pubchem.ncbi.nlm.nih.gov/). Subsequently, an in silico analysis was conducted to assess the physicochemical and ADMET profiles of the compounds. This analysis was performed online through servers like SwissADME (http://www.swissadme.ch/) with searching these characters, pKCSM (http://biosig.unimelb.edu.au/), and a toxicity-related server named Protox-II using credential (http://tox-new.charite.de/), respectively. The SwissADME server evaluates the physicochemical parameters of all selected molecules in this study. These parameters include molar refractivity, hydrogen bond acceptors, log P, hydrogen bond donors, molecular weight, topological polar surface area, as well as drug-likeness score for each molecule. Several pharmacokinetic elucidations of the compounds, such as their absorption into the gastrointestinal tract, skin permeability, water solubility, blood-brain barrier permeability, Caco-2 permeability, unbound fractions, and whether they are substrates for P-glycoproteins, were investigated using the pKCSM server. Additionally, toxicity parameters, including their AMES mutagenicity, chronic toxicity, and oral rat acute toxicity, were assessed using the Protox-II server. Basic toxicity profiles for compounds, including immune toxicity, hepatotoxicity, cytotoxicity, mutagenicity, and carcinogenicity, were predicted with the ProTox II database.

2.3.2. Target genes screening to selected compounds and DFUs:

The selection of targeted genes encoding bioactive compounds was performed using web servers SwissTarget (http://www.swisstargetprediction.ch/) with credential and TargetNet (http://targetnet.scbdd.com/calcnet/index/) with this credential. The UniProt IDs of the targeted genes were converted into a common gene name using the online web server UniProt (https://www.uniprot.org/id-mapping). Subsequently, an online database, GeneCards (https://www.genecards.org/), and DisGeNET (https://www.disgenet.org/), with credentials, were employed to identify genes associated with DFUs. The screening and intersections of common genes of the compounds and DFUs were conducted using the Venny Tool 2.1 with this credential (https://bioinfogp.cnb.csic.es/tools/venny/).

2.3.3. Network construction of interconnection between screened genes:

The web server STRING version 11.5 (https://string-db.org/) was utilized to develop the protein-protein interaction (PPI) network. The common screened genes were identified by setting an optimal interaction score of 0.4. The analysis of gene interactions and the identification of intersecting potential genes led to the creation of a hub gene network and was utilized for further study and evaluation.

2.3.4. Gene ontology and KEGG pathways analysis of hub genes:

The web server ShinyGo with credential (http://bioinformatics.sdstate.edu/go/) was used to predict the Gene Ontology (GO) of hub genes. The false discovery rate (FDR) threshold was set at 0.05, and the common genes found in the intersection were entered into the ShinyGo database with the organism option set to "human." FDR is the ratio of the percentage of false positive results out of all positive results. Hub genes were predicted to have a KEGG pathway from the above web server, and a significant signaling pathway enrichment image was downloaded and explored.

2.3.5. Validation of quality of genes:

The selected hub genes are assessed for their quality using the online web server PROCHECK (https://saves.mbi.ucla.edu/). Protein Data Bank with online search option (https://www.rcsb.org/) is an online service from which PDB format structures can be obtained. The structures that were recovered are then added to the PROCHECK database and examined for individual proteins' Ramachandran plots in order to assess their quality. Using the web server ProSA (https://prosa.services.came.sbg.ac.at/prosa.php), the protein's quality is further evaluated.

2.4 Molecular docking analysis of the screened bioactive molecules on a hub gene:

Finally, the screened hub genes and compounds were tested for their interaction to assess target accuracy. Using the online web server CACTUS (https://cactus.nci.nih.gov/translate/), the canonical SMILES of Moxifloxacin and bioactive chemicals (as ligands) were transformed into PDB formats. The structure of selected human hub genes (as proteins) in PDB format was downloaded from the online server Protein Data Bank with online search option (https://www.rcsb.org/). As both proteins and ligands were converted to PDB format for molecular docking tests in the subsequent study using Autodock Tool 1.5.7 software, the Biovia Discovery Studio Visualizer tool was recently utilized to visualize protein-ligand interactions images in two-dimensional and three-dimensional representations.

3. RESULTS:

3.1 ADMET evaluation of selected compounds:

The profile screened by ADMET for all 31 bioactive compounds was evaluated, as presented in Supplementary Tables S1A–S1C. All compounds were screened based on the criteria of their predictive toxicity and five optimization rules for the drug likeness score (Lipinski’s rules). In this study, we focused on compounds with zero Lipinski’s violation and a probability of toxicity below 0.70. Four compounds meet these criteria, and the physiological profile of selected bioactive compounds is shown in Table 1. They were evaluated for their target site; only lactopiperanol C was found not targeted by the Swiss Target database. Therefore, four bioactive compounds and one reference molecule are selected for further study, namely nimbidiol, caulerpin, lactopiperanol C, phloretin, and moxifloxacin.

3.2 Target genes identification of selected compounds, reference and DFUs:

A comprehensive analysis of 3490 genes was conducted utilizing the Swiss Target prediction and Target Net databases in conjunction with selected bioactive compounds and reference drugs.Additionally, GeneCards and DisGeNET identified 160 genes, which are associated with DFUs.

Using the Venny Tool, a total of 581 common genes were found in selected bioactive compounds, including "nimbidiol," "caulerpin," "phloretin," and "lactopiperanol c." Subsequent screening reported 580 genes were common by both selected and reference compounds. Overall, 17 genes were found to be common among the selected bioactive compounds, reference, and DFUs (Fig. 2A).

3.3 PPI network of hub genes:

Finally, seventeen genes were identified as being involved in a network of protein-protein interaction (PPI), constructed using the online Database ‘STRING’ with a confidence score of 0.400. The network comprises 17 nodes and 44 edges, with 17 of these directly selected and reference compounds for pharmacological evaluations (Fig. 2B). The database identified the presence of generated points in the network, including an average node degree of 5.18, an average local clustering coefficient 0.796, an expected number of edges of 11, as well as a PPI enrichment p-value of 8.23e-14. Overall, we were able to identify and focus on genes significantly; including those directly involved in the network, namely TGBR1, F2, MAPK14, SIRT1, and NOS3, for pharmacological evaluations and identifies them as hub genes for the present study.

Following the hub gene selection, Gene Ontology (GO) was used to examine the pharmacological activities, and the ShinyGO database was utilized for the gene enrichment analysis using KEGG pathways (Figs. 2C and 2D). There, various activity of genes was detected predicting various biological processes for the targeted hub genes, which were also involved in several biological pathways, such as relaxin signaling (FDR: 2.42E-08), AGE-RAGE signaling in diabetic complications (FDR: 2.22E-05), VEGF signaling (FDR: 0.00425), IL-17 signaling (FDR: 1.17E-05), diabetic cardiomyopathy (FDR: 1.17E-05), platelet activation (FDR: 0.000834), and cellular senescence (FDR: 0.001207) in this study.

3.4 Validation of selected receptor:

The target hub genes in the form of its PDB file were analyzed using the PROCHECK server database to evaluate their quality as Ramachandran plots. The percentage of amino acids in the preferred region of the receptor plots was determined to be TGBR1 (100%), F2 (98.4%), MAPK14 (99.3%), SIRT1 (100%), and NOS3 (99.8%), as presented in Supplementary Figures S1A-S1E, respectively.

Figure 2: (A) Intersection of genes associated with ligands, receptor, and DFUs; (B) Protein-protein interaction network of hub genes involved in DFUs; (C) False discovery rate (FDR) for identified genes associated with DFUs and (D) KEGG pathway analysis of diabetic complications.

Furthermore, the quality of the selected receptors was assessed utilizing the ProSA web database. The z-scores of the corresponding map PDB proteins were scientifically validated through NMR spectroscopy (deep blue) and X-ray diffraction (pale blue). The z-scores for the receptors were TGBR1 (-5.46), F2 (-8.24), MAPK14 (-6.66), SIRT1 (-6.76), and NOS3 (-8.58), as depicted in Supplementary Figures S2A-S2E, respectively.

3.5 Molecular docking analysis:

An examination of computational molecular docking was performed to determine the affinities of ligand binding to particular target proteins. All simulations utilized a consistent grid spacing of 0.375 angstrom as well as a uniform grid size of x center:126, y center:126, and z center:126 for all proteins and ligands. The mapping coordinates (x-,y-,z-dimension) for the proteins were as follows: TGBR1 (12.145, 1.093, 12.016), SIRT1 (7.570, 29.268, 78.716), NOS3 (64.690, 31.336, -185.935), MAPK14 (130.809, 127.876, 108.042), and F2 (79.779, -44.678, -23.808). Among the ligands studied, SIRT1 consistently exhibited the most favorable binding affinity (-7.43 kcal/mol) across all sites in the present study, suggesting its potential as a potent ligand for nimbidiol. Nimbidiol exhibited superior binding affinity compared to the reference compounds and remaining bioactive compounds for the target proteins SIRT1, MAPK14, F2, NOS3, and TGBR2. The binding scores for these proteins were -7.43 kcal/mol, -7.36 kcal/mol, -7.31 kcal/mol, -6.62 kcal/mol, and -6.58 kcal/mol, respectively.

Caulerpin also demonstrated promising binding properties against the reference compounds and remaining bioactive compounds of this study (excluding nimbidiol) for the target proteins SIRT1, F2, NOS3, MAPK14, and TGBR2. The binding scores for these proteins were -7.25 kcal/mol, -6.75 kcal/mol, -6.57 kcal/mol, -5.94 kcal/mol, and -5.61 kcal/mol, respectively. However, it exhibited lower potency compared to nimbidiol.

In contrast, phloretin and lactopiperanol c exhibited favorable binding affinities in comparison to the reference compounds for the target protein F2, with binding scores of -5.99 kcal/mol and -5.92 kcal/mol, respectively. Additionally, lactopiperanol c exhibited a higher binding affinity with respect to the reference compound for the TGBR1 receptor, with a binding score of -5.92 kcal/mol. The Biovia discovery tool examined and elucidated various interactions between the receptors and ligands, involving diverse molecular efficacies. The highest interactions, binding scores, and 3D visualizations of receptor-ligand complexes are presented in Table 2 and Fig. 3A-3B and Fig. 4A-4C. The remaining molecules binding affinity and 3D visualizations of receptor-ligand complexes are shown in Supplementary Table 4 and Fig. 3-4.

Table 2: Binding energy of ligands with DFUs receptors

Compound	Protein	Binding energy (kcal/mol)
Nimbidiol	TGBR1	-6.58
	F2	-7.31
	SIRT1	-7.43
	NOS3	-6.62
	MAPK14	-7.36

Figure 3: Molecular docking analysis interaction images of nimbidiol with strong binding affinities with key proteins involved in diabetic foot ulcer (DFU) pathogenesis. (A) SIRT1 and (B) MAPK14.

4. DISCUSSION:

Millions of individuals worldwide are affected by diabetic foot ulcers (DFUs), which significantly impact their physical and mental well-being. Furthermore, DFUs impose substantial burdens on healthcare systems due to their intricate treatment requirements and associated economic costs. Notably, DFUs pose particular challenges in patients with diabetes, a condition characterized by the absence of normal neurological processes in the foot, chronic inflammation, vascular insufficiency, inadequate nutrient provision, and subsequent development of ulcers and gangrene. Due to their high infection incidence and poor wound healing rate, diabetic foot ulcers (DFUs) are a major cause of catastrophic amputations in diabetes patients ^47-48.

The process of mending a wound has many facets, which involve homeostasis, inflammation, proliferation, and remodeling. A mismatch between pro- and anti-inflammatory signals is seen in chronic wounds, where too much inflammation hinders the inflammatory phase's resolution and causes slowed or compromised healing ⁴⁹. In this study, molecular docking analysis was conducted on two plants, neem and giloy, due to their demonstrated efficacy against bacterial and fungal infections, as well as in regulating hyperglycemic levels^50-54.

Figure 4: Molecular docking analysis interaction images of nimbidiol with strong binding affinities with key proteins involved in diabetic foot ulcer (DFU) pathogenesis. (A) F2, (B) NOS3 and (C) TGBR1.

We identified inflammation, neurological disorders, and vascular dysfunction as key issues associated with hyperglycemia and conducted research on the constituents of Neem and Giloy. Neem contains numerous chemical constituents that have been historically recognized for their potential therapeutic properties. Furthermore, Giloy contains potent chemical constituents that have been demonstrated to be effective in treating a variety of diseases and disorders^55-57.

The primary disorders associated with DFUs, including inflammation, neurological disorders, vascular dysfunction, and immune system impairment, was the focus of our research. Our analysis revealed that the selected bioactive molecules from both medicinal plants and moxifloxacin (reference molecule) interacted with five genes associated with the disease. These five genes, TGBR1, SIRT1, MAPK14, F2, and NOS3, play pivotal roles in wound healing. TGBR1 is involved in tissue repair and the extracellular matrix production, SIRT1 regulates insulin signaling, metabolism, inflammation, and cellular mechanisms, MAPK14 reduces inflammation and promotes healing, NOS3 enhances vascular function and blood flow, and F2 regulates thrombin activity, preventing excessive clotting or bleeding and facilitating optimal wound healing.

The mechanisms underlying these processes are suggested by KEGG pathways, including the AGE-RAGE signaling pathway in diabetic complications, the Relaxin signaling pathway, the IL-17 signaling pathway associated with diabetic cardiomyopathy, platelet activation, the VEGF signaling pathway, cellular senescence, lipid and atherosclerosis, and pathways in cancer.

Moxifloxacin was utilized as a reference compound due to its established safety and efficacy against specific infections. It has also demonstrated promising results in the treatment of DFUs. The molecular docking simulation was carried out in order to assess and compare the profiles of bioactive compounds with the reference molecule, thereby identifying potential candidates for the treatment of DFUs.

Molecular docking analysis identified potential candidates with their targeting sites or receptors⁵⁸. Nimbidiol exhibited a potent binding affinity compared to all bioactive and reference molecules. It bound to SIRT1 with a higher score (binding affinity) of -7.31 kcal/mol, MAPK14 with -7.36 kcal/mol, F2 with -7.13 kcal/mol, NOS3 with -6.62 kcal/mol, and TGBR1 with -6.58 kcal/mol. Caulerpin demonstrated the second-highest potential activity against moxifloxacin and other bioactive molecules in this study. Additionally, lactopiperanol c and phloretin emerged as potential candidates in comparison to moxifloxacin, exhibiting affinity for receptors such as TGBR1 (-5.92 kcal/mol) and F2 (-5.89 kcal/mol) for lactopiperanol c and F2 (-5.99 kcal/mol) for phloretin. However, their affinity was lower compared to nimbidiol and caulerpin.

Furthermore, SIRT1, NOS3, and MAPK14 levels were lower in both lactopiperanol c and phloretin compared to moxifloxacin and the bioactive molecules nimbidiol and caulerpin. Molecular docking analysis of nimbidiol revealed strong binding affinities with key proteins involved in diabetic foot ulcer (DFU) pathogenesis. As shown in Figure 3A-3B and Figure 4A-4C, nimbidiol exhibited the highest binding affinity to SIRT1 (Fig. 3A), MAPK14 (Fig. 3B), F2 (Fig. 4A), NOS3 (Fig. 4B), and TGBR1 (Fig. 4C), suggesting its potential therapeutic efficacy for DFU. Consequently, further research is essential to verify and assess the potential activity of nimbidiol and caulerpin. In vivo studies could be employed to evaluate the efficacy and safety of bioactive compounds of the present study in animal models. Additionally, clinical trials may also be required to evaluate the effectiveness of these bioactive compounds in human patients with DFUs.

5. CONCLUSION:

The systematic molecular docking study successfully investigated the potential of compounds from T. cordifolia and A. indica for treating DFUs. By targeting key hub genes involved in various biological pathways related to DFUs, including relaxin signaling (FDR: 2.42E-08), AGE-RAGE signaling (FDR: 2.22E-05), VEGF signaling (FDR: 0.00425), and platelet activation (FDR: 0.000834), the FDR values indicate the statistical significance of the identified pathways in relation to DFUs. Among the selected compounds, nimbidiol emerged as a standout, exhibiting strong binding affinities to all target proteins. Its superior binding characteristics compared to moxifloxacin and other bioactive molecules suggest its potential as a promising therapeutic agent for DFUs. Specifically, nimbidiol demonstrated binding affinities of -7.43 kcal/mol to SIRT1, -7.36 kcal/mol to MAPK14, -7.31 kcal/mol to F2, -6.62 kcal/mol to NOS3, and -6.58 kcal/mol to TGBR1. Additionally, caulerpin demonstrated favorable binding properties, ranking second in terms of affinity score. Other compounds, such as lactopiperanol C and phloretin, also exhibited potential binding to specific receptors, suggesting their potential roles in modulating DFU-related pathways. Although this study sheds light on how these drugs interact molecularly with target proteins, more in vivo research is required to confirm their effectiveness and safety profiles in the treatment of diabetic foot ulcers (DFUs). The identified hub genes, particularly SIRT1 and MAPK14, warrant further investigation as potential therapeutic targets. Overall, the potentials of this study offer a promising foundation for future pharmaceutical research and development of novel therapeutic strategies for DFUs.

6. ACKNOWLEDGEMENTS:

The author thankfully acknowledge to Dr. Sunil Kumar Mishra, Pharmaceutical Engineering and Technology, IIT (BHU) Varanasi-221 005, Uttar Pradesh, India, for guidance during the work. The author is also thankful to Dr. Anand Kumar Singh, Head of Department of Chemistry, PG College Mariahu, Jaunpur (222161), Uttar Pradesh, India, for key notes during the work.

7. CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

8. REFERENCE:

1. Li Y, Leng Y, Liu Y, Zhong J, Li J, Zhang S, Li Z, Yang K, Kong X, Lao W, Bi C. Advanced multifunctional hydrogels for diabetic foot ulcer healing: Active substances and biological functions. Journal of Diabetes. 2024; Apr;16(4): e13537.

2. Zheng H, Na H, Yao J, Su S, Han F, Li X, Chen X. 16S rRNA seq-identified Corynebacterium promotes pyroptosis to aggravate diabetic foot ulcer. BMC Infectious Diseases. 2024; Apr 1; 24(1): 366.

3. Al Mamun A, Shao C, Geng P, Wang S, Xiao J. The Mechanism of Pyroptosis and Its Application Prospect in Diabetic Wound Healing. Journal of Inflammation Research. 2024; Dec 31: 1481-501.

4. Yang C, Wang D. Antibiotic bone cement accelerates diabetic foot wound healing: Elucidating the role of ROCK1 protein expression. International Wound Journal. 2024 Apr;21(4):e14590.

5. Kim E, Seo SH, Hwang Y, Ryu YC, Kim H, Lee KM, Lee JW, Park KH, Choi KY. Inhibiting the cytosolic function of CXXC5 accelerates diabetic wound healing by enhancing angiogenesis and skin repair. Experimental and Molecular Medicine. 2023 Aug;55(8):1770-82.

6. Abbas HA. Diabetic foot infection. Research journal of pharmacy and technology. 2015;8(5):575-9.

7. Abbas HA, El-Sayed MA, Al-Kadi LM, Gad AI. Diabetic foot infections in Zagazig University Hospital: bacterial etiology, antimicrobial resistance and biofilm formation. Research Journal of Pharmacy and Technology. 2014;7(7):783-8.

8. Han Z, Li A, Xue Z, Guan SB, Yin G, Zheng X. Eugenol-loaded polyurethane gelatin dressing for efficient angiogenesis and antibacterial effects in refractory diabetic wound defect healing. International Journal of Biological Macromolecules. 2024 Jun 1;271:132619.

9. Xie J, Liu X, Wu B, Chen B, Song Q, Guan Y, Gong Y, Yang C, Lin J, Huang M, Tan X. Bone transport induces the release of factors with multi-tissue regenerative potential for diabetic wound healing in rats and patients. Cell Reports Medicine. 2024 Jun 18;5(6).

10. Shin SS, Hwang B, Muhammad K, Gho Y, Song JH, Kim WJ, Kim G, Moon SK. Nimbolide represses the proliferation, migration, and invasion of bladder carcinoma cells via Chk2‐mediated G2/M phase cell cycle arrest, altered signaling pathways, and reduced transcription factors‐associated mmp‐9 expression. Evidence‐Based Complementary and Alternative Medicine. 2019;2019(1):3753587.

11. Chinnasamy G, Chandrasekharan S, Koh TW, Bhatnagar S. Synthesis, characterization, antibacterial and wound healing efficacy of silver nanoparticles from Azadirachta indica. Frontiers in Microbiology. 2021 Feb 19;12:611560.

12. Maan P, Yadav KS, Yadav NP. Wound healing activity of Azadirachta indica A. juss stem bark in mice. Pharmacognosy Magazine. 2017 Jul; 13(Suppl 2): S316.

13. Naveed N, Murthykumar K, Soundarajan S, Srinivasan S. The use of neem in oral health. Research Journal of Pharmacy and Technology. 2014; 7(9): 1060-4.

14. Bose S, Shinde H, Karikalan K, Lalitha P, Mandal AK. Antibacterial, antiinflamatory, and antiproliferative activity of silver nanoparticles synthesized from leaf extract of Azadirachta indica A. juss. Research Journal of Pharmacy and Technology. 2016; 9(12): 2422-6.

15. Sharma A, Saggu SK, Mishra R, Kaur G. Anti-brain cancer activity of chloroform and hexane extracts of Tinospora cordifolia Miers: an in vitro perspective. Annals of Neurosciences. 2019 Jan; 26(1): 10-20.

16. Jeyachandran R, Xavier TF, Anand SP. Antibacterial activity of stem extracts of Tinospora cordifolia (Willd) Hook. f and Thomson. Ancient Science of Life. 2003 Jul 1; 23(1): 40-3.

17. Philip S, Tom G, Balakrishnan Nair P, Sundaram S, Velikkakathu Vasumathy A. Tinospora cordifolia chloroform extract inhibits LPS-induced inflammation via NF-κB inactivation in THP-1cells and improves survival in sepsis. BMC Complementary Medicine and Therapies. 2021 Dec; 21: 1-3.

18. Alsuhaibani S, Khan MA. Immune‐stimulatory and therapeutic activity of Tinospora cordifolia: double‐edged sword against salmonellosis. Journal of Immunology Research. 2017; 2017(1): 1787803.

19. Biswasroy P, Panda S, Das C, Das D, Kar DM, Ghosh G. Tinospora cordifolia-A plant with Spectacular natural immunobooster. Research Journal of Pharmacy and Technology. 2020; 13(2): 1035-8.

20. Barlaskar WH, Sharma NR, Kumar G, Kataria S. Effect of Host Trees on Berberine Content in Stem of Tinospora cordifolia Climber: A Preliminary Study. Research Journal of Pharmacy and Technology. 2017; 10(3): 818-22.

21. Suganya J, Viswanathan T, Radha M, Marimuthu N. In silico Molecular Docking studies to investigate interactions of natural Camptothecin molecule with diabetic enzymes. Research Journal of Pharmacy and Technology. 2017; 10(9): 2917-22.

22. Suganya J, Manoharan S, Radha M, Singh N, Francis A. Identification and Analysis of Natural Compounds as Fungal Inhibitors from Ocimum sanctum using in silico Virtual Screening and Molecular Docking. Research Journal of Pharmacy and Technology. 2017; 10(10): 3369-74.

23. Sharma A, Kaur G. Tinospora cordifolia as a potential neuroregenerative candidate against glutamate induced excitotoxicity: an in vitro perspective. BMC Complementary and Alternative Medicine. 2018 Dec; 18: 1-7.

24. Huang L, Cai HA, Zhang MS, Liao RY, Huang X, Hu FD. Ginsenoside Rg1 promoted the wound healing in diabetic foot ulcers via miR-489–3p/Sirt1 axis. Journal of Pharmacological Sciences. 2021 Nov 1; 147(3): 271-83.

25. Letonja J, Završnik M, Makuc J, Šeruga M, Peterlin A, Cilenšek I, Petrovič D. Sirtuin 1 rs7069102 polymorphism is associated with diabetic nephropathy in patients with type 2 diabetes mellitus. Bosnian Journal of Basic Medical Sciences. 2021 Oct; 21(5): 642.

26. Hu YJ, Song CS, Jiang N. Single nucleotide variations in the development of diabetic foot ulcer: A narrative review. World Journal of Diabetes. 2022 Dec 12; 13(12): 1140.

27. Zhang J, Lin J, Liang B, Chen L, Yang X, Li M. Fibrinogen function indexes are potential biomarkers for evaluating the occurrence and severity of diabetic foot. Diabetology and Metabolic Syndrome. 2022; Dec 2; 14(1): 182.

28. Li XH, Guan LY, Lin HY, Wang SH, Cao YQ, Jiang XY, Wang YB. Fibrinogen: a marker in predicting diabetic foot ulcer severity. Journal of Diabetes Research. 2016; 2016(1): 2358321.

29. Korkmaz P, Koçak H, Onbaşı K, Biçici P, Özmen A, Uyar C, Özatağ DM. The role of serum procalcitonin, interleukin‐6, and fibrinogen levels in differential diagnosis of diabetic foot ulcer infection. Journal of Diabetes Research. 2018; 2018(1): 7104352.

30. Du G, Chen J, Zhu X, Zhu Z. Bioinformatics analysis identifies TGF-β signaling pathway-associated molecular subtypes and gene signature in diabetic foot. IScience. 2024 Mar 15;27(3).

31. Patel AS, Patel SA, Fulzele PR, Mohod SC, Chandak M, Patel SS. Evaluation of the role of propolis and a new herbal ointment in promoting healing of traumatic oral ulcers: an animal experimental study. Contemporary Clinical Dentistry. 2020 Apr 1; 11(2): 121-5.

32. Castillo AL, Osi MO, Ramos JD, De Francia JL, Dujunco MU, Quilala PF. Efficacy and safety of Tinospora cordifolia lotion in Sarcoptes scabiei var hominis-infected pediatric patients: A single blind, randomized controlled trial. Journal of Pharmacology and Pharmacotherapeutics. 2013 Mar; 4(1): 39-46.

33. Nasrine A, Narayana S, Ahmed MG, Sultana R, Noushida N, Salian TR, Almuqbil M, Almadani ME, Alshehri A, Alghamdi A, Alshehri S. Neem (Azadirachta Indica) and silk fibroin associated hydrogel: Boon for wound healing treatment regimen. Saudi Pharmaceutical Journal. 2023 Oct 1; 31(10): 101749.

34. Fiaschini N, Carnevali F, Van der Esch SA, Vitali R, Mancuso M, Sulli M, Diretto G, Negroni A, Rinaldi A. Innovative Multilayer Electrospun Patches for the Slow Release of Natural Oily Extracts as Dressings to Boost Wound Healing. Pharmaceutics. 2024 Jan 24; 16(2): 159.

35. Veerendrakumar P, Neralla M, Baskar V, Satheesh T. Comparative Extraction and Bioactive Potential of the Leaf Extracts of Azadirachta indica for Combatting Postoperative Head and Neck Infections: An In Vitro Study. Cureus. 2023 Dec; 15(12).

36. Ezhilarasu K, Kasiranjan A, Priya S, Kamaraj A. The Antibacterial Effect of Tinospora Cordifolia (Guduchi) and Its Role in Combating Antimicrobial Resistance. Medeniyet Medical Journal. 2023 Sep 1; 38(3): 149-58.

37. Mishra A, Kumar S, Pandey AK. Scientific validation of the medicinal efficacy of Tinospora cordifolia. The Scientific World Journal. 2013;2013(1):292934.

38. Faloye KO, Adesida SA, Oguntimehin SA, Adewole AH, Omoyeni OB, Fajobi SJ, Ugwo JP, Asiyanbola ID, Bamimore VO, Fakola EG, Oladiran OJ. LC-MS analysis, computational investigation, and antimalarial studies of Azadirachta indica fruit. Bioinformatics and Biology Insights. 2023 Feb; 17: 11779322231154966.

39. Borkar S, Roy M, Jadhao S, Gade N, Dutta G, Hirpurkar S, Mishra O. High resolution liquid chromatography-mass spectrometry analysis (q-tof-ms) and free radical scavenging activity of extract of azadirachta indica leaves.

40. Barrek S, Paisse O, Grenier-Loustalot MF. Analysis of neem oils by LC–MS and degradation kinetics of azadirachtin-A in a controlled environment: Characterization of degradation products by HPLC–MS–MS. Analytical and Bioanalytical Chemistry. 2004 Feb; 378: 753-63.

41. Mohan MC, Abhimannue AP. Identification and characterization of Berberine in tinospora cordifolia by liquid chromatography Quadrupole time of flight mass spectrometry (LC MS/MS q-tof) and evaluation of its anti inflammatory potential. Pharmacognosy Journal. 2017; 9(3).

42. Reddi KK, Tetali SD. Dry leaf extracts of Tinospora cordifolia (Willd.) Miers attenuate oxidative stress and inflammatory condition in human monocytic (THP-1) cells. Phytomedicine. 2019 Aug 1;61:152831.

43. Royani A, Hanafi M, Julistiono H, Dinoto A, Lotulung PD, Manaf A. The potential of Tinospora cordifolia extracts as antibacterial material against Pseudomonas aeruginosa. Trends in Sciences. 2023; 20(1): 3884-.

44. Schaper NC, Dryden M, Kujath P, Nathwani D, Arvis P, Reimnitz P, Alder J, Gyssens IC. Efficacy and safety of IV/PO moxifloxacin and IV piperacillin/tazobactam followed by PO amoxicillin/clavulanic acid in the treatment of diabetic foot infections: results of the RELIEF study. Infection. 2013 Feb; 41: 175-86.

45. Lipsky BA, Giordano P, Choudhri S, Song J. Treating diabetic foot infections with sequential intravenous to oral moxifloxacin compared with piperacillin–tazobactam/amoxicillin–clavulanate. Journal of Antimicrobial Chemotherapy. 2007 Aug 1; 60(2): 370-6.

46. Gyssens IC, Dryden M, Kujath P, Nathwani D, Schaper N, Hampel B, Reimnitz P, Alder J, Arvis P. A randomized trial of the efficacy and safety of sequential intravenous/oral moxifloxacin monotherapy versus intravenous piperacillin/tazobactam followed by oral amoxicillin/clavulanate for complicated skin and skin structure infections. Journal of Antimicrobial Chemotherapy. 2011 Nov 1; 66(11): 2632-42.

47. Wang T, Li X, Tao Y, Wang X, Li L, Liu J. METTL3-mediated NDUFB5 m6A modification promotes cell migration and mitochondrial respiration to promote the wound healing of diabetic foot ulcer. Journal of Translational Medicine. 2024 Jul 9;22(1):643.

48. Xu J, Gao J, Li H, Zhu Z, Liu J, Gao C. The risk factors in diabetic foot ulcers and predictive value of prognosis of wound tissue vascular endothelium growth factor. Scientific Reports. 2024 Jun 19; 14(1): 14120.

49. Hu K, Liu L, Tang S, Zhang X, Chang H, Chen W, Fan T, Zhang L, Shen B, Zhang Q. MicroRNA-221-3p inhibits the inflammatory response of keratinocytes by regulating the DYRK1A/STAT3 signaling pathway to promote wound healing in diabetes. Communications Biology. 2024 Mar 9;7(1):300.

50. Lakkim V, Reddy MC, Lekkala VV, Lebaka VR, Korivi M, Lomada D. Antioxidant efficacy of green-synthesized silver nanoparticles promotes wound healing in mice. Pharmaceutics. 2023 May 17; 15(5): 1517.

51. Suresh MX, Mahalakshmi JA, Vinisha G. Computational Modeling of Novel Drug Targets ftsE and mpt83 for MDRTB and Molecular Docking of Selected Compounds from Medicinal Plants. Research Journal of Pharmacy and Technology. 2018; 11(4): 1413-20.

52. Nigussie D, Davey G, Legesse BA, Fekadu A, Makonnen E. Antibacterial activity of methanol extracts of the leaves of three medicinal plants against selected bacteria isolated from wounds of lymphoedema patients. BMC Complementary Medicine and Therapies. 2021 Dec; 21: 1-0.

53. Malve H, More D, More A. Effects of two formulations containing Phyllanthus emblica and Tinospora cordifolia with and without Ocimum sanctum in immunocompromised mice. Journal of Ayurveda and Integrative Medicine. 2021 Oct 1;12(4):682-8.

54. Adelakun AO, Awosika A, Adabanya U, Omole AE, Olopoda AI, Bello ET. Antimicrobial and Synergistic Effects of Syzygium cumini, Moringa oleifera, and Tinospora cordifolia Against Different Candida Infections. Cureus. 2024 Jan; 16(1).

55. Xiao Y, Qian J, Deng X, Zhang H, Wang J, Luo Z, Zhu L. Macrophages regulate healing-associated fibroblasts in diabetic wound. Molecular Biology Reports. 2024 Dec; 51(1): 203.

56. Tong J, Zhang J, Xiang L, Li S, Xu J, Zhu G, Dong J, Cheng Y, Ren H, Liu M, Yue L. Continuous intrafemoral artery infusion of urokinase improves diabetic foot ulcers healing and decreases cardiovascular events in a long-term follow-up study. BMJ Open Diabetes Research and Care. 2024 Jan 1; 12(1): e003414.

57. Li D, Peng H, Qu LE, Sommar P, Wang A, Chu T, Li X, Bi X, Liu Q, Serezal IG, Rollman O. miR-19a/b and miR-20a promote wound healing by regulating the inflammatory response of keratinocytes. Journal of Investigative Dermatology. 2021; Mar 1; 141(3): 659-71.

58. Bhardwaj K, Khurana N, Sutte A, Khatik G. Identification of Dipeptidyl peptidase-4 (DPP-4) inhibitors as Potential Antidiabetic agents using Molecular docking study. Research Journal of Pharmacy and Technology. 2020; Nov 1; 13(11): 5257-62.

Received on 10.11.2024 Revised on 08.03.2025

Accepted on 18.05.2025 Published on 08.11.2025

Available online from November 13, 2025

Research J. Pharmacy and Technology. 2025;18(11):5416-5424.

DOI: 10.52711/0974-360X.2025.00781

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

S. No.	Ligands	MF	MW (g/mol)	HBA	HBD	BBB	DL (Lipinski's violation)
1	Caulerpin	C24H18N2O4	398.41	4	2	0.169	Yes; 0
2	Lactopiperanol c	C16H26O4	282.38	4	2	0.039	Yes; 0
3	Nimbidol	C17H22O3	274.35	3	2	0.065	Yes; 0
4	Phlorectin	C15H14O5	274.27	4	4	-0.927	Yes; 0