INTRODUCTION:

Endothelial dysfunction

Endothelium, the thin layer that lines blood vessels and the heart, can be damage, resulting in endothelial dysfunction and is unable to carry out its usual roles. Vascular endothelial cells play a crucial role in maintaining physiological balance and serve as a protective barrier within blood vessel walls.¹ Endothelial dysfunction is believed to contribute significantly to atherosclerosis, a major cause of mortality in many Western countries, and is recognized as a key cardiovascular risk factor.

The presence of oxidative stress leads to the generation of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), hydroxyl (OH−), superoxide (O2−), and hypochlorite (OCl−). These ROS act as important signaling molecules at lower physiological levels, influencing inflammation and cell survival. However, excessive ROS production in endothelial cells is primarily responsible for initiating the formation of atheromatous plaques, a critical aspect of atherosclerosis pathophysiology.Additionally, ROS-induced endothelial dysfunction is implicated in several key events related to the development of atherosclerosis.^2,3,4 Reports indicate that reactive oxygen species play multiple roles in activating mitogen-activated protein kinases (MAPKs) and facilitating the movement of nuclear factor (NF)-κB from the cytosol into the nucleus. These activities are vital mediators in numerous pathological conditions. Specifically ROS can initiate the phosphorylation of P38 MAPK in endothelial cells, resulting in increased transcriptional and translational production of adhesion molecules and inflammatory cytokines.⁵ Excessive reactive oxygen species (ROS) can harm the outer mitochondrial membrane, leading to increased permeability. This change results in the release of various apoptosis-related proteins and proteases into the cytosol, which accelerates apoptosis and ultimately leads to cell death by cleaving essential cellular proteins. It is suggested that an effective strategy for preventing vascular diseases involves reducing oxidative damage and inflammatory responses in endothelial cells by inhibiting the ROS-mediated MAPK/NF-κB signaling pathway and addressing mitochondrial dysfunction through pharmacological interventions. This study aimed to evaluate the endothelial protective effects of herbal extracts by employing molecular docking after characterization of the extracts. By analyzing the binding affinities and bioactive profiles of these herbal extracts, we seek to highlight their potential therapeutic benefits in managing endothelial dysfunction associated with cardiovascular diseases.

Plants utilized for research:

Gurmar:

Gymnema sylvestre, often referred as gurmar or the "sugar destroyer," is significant in the management of metabolic disorders, with recent research underscoring its beneficial effects on endothelial dysfunction. This type of damage is frequently a precursor to cardiovascular diseases and is associated with metabolic issues such as type 2 diabetes.⁶ The active compounds in gurmar, particularly gymnemic acid, thought to provide protective benefits to blood vessels. Nitric oxide plays a crucial role in regulating vascular tone and ensuring endothelial health, and its diminished availability is a key indicator of endothelial dysfunction, which can lead to constricted and stiffer blood vessels. Studies suggest that gymnemic acids can boost nitric oxide production, facilitating blood vessel dilation and enhancing circulation.⁷ Furthermore, investigations into the effects of gymnemic acid in gurmar have revealed their connection to the activation of endothelial nitric oxide synthase (eNOS). Oxidative stress significantly contributes to endothelial dysfunction, especially in diabetes, where elevated glucose levels exacerbate oxidative damage to endothelial cells. Gurmar's antioxidant properties help shield the endothelium from oxidative stress by neutralizing reactive oxygen species, and research has shown that it enhances the levels of crucial antioxidant enzymes like superoxide dismutase and catalase, which are vital for mitigating oxidative stress.⁸Experimental studies have also demonstrated that extracts of gurmar can lower lipid peroxidation and other oxidative markers that adversely affect endothelial health.^9,10

Grapes:

Grapes; VitusVinefera, are rich in polyphenols such as resveratrol and flavonoids, have shown promise in addressing endothelial dysfunction, precursor to cardiovascular diseases.¹¹ Recent research indicates that these bioactive compounds can enhance endothelial function. Polyphenols found in grapes are known to stimulate the production of nitric oxide, leading to vasodilation, reduced blood pressure, and improved blood flow.¹² Additionally, these compounds help mitigate oxidative stress, a factor that contributes to endothelial dysfunction, by lowering the concentration of reactive oxygen species that can damage endothelial cells. Studies have also suggested that grape polyphenols may reduce the apoptosis of endothelial cells, thereby maintaining their structure and functionality. Some trials have indicated that resveratrol-rich grapes can elevate aortic endothelial nitric oxide synthase (eNOS), an enzyme crucial for nitric oxide production, further contributing to vascular health.^13,14

Curry leaves:

Curry leaves (Murraya koenigii) are commonly utilized in culinary practices and have demonstrated potential health benefits for various ailments. Research suggests that these leaves help in the prevention or management of conditions such as cardiovascular disease, diabetes, and cancer. Preceding studies reported theat inclusion of curry leaves improved the plasma lipid profile, and in obese mice, it positively affected glycemic levels and exhibited cholesterol-lowering effects. Some studies also indicate that it possess anti-oxidative and anti-lipid peroxidative properties, suggesting they may help protect against oxidative stress.^15,16,17Some studies suggest that these leaves possess anti-oxidative and anti-lipid peroxidative properties, indicating that they may help protect against oxidative stress.¹⁸^,¹⁹

Garlic:

Garlic, rich in bioactive compounds such as allicin, S-allyl cysteine, and sulfur-containing compounds, plays a significant role in addressing endothelial dysfunction.^20,21 Recent research highlights garlic's potential to enhance endothelial function through Nitric oxide (NO) production and its deficiency is often linked to atherosclerosis, characterized by vasoconstriction and inflammation. Allicin has been shown to boost NO production, promoting vasodilation and contributing to lower blood pressure. Studies indicate that garlic extract enhances NO availability by stimulating the endothelial nitric oxide synthase enzyme, particularly benefiting high-risk individuals with hypertension and type 2 diabetes, who frequently experience endothelial dysfunction. Furthermore, garlic has been associated with increased NO levels and reduced arterial stiffness, aiding in the management of early vascular disease signs. The presence of reactive oxygen species (ROS) is a key factor in the development of oxidative stress, leading to endothelial damage and a reduction in nitric oxide (NO) levels. Fortunately, garlic offers a natural solution, as its antioxidant capabilities help mitigate oxidative stress and protect the endothelium. Additionally, garlic exhibits anti-inflammatory properties, addressing elevated inflammatory cytokines like IL-6 and TNF-α, which are common in individuals at risk for cardiovascular disease.^22,23

Extraction procedure:

Selected plants were identified, collected and authenticated from renowned botanist Prof. Dr. Vijay Malik Head Department of Botany, Chaudhary Charan Singh University Meerut Ref: Bot/PB/368. Leaves of Gymnema Sylvestre, Murraya koenigii, bulbs of Allium sativum were dried and were coarsely powdered, individual plant powder was defat by petroleum ether and the extract was primed by Hydroalcoholic, water and ethanol in the ratio (3:7) using Soxhlet apparatus and the Berries of Vitis vinifera were macerated. The percentage yield of Gymnema Sylvestre, Murraya koenigii, Allium sativum and Vitis vinifera were calculated and found to be 12.35, 14, 10.82 and 9.5 respectively.^24,25,26,27

Characterization by IR, NMR and Mass spectroscopy:

The plant extract obtained after the extraction procedure were subjected to the characterization with the aid of IR, NMR and Mass spectroscopy and the important information regarding the individual plant extracts for IR spectroscopy findings are given in table 1 which confirms that the presence of expected constituents in the extract by identification of the suitable functional groups. All the chemical constituents in the plants are found in the extract by utilizing the all the characterization technique.^28,29

¹H NMR: δ 3.51-3.63 (4H, 3.57 (d, J = 6.3 Hz), 3.57 (d, J = 6.3 Hz), 3.57 (d, J = 6.3 Hz)), 4.89-5.04 (2H, 4.97 (dd, J = 17.3, 2.2 Hz), 4.97 (dd, J = 17.3, 2.2 Hz)), 5.07-5.20 (2H, 5.13 (dd, J = 10.6, 2.2 Hz), 5.13 (dd, J = 10.6, 2.2 Hz)), 6.45-6.63 (2H, 6.54 (ddt, J = 17.3, 10.6, 6.3 Hz), 6.54 (ddt, J = 17.3, 10.6, 6.3 Hz)), 9.00 (2H, d, J = 3.2 Hz).

¹H NMR: δ 1.43 (3H, s), 1.49-1.59 (6H, 1.54 (s), 1.54 (s)), 1.89 (2H, t, J = 7.3 Hz), 2.05 (2H, td, J = 7.3, 7.1 Hz), 5.27 (1H, t, J = 7.1 Hz), 5.78 (1H, d, J = 10.0 Hz), 6.29 (1H, d, J = 10.0 Hz), 6.59 (1H, d, J = 8.8 Hz), 7.16-7.32 (2H, 7.23 (ddd, J = 8.1, 6.6, 1.5 Hz), 7.25 (ddd, J = 8.0, 6.6, 1.8 Hz)), 7.60-7.81 (3H, 7.66 (ddt, J = 8.0, 1.5, 0.5 Hz), 7.67 (dd, J = 8.8, 0.5 Hz), 7.74 (ddd, J = 8.1, 1.8, 0.5 Hz)).

¹H NMR: δ 6.76 (1H, dt, J = 8.2, 2.7 Hz), 6.93 (2H, ddd, J = 8.3, 2.7, 0.5 Hz), 7.00-7.47 (7H, 7.07 (d, J = 13.9 Hz), 7.18 (d, J = 13.9 Hz), 7.20 (ddd, J = 8.3, 1.9, 0.5 Hz), 7.28 (ddd, J = 8.2, 7.7, 0.5 Hz), 7.34 (ddd, J = 2.7, 1.7, 0.5 Hz), 7.40 (ddd, J = 7.7, 2.7, 1.7 Hz))

m/z: 806.45 (100.0%), 807.45 (46.5%), 808.45 (10.6%), 808.45 (2.9%), 809.45 (1.3%), m/z: 228.08 (100.0%), 229.08 (15.1%), 230.09 (1.1%), m/z: 331.19 (100.0%), 332.20 (24.9%), 333.20 (2.7%), m/z: 162.02 (100.0%), 164.01 (9.0%), 163.02 (6.5%), 163.02 (1.6%)

The NMR data suggest that the plant extract consists of a large number of components: oxygen-containing non-saturated hydrocarbons which may be sugar or glycosides as well as aromatic compounds which may include phenol or polyphenol. The peaks are manifested at around 6.93 and 7-9ppm, which point to the presence of bioactive phenolic compound which gives the idea that extract has anti-oxidant qualities. Furthermore, the signals around 5-4ppm pertain to sugar-like structures indicating glycosidic linkages.

This Mass data analysis of the four plant samples shows that there are several molecular ion peaks, which points to variety of compounds unique to different plant. The observed base peaks as well as their relative intensity contain valuable information regarding the molecular structure of the analyzed compounds. Molecular ion observed at m/z 806.45 with another three peaks at m/z 807.45, 808.45, and 809.45 indicating the presence of complex framework belonging to glycosylated flavonoids or polyphenolic compounds usual in plants. Molecular ion at m/z 228.08 is flanked by low intensity peaks at m/z 229.08 and 230.09. This distribution suggests a less extensive conjugate, likely aromatic. This mass range is in agreement with the structures of alkaloids or nitrogen-containing plant secondary metabolites. Molecular ion at m/z 331.19 and two other ions at m/z 332.20 and 333.20, correlated with the complex plant phenolics or flavonoid like structures .

Molecular ion at m/z 162.02, with minor peaks at m/z 163.02 & 164.01 corresponds to compounds having simple or only slightly more complex structure characteristic for aroma compounds or for carbohydrates.

Characterization by HPTLC:

Sample Preparation or sampling technique and the analytical Method used:

Sample Solvent: All samples were dissolved in methanol a solution which can dissolve many of the plant metabolites consisting of both polar as well as semi polar compounds.

Mobile Phase Composition: The mobile phase was a solution containing toluene, chloroform, ethyl acetate, formic acid, n-butanol and methanol in a ratio of 3:2:4.9:0.1:0:0.5:0.5 respectively. This particular composition provides an optimal degree of polarity, improving the ability of the mobile phase to separate compounds having different hydrophobic and hydrophilic characteristics.

Stationary Phase and Development Conditions:

Stationary Phase: In the present investigation, HPTLC Silica gel 60 F₂₅₄ was employed as the stationary phase. The advantage of silica is the selectivity it displays towards virtually all classes of plant metabolites with polar chemical moieties.

Application and Track Setup: The tracks were allocated according to the sample with the volume of the sample being standardised at 4µL for 3 samples and 5µL for 1 sample which assists in comparing the samples uniformly.

Development Parameters: Saturation time was fixed at 20 minutes due to the fact that, this is the normal time that allows plates to go to their required saturation point making the results easily reproducible. Room temperature drying allows preserving most of the compounds of analysed samples without thermal degradation.

Detection and Scanning Conditions:

Detection Wavelengths:

The analysis utilized two wavelengths, 254nm and 366 nm, covering UV-active compounds, likely capturing glycosides, phenolic compounds, flavonoids, and other conjugated systems. The choice of these wavelengths is appropriate for capturing various secondary metabolites.

Scan Parameters:

Scans were conducted at 20 mm/s with a deuterium and tungsten lamp source, ensuring high-resolution images and sensitive peak detection across a broad absorbance range.

In- Silico (Docking studies):

Protein docking done for plant extracts enables identification of the kind of interaction geometry between bioactive molecules and selected protein receptors, which can aid in understanding of putative therapeutic effects.

Preliminary checks and measurement protocols which forms the basis of docking studies.

Software and Tools:

Molecular Docking Software:

The current research utilizes Schrödinger Suite for docking

Molecular Modeling Software:

ChemDraw is used for drawings chemical structure of plant extracts

Protein Data Bank (PDB):

A possible method for obtaining the 3D structures of target proteins. Proteins that have crystal structures of relatively high-resolution are utilized in this study.

Preparation of Ligands³⁴

Structure Collection: It is crucial to determine possible compounds in the plant extract giving it the ability to interact with cellular structures and exert various effects. Experimentally the active components are isolated and aided with a literature study.

Ligand Optimization:

By the help of the molecular modeling, it is possible to calculate the energy of ligands and adjust the correct stereochemistry. The ligands are taken in formats, which can be easily understood by any docking software, like PDBQT or MOL2 format. In the process of docking, preparation of protein targets is an important step.

Protein Selection:

Proteins are selected considering its role in the disease This, of course, needs a background check or literature research.

Protein Optimization:

Protein structure from Protein Data Bank, was retrieved, for refining the structure and preparation for docking the following steps was executed: water molecules were removed, added hydrogens and optimized the structure by adding missing atoms and/or side chains. The preparations were done using Schrödinger Suite.

Active Site Identification:

Binding site was ascertained from literature, Sketch the boundary around the active site and consider it to be the grid box.^35,36

Docking Procedure:

Step 1: Ligand Preparation:

Gather the structure of bioactive compounds that exist in plant extracts. Docking require different format of the structure file and software for example Open Babel is utilized for conversion in the needed format. Reduce the energy employing molecular modeling software to align it for docking purpose.

Step 2: Protein Preparation:

The structure of the protein is taken from the Protein Data Base (PDB). Desolvate the crystal structure and go on to exclude ligands, ions, and water molecules from the structure. Structure can be optimized by placing polar hydrogens and Kollman charges. The structure should be saved in a format necessary for the usage utilizing docking software. The structure is saved in required format by docking software.

Step 3: Docking Simulation:

Place the three dimensional grid box around the active or binding site using the graphical user interface in the docking software. Setting of the grid center and the size of the grid should be determined with reference to the binding site. The protein and ligand files should be prepared and uploaded on the docking software to perform the simulation.

Step 4: Analysis of Results:

Docking poses and the binding scores are assessed for the determination of suitable binding position. Hydrogen bonding, hydrophobic interactions or π-π stacking can create special interfaces and can be analysed. It is therefore, evident that the best binding conformation commonly has the lowest value of binding energy score concurring with the stable conformation of the ligand-protein complex.^37,38(Table 3)

RESULTS AND DISCUSSION:

The purpose of this research was to propose the significance of using the combination of grapes, garlic, gurmar, and curry leaves in treating endothelial dysfunction by characterization of extract by using HPTLC, IR, NMR, and mass spectroscopy specifically through molecular docking. Table 2 gives the quantitative analysis associated with bioactive compounds present in the herbal extracts.

The results of the mass spectrometry not only showed presence of bioactive compounds but also their quantity, molecular details and possible combined effect. These findings back up the use of the extracts in the therapeutic attention of endothelial dysfunction, relating to antioxidative and anti-inflammatory effects. Future studies could build on this work to extrapolate the pharmacokinetics of these compounds, including factors that predict the metabolism and bioavailability of given compounds in living systems. The presence of multiple bioactive compounds, including those indicated at m/z 807.45 and 808.45, may act coherently to enhance extract’s antioxidative and anti-inflammatory properties. For instance, the combination of flavonoids with polyphenols can attenuate ROS scavenging activity and greater endothelial protection.

HPTLC analysis:

Analyzing Peak Shapes and Their Areas:

Peak Shapes:

The height and shape of the peaks which can be obtained through HPTLC might be useful in determining not only the purity of compounds but also their identity. Symmetrical peaks are typical for well resolved and single compounds whereas broader or asymmetrical peaks may characterize two compounds or substances mixed together or impurities.

Sample 1:

Presents well-defined and sharp peaks, especially between Rf values of 0.618-0.647 which gives the indication of the glycosides are relatively pure or highly concentrated. From the available literature the phenolic acids and flavonoids have possess similarities in established HPTLC profile characteristics.

Sample 2:

Produces distinct and defined peaks that might indicate compounds with similar molecular formulae or similar Phenolic compounds present often in plant extracts.

Sample 3:

It consists of broad peaks embodying significant area and height. This pattern could be attributable to the presence of such compounds dominantly alkaloids and compounds like tannins, or saponin, which are most frequently noted in plants with intertwined polarities.

Sample 4: Comprises defined peak with fairly reasonable sharpness, suggesting the existence of intricate primary and secondary metabolites of varying polarity substances, which might be organosulfur compounds, phenolic acid or alkaloids.

Peak Areas:

The area under the peaks is proportional to the concentration of the compound in question.

Sample 1:

The largest area of peak corresponds to the second peak ranging RF 0.805-0.984, also the first peak is distinct at 0.618- 0.647 Rf range, may suggest the presence of a considerable compound of Triterpenoid glycosides, polypeptides and so on may possessing Hypolipidemic, Hypoglycemic and antioxidant activities.

Sample 2:

The Histogram depicts four large peaks with Rf range 0.515-0.658, 0.671-0.781, 0.781-0.865 and 0.865-0.982 respectively. Which indicated presence of Polyphenols, flavonoids and stilbenes Resveratrol, betulinic acids, possessing the potent antioxidant activity.

Sample 3:

Especially, there are six obvious peak with Rf ranging 0.065-0.137, 0.136-0.205, 0.274-0.329, 0.550-0.645, 0.645-0.815, 0.815-0.994 respectively. value that indicates a high content of a compound such as alkaloids such as Mahanimbine, Mahanine, flavonoids such as Quercetin, Catechin, Rutin etc. which has been reported to possess Hypolipidemic, anti-hypertensive, antioxidant and anti- inflammatory activities.

Sample 4:

In the present HPTLC elution profile, four large peaks with distinct Rf value range 0.160-0.269, 0.532-0.658, 0.663-0.802 and 0.803-0.985 respectively. Major compound probably organosulfur compounds, alkaloid or polyphenol which may be responsible for the potential therapeutic uses, specifically in anti-cancer or anti-inflammatory treatment regimes.

2. Identification of compound and Its therapeutic Relevance judged from Literature Data:

Research articles on HPTLC profiling of related plants mentioned in this study offer probable compound identifications in relation to similar Rf values and peak shapes at 254nm and 366nm. By comparing observed data with previous studies, we can speculate on specific bioactive compounds:

Phenolic Compounds and Flavonoids:

Common Peaks in 0.6–0.9 Rf Range:

Polyphenols and Flavonoids found in the vast majority of plant extracts that gives HPTLC peaks in this area when scanned at either 254 or 366nm includes Resveratrol, quercetin, kaempferol and catechin. These compounds are investigated due to their antioxidant and anti-inflammatory effects; furthermore, resveratrol and quercetin demonstrate the most promising results in cardiovascular uses.

Sample 2 and Sample 4:

The significant regions in these samples can be estimated for polyphenols and flavonoid content based on the literature parameters described earlier and referred bioactivities as antioxidant, anti-diabetic, and anti-inflammatory ones.

Tannins and Saponins:

Broad Peaks in Sample 3:

Through a consideration of the results to this sample, one may attribute them to either tannins or saponins, both recognized for their wound healing as well as the antimicrobial effects. As can be seen in literature, the tannins have similar elution and broad intense peaks of Rf in this range.

Alkaloids:

Peak third Rf range 0.27-0.329 in Sample 3: It co-ordinates with Rf values known for alkaloids such as mahanimbine, and other range identifies Mahanine, koenimbine, Murrayanol, berberine or other related bioactive nitrogenous compounds.

Phenolic Acids:

Sample 2 with High Rf Peaks in the 0.6–0.8 Range: Other compound classes, especially phenolic acids; ferulic and caffeic acids fall in this Rf category; and have been associated with therapeutic potential in managing oxidative stress and skin protection.

Glycosides:

Peak first of Sample 1 with Rf range 0.618-0.647 represents triterpenoids such as Gymnemic acid, Oleanane possessing Hypolipidemic, hypoglycemic, anti-inflammatory activity.

The investigation of four plant extracts through HPTLC differentiated the profile of every plant, which indicates the presence of different classes of bioactive constituents that can be therapeutically beneficial. Some observations include pointed and sharp peaks in Sample 1 which suggest triterpenoids compounds such as gymnemic acid or Oleanane having benefits as hypolipidemic, hypoglycemic, antioxidants and anti-inflammatory compounds. Sample 2 could have polyphenols and phenolic acids including Resveratrol, betulinic acids ferulic and caffeic acids that in part play a role in skin defence and oxidative stress and inflammation. If we concentrate on broad peaks, Sample 3 may contain tannins or saponins, related to wound healing and antimicrobial activity, and small peaks can be interpret as carbazole alkaloid exhibiting hypolipidemic, anti inflammatory, hypotensive activity and Sample 4 contains organosulfur such as Allicine, and alkaloids like quercetine which are helpful in treatment of inflammation, hypertension, hyperlipidemia and treatment of cancer and neuroprotection.

These compound identifications are backed up by literature comparisons using Rf values and also peak characteristics of each sample with therapeutic implications emphasized.

In Silico Studies:

The docking study established strong-binding affinities of bioactive compounds in the extracts to the protein receptors accountable for endothelial dysfunction. Active constituents like, gymnemic acid, resveratrol, mahanimbine and allicine were found to exercise considerable receptor-binding affinities that are relevant to hypoglycemic, anti-inflammatory, hypolipidemic, anti-hypertensive and antioxidant properties. More precisely In- Silico investigation confirms the hypoglycemic activity of gymnemic acid and Mahanimbine with binding energy of -6.8kcal/mol and -6.6kcal/mol with PDB Id 5XCB, anti- inflammatory activity is affirm for Gymnemic acid and allicine with binding energy -5.9kcal/mol and -6.9kcal/mol with PDB Id 1CX2, Anti-hypertensive pursuit in Gymnemic acid, mahanimbine and allicine with binding energy -7.3 kcal/mol, -6.6kcal/mol and -7.1kcal/mol with PDB Id 6JOD, antioxidant activity was countered in with resveratrol, mahanimbine and allicine with binding energy of -5.9 v, -6.8 v and -6.6kcal/mol respectively, lastly investigation for hypolipidemic activity was confirm with allicine with binding energy -6.7kcal/mol on PDB Id 1R31. The data on the impact of these compounds on the effective binding indicated their possible use in decreasing the levels of hyperglycemia, inflammation, hypertension, oxidative stress, hyperlipidemia and also help in increasing NO production for endothelia support.

Taken together, the data confirm the hypothesis that the bioactive compounds of the investigated herbal extracts possess multiple pharmacological effects, such as antioxidant, anti-inflammatory, hypoglycemic, hypolipidemic and pro-NO effects, which can be helpful for the treatment of endothelial dysfunction. study foster with the IR spectroscopy analysis ratify the existence of different functional group indicating different bioactive compounds.

CONCLUSION:

Therefore, the evidence derived from the present research indicates that the combination of the studied herbal extracts has potent endothelial protective effects because of their antioxidant and inflammation inhibitory activities that may be useful in the management of cardiopathy. To validate and explore the richness of these materials for therapeutic uses, additional in vivo studies are warranted. The extracts herein contained constituents such as gymnemic acid, resveratrol, mahanimbine and allicin that showed robust receptor binding affinities for receptors belonging to signaling pathways involved in oxidative stress, inflammation, diabetes, hypertension, hyperlipidemia, and nitric oxide production through docking analysis. This binding should be a major way that these compounds assist in maintaining endothelial cells health as they would modulate NO, diminish oxidative stress, and inflammation. Purity of isolated compounds was verified using different characterization techniques HPTLC, IR, NMR, mass spectrometry, and the outcomes illustrated that Gymnemic acid, Resveratrol, Mahanimbine, Allicine, Quercetin, Catechin, Tannins and Saponins, whose presence justified the therapeutic uses of each plant extract, do exert beneficial pharmacological effects. These compounds have been reviewed extensively for cardiovascular effects ranging from vasodilation to anti inflammation and inhibition of plaque formation.

REFERENCES:

1. Goyal S, Bansal VK, Goswami DS, Kumar S. Vascular Endothelial Dysfunction: Complication of Diabetes Mellitus and Hyperhomocysteinemia. Research Journal of Pharmacy and Technology. 2010; 3(3): 657-64.

2. Xu SW, Ilyas I, Weng JP. Endothelial dysfunction in COVID-19: an overview of evidence, biomarkers, mechanisms and potential therapies. Acta Pharmacologica Sinica. 2023; Apr; 44(4): 695-709. https://doi.org/10.1038/s41401-022-00998-0

3. Wang M, Li Y, Li S, Lv J. Endothelial dysfunction and diabetic cardiomyopathy. Frontiers in endocrinology. 2022 Apr 7, 13: 851941. https://doi.org/10.3389/fendo.2022.851941

4. Takeda Y, Matoba K, Sekiguchi K, Nagai Y, Yokota T, Utsunomiya K, Nishimura R. Endothelial dysfunction in diabetes. Biomedicines. 2020 June, 8: 182. https://doi.org/10.3390/biomedicines8070182.

5. Maneta E, Aivalioti E, Tual-Chalot S, EminiVeseli B, Gatsiou A, Stamatelopoulos K, Stellos K. Endothelial dysfunction and immunothrombosis in sepsis. Front Immunol. 2023, 14: 1144229. https://doi.org/10.3389/fimmu.2023.1144229

6. Nirmala S, Ravichandiran V, Vijayalakshmi A, Nadanasabapathi P. Protective effect of Gymnemic acid isolated from Gymnema sylvestre leaves coated Chitosan reduced gold nanoparticles in hyperlipedimia and Diabetes Induced vascular tissue damage in Rats. Research Journal of Pharmacy and Technology. 2018; 11(3): 1193-206. 10.5958/0974-360X.2018.00222.6

7. Subramanian S, Dowlath MJ, Karuppannan SK, Arunachalam KD. Effect of solvent on the phytochemical extraction and GC-MS analysis of Gymnema sylvestre. Pharmacognosy Journal. 2020; 12(4). 10.5530/pj.2020.12.108

8. US MR, Sundaram CS, Srinivasan S. Gymnemic acid mitigates hyperglycemia by attenuating the hepatic glucose metabolic enzymes in high fat diet fed-low dose streptozotocin-induced experimental rodents. Research Journal of Pharmacy and Technology. 2020; 13(2): 719-26. US MR, Sundaram CS, Srinivasan S. Gymnemic acid mitigates hyperglycemia by attenuating the hepatic glucose metabolic enzymes in high fat diet fed-low dose streptozotocin-induced experimental rodents. Research Journal of Pharmacy and Technology. 2020; 13(2): 719-26.

9. Sandech N, Jangchart R, Komolkriengkrai M, Boonyoung P, Khimmaktong W. Efficiency of Gymnema sylvestre-derived gymnemic acid on the restoration and improvement of brain vascular characteristics in diabetic rats. Experimental and Therapeutic Medicine. 2021 Oct; 22(6): 1420. https://doi.org/10.3892/etm.2021.10855

10. Khan KA, Dobani S, Shareef MA. Molecular docking and preclinical studies of Gymnema sylvestre on endothelial nitric oxide synthase (ENOS) in type-2 diabetes related complications. Journal of Young Pharmacists. 2014; 6(4): 25. 10.5530/jyp.2014.4.5

11. Chaves AA, Joshi MS, Coyle CM, Brady JE, Dech SJ, Schanbacher BL, Baliga R, Basuray A, Bauer JA. Vasoprotective endothelial effects of a standardized grape product in humans. Vascular pharmacology. 2009 Jan 1; 50(1-2): 20-6. https://doi.org/10.1016/j.vph.2008.08.004

12. Ashoori M, Soltani S, Kolahdouz-Mohammadi R, Moghtaderi F, Clayton Z, Abdollahi S. The effect of whole grape products on blood pressure and vascular function: A systematic review and meta-analysis of randomized controlled trials. Nutrition, Metabolism and Cardiovascular Diseases. 2023 Oct 1; 33(10): 1836-48. https://doi.org/10.1016/j.numecd.2023.05.001

13. Zhu FQ, Hu J, Lv FH, Cheng P, Gao S. Effects of oligomeric grape seed proanthocyanidins on L‐NAME‐induced hypertension in pregnant mice: Role of oxidative stress and endothelial dysfunction. Phytotherapy Research. 2018; Sep; 32(9): 1836-47. https://doi.org/10.1002/ptr.6119

14. Bijak M, Kolodziejczyk-Czepas J, Ponczek MB, Saluk J, Nowak P. Protective effects of grape seed extract against oxidative and nitrative damage of plasma proteins. International Journal of Biological Macromolecules. 2012; Oct 1; 51(3): 183-7. https://doi.org/10.1016/j.ijbiomac.2012.05.009

15. Firdaus SB, Ghosh D, Chattyopadhyay A, Dutta M, Paul S, Jana J, Basu A, Bose G, Lahiri H, Banerjee B, Pattari S. Protective effect of antioxidant rich aqueous curry leaf (Murraya koenigii) extract against gastro-toxic effects of piroxicam in male Wistar rats. Toxicology reports. 2014 Jan 1; 1: 987-1003. https://doi.org/10.1016/j.toxrep.2014.06.007

16. Balakrishnan R, Vijayraja D, Jo SH, Ganesan P, Su-Kim I, Choi DK. Medicinal profile, phytochemistry, and pharmacological activities of Murraya koenigii and its primary bioactive compounds. Antioxidants. 2020 Jan 24; 9(2): 101. https://doi.org/10.3390/antiox9020101

17. Phatak RS, Khanwelkar CC, Matule SM, Datkhile KD, Hendre AS. Antihyperglycemic activity of murraya koenigii leaves extract on blood sugar level in streptozotocin-nicotinamide induced diabetes in rats. Biomedical and Pharmacology Journal. 2019; Jun 25; 12(2): 597-602. https://dx.doi.org/10.13005/bpj/1679

18. Ramnath GM, Jayaraman S, Thirugnanasampandan R, Gunasekaran B, Gopalakrishnan A. Chemical composition analysis and evaluation of antioxidant, antiacetylcholinesterase and cytotoxicity of Murraya koenigii (l.) Spreng fruit oil. Journal of microbiology, Biotechnology and Food Sciences. 2023; Aug 2; 13(3): e6135. https://doi.org/10.55251/jmbfs.6135

19. Sharma A, Nagraik R, Venkidasamy B, Khan A, Dulta K, Kumar Chauhan P, Kumar D, Shin DS. In vitro antidiabetic, antioxidant, antimicrobial, and cytotoxic activity of Murraya koenigii leaf extract intercedes ZnO nanoparticles. Luminescence. 2023 Jul; 38(7): 1139-48. https://doi.org/10.1002/bio.4244

20. Jasim FA, Al-Hilu HS. Chemical Datasets, Antioxidant, Free Radicals Scavenger activities estimate in Aqueous Garlic (Allium sativum) extract. Research Journal of Pharmacy and Technology. 2021; 14(10): 5157-62. 10.52711/0974-360X.2021.00897

21. Subroto E, Cahyana Y, Tensiska M, Lembong F, Filianty E, Kurniati E, Wulandari D, Saputra R, Faturachman F. Bioactive compounds in garlic (Allium sativum L.) as a source of antioxidants and its potential to improve the immune system: a review. Food Res. 2021 Dec; 5(6): 1-1. https://doi.org/10.26656/fr.2017.5(6).042

22. Quintero-Fabián S, Ortuño-Sahagún D, Vázquez-Carrera M, López-Roa RI. Alliin, a garlic (Allium sativum) compound, prevents LPS‐induced inflammation in 3T3‐L1 adipocytes. Mediators of inflammation. 2013; 2013(1): 381815. https://doi.org/10.1155/2013/381815

23. Buendia AS, Rojas JG, García-Arroyo F, Trejo OE, Sánchez-Muñoz F, Argüello-García R, Sánchez-Lozada LG, Bojalil R, Osorio-Alonso H. Antioxidant and anti-inflammatory effects of allicin in the kidney of an experimental model of metabolic syndrome. PeerJ. 2023; Sep 27; 11: e16132. https://doi.org/10.7717/peerj.16132

24. Waseem M, Majeed Y, Nadeem T, Naqvi LH, Khalid MA, Sajjad MM, Sultan M, Khan MU, Khayrullin M, Shariati MA, Lorenzo JM. Conventional and advanced extraction methods of some bioactive compounds with health benefits of food and plant waste: A comprehensive review. Food Frontiers. 2023; Dec; 4(4): 1681-701. https://doi.org/10.1002/fft2.296

25. Nawaz H, Shi J, Mittal GS, Kakuda Y. Extraction of polyphenols from grape seeds and concentration by ultrafiltration. Separation and Purification Technology. 2006; Mar 1; 48(2): 176-81. https://doi.org/10.1016/j.seppur.2005.07.006

26. Del Valle JM, Mena C, Budinich M. Extraction of garlic with supercritical CO2 and conventional organic solvents. Brazilian Journal of Chemical Engineering. 2008; 25: 532-42. https://doi.org/10.1590/S0104-66322008000300011

27. Patil SS, Deshannavar UB, Ramasamy M, Emani S, Khalilpoor N, Issakhov A. Study of extraction kinetics of Total polyphenols from curry leaves. International Journal of Chemical Engineering. 2021; 2021(1): 9988684. https://doi.org/10.1155/2021/9988684

28. Sasidharan S, Chen Y, Saravanan D, Sundram KM, Latha LY. Extraction, isolation and characterization of bioactive compounds from plants’ extracts. African journal of traditional, Complementary and Alternative Medicines. 2011; 8(1). 10.4314/ajtcam.v8i1.60483

29. Fonmboh DJ, Abah ER, Fokunang TE, Herve B, Teke GN, Rose NM, Borgia NN, Fokunang LB, Andrew BN, Kaba N, Bathelemy N. An overview of methods of extraction, isolation and characterization of natural medicinal plant products in improved traditional medicine research. Asian J Res Med Pharm Sci. 2020 Aug; 9(2): 31-57. 10.9734/AJRIMPS/2020/v9i230152

30. Srinivasan K, Perumal K. Study on Extraction and Purification of Gymnemic Acid from Gymnema sylvestre R. Br. International Journal of Pharmacy and Biological Sciences. 2019; Jan, 9(1): 305-311. DOI: https://doi.org/10.21276/ijpbs.2019.9.1.40

31. Lotz A, Milz B, Spangenberg B. A new and sensitive TLC method to measure trans-resveratrol in red wine. Journal of Liquid Chromatography & Related Technologies. 2015; Jul 3; 38(11): 1104-8. https://doi.org/10.1080/10826076.2015.1028288

32. Mukhopadhyay N, Ahmed R, Mishra K, Sandbhor R, Sharma RJ, Kaki VR. A validated, precise TLC-densitometry method for simultaneous quantification of mahanimbine and koenimbine in marketed herbal formulations. Future Journal of Pharmaceutical Sciences. 2024; Feb 20; 10(1): 23.https://doi.org/10.1186/s43094-024-00591-8

33. Nair S, Mukne A. Assessment of chemical stability of constituents in thiosulfinate derivative-rich extract of garlic by a validated HPTLC method. Indian Journal of Pharmaceutical Sciences. 2017 May 1; 79(3).

34. Lutfiya AS, Priya S, Manzoor MA, Hemalatha S. Molecular docking and interactions between vascular endothelial growth factor (VEGF) receptors and phytochemicals: An in-silico study. Biocatalysis and Agricultural Biotechnology. 2019; Nov 1; 22: 101424. https://doi.org/10.1016/j.bcab.2019.101424

35. Abdelli I, Hassani F, Bekkel Brikci S, Ghalem S. In silico study the inhibition of angiotensin converting enzyme 2 receptor of COVID-19 by Ammoides verticillata components harvested from Western Algeria. Journal of Biomolecular Structure and Dynamics. 2021; Jun 13; 39(9): 3263-76. https://doi.org/10.1080/07391102.2020.1763199

36. Asiamah I, Obiri SA, Tamekloe W, Armah FA, Borquaye LS. Applications of molecular docking in natural products-based drug discovery. Scientific African. 2023; July. https://doi.org/10.1016/j.sciaf.2023.e01593

37. El-Behairy MF, Fayed MA, Ahmed RM, Abdallah IA. Computational drug reproposing to identify SARS-CoV-2 mpro inhibitors: Molecular docking, admet analysis, and in-silico/in-vitro toxicity study. Universal Journal of Pharmaceutical Research. 2022 Nov. 10.22270/ujpr.v7i5.837

38. Pinzi L, Rastelli G. Molecular docking: shifting paradigms in drug discovery. International Journal of Molecular Sciences. 2019; Sep 4; 20(18): 4331. https://doi.org/10.3390/ijms20184331

Received on 19.12.2024 Revised on 21.03.2025

Accepted on 02.06.2025 Published on 16.03.2026

Available online from March 18, 2026

Research J. Pharmacy and Technology. 2026;19(3):1317-1326.

DOI: 10.52711/0974-360X.2026.00189

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

S. No	Extract	Reported peak (cm ^-1)	Observed peak ( cm ^-1)	Inference
1	Extract 1	3400-3300 cm-1	3290.61	O-H stretch (Phenolic)
		1260-1000	1022.68	-C-H stretch (Aromatic)
		1500-1600	1600	C=C stretching (Phenol)
2	Extract 2	Around 1050 cm-1	1033.56	S=O stretch (Phenolic)
		Greater than 3000	3281.35	=C-H stretch (Alkene)
		1660-1600	1628.43	C=C stretching (Alkene)
3	Extract 3	3400-3300 cm-1	3289.95	O-H stretch (Alcohol)
		1730-1600	1602.20	=C=O stretch (Acid)
		1260-1000	1026.20	C-C stretching (Alcohol)
4	Extract 4	3400-3200 cm-1	3269.72	N-H stretch (Amines)
		900-690	1039	=C-H oop bending (Acid)
		1440-1220	1387.16.20	C-O-H bending (Alcohol)
		1660-1600	1599.70	C=C stretch(alkene)

Compound	Source Plant	Concentration (mg/g of extract)	Analytical Method	Major Activity
Resveratrol	Grapes (Vitis vinifera)	15.2	HPTLC, MS	Antioxidant, vasodilator
Allicin	Garlic (Allium sativum)	18.7	HPTLC, MS	NO synthesis, vasodilation, anti-inflammatory
Gymnemic Acid	Gurmar (Gymnema sylvestre)	12.5	HPTLC, NMR	NO enhancement, anti-inflammatory
Mahanimbine	Curry leaves (Murraya koenigii)	9.3	HPTLC, MS	Hypolipidemic, antioxidant
Catechin	Grapes (Vitis vinifera)	7.8	HPTLC, NMR	Antioxidant, ROS reduction
Quercetin	Grapes (Vitis vinifera)	5.4	HPTLC, MS	Anti-inflammatory, antioxidant
S-allyl cysteine	Garlic (Allium sativum)	4.9	HPTLC, MS	ROS reduction, NO availability

S. No	Compound	Activity	Receptor	Binding Energy	PDB Id
1	Gymnemic acid	Hypoglycemic	Alpha glucosidase	-6.8	5ZCB
		Anti-inflammatory	Cyclooxygenase-2	-5.9	1CX2
		Anti- hypertensive	AT-II receptor	-7.3	6JOD
2	Reveratrol	Antioxidant	Human heme oxygenase 1	-5.9	1N3U
3	Mahanimbine	Anti- hypertensive	AT-II receptor	-6.6	6JOD
		Antioxidant	Human heme oxygenase 1	-6.8	1N3U
		Hypoglycemic	Alpha glucosidase	-6.6	5ZCB
4	Allicine	Anti hypertensive	AT-II receptor	-7.1	6JOD
		Anti-inflammatory	Cyclooxygenase-2	-6.9	1CX2
		Hypolipidemic	HMG-CoA reductase	-6.7	1R31
		Antioxidant	Human heme oxygenase 1	-6.6	1N3U