Unlocking the potential of Trigonella foenum-graecum L. plant leaf extracts against diabetes-associated hypertension: A proof of concept by in silico studies

1 Introduction

A metabolic disorder is a non-communicable disease affecting the people of all age groups due to disruptions in normal glucose levels, insulin sensitivity, cardiovascular functions, cholesterol levels, and renal functions [1]. Diabetes mellitus is one of the metabolic disorders that pose a significant threat to human health globally and is characterized by an increase in blood glucose level due to a decrease in the insulin secretion or uptake of insulin by the cells related to the resistance mechanisms. The number of diabetic patients is rising more in low- and middle-income countries than in high-income countries. According to the International Diabetes Federation (IDF) Diabetes Atlas 11th Edition (2025), approximately 589 million adults between age 20 and 79 suffered from diabetes worldwide in 2024, representing about 1 in 9 adults, and the number is expected to rise to 853 million by 2050 [2]. Also, diabetes mellitus is associated with secondary complications such as diabetic nephropathy, diabetic retinopathy, and diabetic neuropathy. Diabetes-associated high BP and hyperlipidemia is another disease cluster difficult to treat with single-drug therapy. The physicians have opted for multidrug therapy that further leads to adverse side effects [3]. Hence, there is a need to search for alternative sources of drugs that possess fewer or no side effects. In this context, medicinal herbs have attracted attention for the treatment of diabetes and associated complications due to less severe side effects. These natural products (NPs) obtained from marine plants, microorganisms, and animal sources have a history of developing new therapeutic agents. These NPs have unique structural characteristics such as a high number of sp3 carbon atoms and chiral centers that provide them the unique ability to interact with biological receptors, thereby exhibiting specific biological functions [4].

Trigonella foenum-graecum L., commonly referred to as fenugreek or methi, is an angiosperm plant classified under the Fabaceae family. Over the course of history, it has been employed in the Indian subcontinent for various purposes, including as a condiment and a medicinal remedy. It is commonly utilized as a traditional herb in Western countries and is composed of several aromatic compounds that give food their unique color and characteristics [5]. Fresh and desiccated leaves are utilized as ingredients in Indian cuisine, although fresh leaves are traditionally added into salads [6,7]. Researchers have reported that the seeds and leaves of T. graecum possess several medicinal properties including anti-inflammatory, anti-spasmodic, anticancer, astringent, carminative, emollient, expectorant, febrifuge, anti-obesity, anti-ulcer, skeletal muscle relaxant, and uterine tonic effects, as well as the ability to lower triglycerides in both humans and animals [8,9,10,11,12]. Several studies have reported its antidiabetic effects, including T. graecum L. seed powder improved lipid metabolism in type II diabetic patients with significant reduction in total cholesterol, triglyceride, and low-density lipoprotein (LDL) levels and an increase in the high-density lipoprotein (HDL) level [13]. Several mechanisms of antidiabetic effects of fenugreek include increased glucose transporter type 4 (GLUT-4) translocation and hexokinase activity, decreased fructose 1,6-bisphosphatase and glucose-6-phosphatase activities, inhibition of α-amylase, protection of β-cells, and increased insulin release [14]. The methanolic extract of fenugreek seeds was also reported to have antihypertensive effects in nephrectomized deoxycorticosterone acetate-salt-induced and fructose-induced hypertensive rats. Mechanism of action studies involved the serotonergic antagonistic effects [15]. In another study, fenugreek along with amlodipine, a calcium ion channel blocker, showed better control of blood pressure (BP) and improved response of amlodipine in hypertensive rat models [16]. An extensive array of phytochemicals, including coumarins (methyl coumarin, trimethyl coumarin, and trigocoumarin), alkaloids (fenugreekine, trigonelline, and choline), flavonoids (naringenin, kaempferol, isovitexin, quercetin, and luteolin), and phenolic compounds (catechin, vanillic acid, syringic acid, gentisic acid, and chlorogenic acid) are present in this plant [17,18]. Galactomannan is another main constituent of fenugreek reported for its antidiabetic effects [19]. All these compounds exhibit various pharmacological properties via multiple mechanisms. In recent years, in silico approaches have emerged as powerful tools in the early stages of drug discovery. Molecular docking, a computational technique that predicts the preferred orientation of a molecule when bound to a target receptor, is widely used to assess the binding affinity and interaction profiles of phytochemicals with key biological targets [20].

Additionally, ADMET (absorption, distribution, metabolism, excretion, and toxicity) prediction models help evaluate the pharmacokinetic and safety profiles of these molecules, reducing the risk of late-stage failures in drug development. Incorporating such computational tools alongside experimental evaluations provides a comprehensive understanding of a compound’s pharmacological potential and supports the rational design of novel therapeutic agents. Therefore, the integration of in silico techniques in this study enhances the reliability and efficiency of identifying effective anti-diabetic and anti-hypertensive compounds from T. graecum L.

In the present investigation, the study was undertaken to evaluate the pharmacological effects of hydroethanolic and chloroform extracts of T. graecum L. leaves in a diabetes-associated hypertension rat model. Further, a molecular docking study of identified compounds was also carried out on target receptors.

2 Materials and methods

3 Results

3.2 Determination of total phenolic content

The total phenolic content was calculated using the linear equation y = mx + c, R ² = 0.999, obtained from the calibration curve (Figure 1) of gallic acid, where y is the absorbance and x is the amount of gallic acid. It was found to be 253 µg/ml as GAEs.

Figure 1

Estimation of total phenol content in the hydroethanolic extract of T. graceum L.

3.3 Reducing power assay

Reducing power is an indicator of the electron-donating ability of the compound used for testing the antioxidant or radical scavenging ability of the extracts. It is used to evaluate the ability of extracts to reduce Fe³⁺ to Fe²⁺. Figure 2a shows the extracts’ reducing power increasing with concentration. The reducing power of hydroethanolic extract (Figure 2b) was found to be 1.32 μg/ml compared to the standard compound at 1.686 μg/ml. The concentration-dependent percentage inhibition of hydroethanolic extracts ranged from 73.4 to 94.3% with an IC₅₀ value of 53.63 μg/ml, while for chloroform extracts (Figure 2c), the percentage inhibition was from 82.4 to 94.3% with an IC₅₀ value of 64.03 μg/ml.

Figure 2

(a) Reducing power assay and (b) DPPH assay of hydroethanolic and (c) chloroform extracts of T. graceum L.

3.4 HPLC quantitative analysis of extract

The presence of quercetin was detected in both the extracts with retention times of 5.6 and 5.8 s for chloroform and hydroethanolic extracts, respectively (Figure 3a and b), whereas it was 5.59 s for the standard (Figure 3d). In addition, the overlay spectra of quercetin and the extracts at the same retention time are illustrated in Figure 3c. Quercetin concentration was found to be higher in the chloroform extract than in the hydroethanolic extract.

Figure 3

HPLC chromatogram of the (a) chloroform extract and (b) hydroalcoholic extract. (c) Standard Quercetin at 360 nm. (d) Overlay spectra of T. graecum L.

3.6 BP parameter

Administration of dexamethasone was noted to significantly increase the BP levels at the 7th day in all groups except for the normal control group (Figure 4). On day 15, two higher doses of hydroethanolic and chloroform extracts showed substantially significant (p < 0.001) reduction in systolic BP levels by 13.8 and 26.4 mm Hg and 14.8 and 26.5 mm Hg, respectively, as compared to the positive control group, whereas diastolic BP levels did not find a significant reduction in BP levels. On the 30th day, the reduction of systolic BP level was maintained for both different extracts (200 and 400 mg/kg), and in the diastolic BP level, only 400 mg/kg showed a less significant reduction as compared to the positive control group. Ramipril as a standard drug showed the highest significant reduction (p < 0.0001) in both BP levels as compared to the positive control group.

Figure 4

Effect of hydroethanolic and chloroform extracts of T. graecum L. on BP levels (mm Hg) after 15 and 30 day intervals.

3.9 Histopathological findings

The lengthy microvilli remained intact, and the proximal tubular cells exhibited typical cellular behavior in the normal control group (Figure 5). We observed that podocyte pedicles and interpedicular diaphragms possessed characteristic morphologies within the glomerulus. Rats that received diabetes treatment showed much worse damage to their kidney structures, including more cell buildup in the spaces between cells, scarring in the glomeruli, and swelling in the surrounding areas. We observed a noticeable improvement after administering two distinct concentrations of both chloroform and hydroethanolic extracts (200 and 400 mg/kg).

Figure 5

Histopathology of rat kidney tissues after treatment with different extracts of T. graecum L. In the normal group, the arrow represents the normal structure of glomeruli. In the positive control group, the arrow shows hemorrhage and tubular damage due to proteinuria; in the ramipril-treated group, the arrow shows mild inflammation with the normal structure of glomeruli; in other extract-treated groups, the arrow shows recovered glomeruli with some distorted one. RBCs are seen with hemorrhage in the hydroethanolic extract-treated group (100 mg/kg).

3.10 Analysis of quercetin in serum on the 45th day using HPLC-DAD

Quercetin was absent in the serum of the normal control group, as depicted in Figure 6. The retention time of the standard quercetin was found to be 10.49, indicating the presence of quercetin at a 93.22% level. The retention time of chloroform extract-treated group was on par with the hydroethanolic extract-treated group (400 mg/kg) on the 45th day: 10.74 (70.23% concentration) and 10.59 (95.27% concentration), respectively.

Figure 6

Estimation of quercetin in serum in different extract-treated groups. (a) Normal control. (b) Standard quercetin. (c) Chloroform extract showing quercetin. (d) Hydroethanolic extract showing quercetin.

3.11 In silico results

3.11.1 ADME/T Studies

The supplementary tables (Tables S1 and S2) provide a comprehensive overview of the predicted ADME/T properties, as well as their corresponding values and probabilities for the two compounds: ramipril and quercetin. ADME/T studies showed comparable absorptive properties for ramipril and quercetin. However, ramipril exhibited higher hepatotoxicity and AMES mutagenesis probabilities. Bioavailability radar suggested a better safety profile for quercetin (Figure S1). Boiled egg plot results indicated that neither drug could cross the BBB but had high GI absorption (Figure S2).

3.11.2 Docking results

We performed docking between proteins of the RAS pathway and both drugs (quercetin and ramipril) to evaluate the affinity of quercetin toward the RAS pathway. The affinity scores are presented in Table S3.

3.11.2.1 Interaction of quercetin and ramipril with ACE

The binding affinities of quercetin and ramipril toward ACE were found to be −7.42 and −6.25 kcal/mol, respectively, indicating that quercetin has a stronger affinity for ACE than ramipril. Figure 7 displays the 3D and 2D molecular interactions, respectively, of quercetin and ramipril with ACE (PDB ID: 1O86). Quercetin was observed to form a complex with ACE through conventional hydrogen bonding, pi–sulfur, pi–cation, and van der Waals interactions. The key amino acid residues involved in these interactions include Tyr146, Leu161, Glu349, and Arg348 (hydrogen bonding); Ser147 (pi–lone pair); Cys352 and Cys370 (pi–sulfur); Lys343 (pi–cation); and Phe512, His353, Glu143, Val351, Val350, Pro344, Gln160, Glu162, Thr150, Aal149, and Trp185 (van der Waals). In contrast, ramipril forms conventional hydrogen bonds, salt bridges, alkyl bonds, and van der Waals interactions with ACE. The parts of ACE that interact with ramipril include Tyr135 and Arg124 (which form hydrogen bonds); with Leu139, Tyr62, Ala89, Ala125, and Ile88 (which form alkyl bonds); with Glu123 (which forms salt bridges); and Leu122, Thr92, Asn85, Trp220, Ile204, Tyr200, and Ser517 (which have van der Waals interactions). These findings suggest that while both ligands bind to ACE through a variety of interactions, quercetin involves additional pi-interactions, while ramipril features alkyl and salt bridge interactions, which may contribute to its effectiveness as an ACE inhibitor.

Figure 7

Illustration of the protein–ligand interaction of quercetin and ramipril with targets involved in the RAS pathway. (a) 3D structures of the ACE_quercetin and ACE_ramipril complexes; (b) 2D structures of ACE_quercetin and ACE_ramipril complexes; (c) 3D structures of renin_quercetin and renin_ramipril complexes; (d) 2D structures of renin_quercetin and renin_ramipril complexes; (e) 3D structures of AT1R_quercetin and AT1R_ramipril complexes; (f) 2D structures of AT1R_quercetin and AT1R_ramipril complexes; (g) 3D structures of AT2R_quercetin and AT2R_ramipril complexes; (h) 2D Structures of AT2R_quercetin and AT2R_ramipril complexes; (i) 3D structures of ACE2_quercetin and ACE2_ramipril complexes; (j) 2D structures of ACE2_quercetin and ACE2_ramipril complexes.

3.11.2.2 Interaction of quercetin and ramipril with renin

Quercetin binds to renin more strongly than ramipril, with binding affinities of −8.1 and −5.74 kcal/mol, respectively. Figure 7 shows the 3D and 2D ways that quercetin and ramipril interact with renin (PDB ID: 2V0Z). We found that renin and quercetin form a complex through conventional hydrogen bonding, pi–pi stacking, pi–alkyl, unfavorable donor–donor, and van der Waals interactions. The amino acid residues of renin involved in these interactions with quercetin were Gly209, Ser227, Asp208, and Leu204 (conventional hydrogen bonds); Trp299 (pi–pi stacking); Cys210, Cys206, and Leu205 (pi–alkyl); Ser224 (unfavorable donor–donor); and Lys230, Pro297, Thr298, and Gly223 (van der Waals). On the other hand, ramipril was observed to form conventional hydrogen bonds, pi–sigma, pi–pi T-shaped, carbon–hydrogen bonds, alkyl, and van der Waals interactions with renin. The amino acid residues of renin involved in interactions with ramipril were Thr77, Gln12 (conventional hydrogen bonding); Val120 (pi–sigma); Tyr75 (pi–pi T-shaped); Gly217 (carbon–hydrogen bonds); Leu114 and Aal115, and Pro111 (alkyl); and Gly34, Asp32, Val30, Asp215, Ser76, Phe117, Met289, Ser219, Phe112, and Trp39 (van der Waals).

3.11.2.3 Interaction of quercetin and ramipril with AT1R

The binding affinities of quercetin and ramipril toward AT1R were −6.2 and −6.43 kcal/mol, respectively, indicating that both compounds have comparable affinity toward AT1R, with quercetin showing a slightly better affinity than ramipril. Figure 7 shows the 3D and 2D images of how quercetin and ramipril interact with the AT1R (PDB ID: 4ZUD). Quercetin was found to form a complex with AT1R through conventional hydrogen bonds, pi–sigma, pi–alkyl, pi–pi T-shaped, and van der Waals interactions. The amino acid residues of AT1R involved in these interactions with quercetin were Glu1004, Tyr99, and Leu204 (conventional hydrogen bond); Leu1003 (pi–sigma); Ala1043, Pro1045, Asn168, Thr1044, Lys102, Asn98, Gly97, Asp1002, and Asp1005 (van der Waals); Tyr99 (pi–pi T-shaped); and Pro1046 and Leu1003 (pi–alkyl). In contrast, ramipril formed interactions via conventional hydrogen bonds, pi–sigma, pi–pi stacked, van der Waals, alkyl, and pi–alkyl interactions. The amino acid residues of renin interacting with ramipril were Phe261, Leu197, Gly196, Asn200, Gly203, Tyr113, Trp253, Ser109, Val108, and Tyr292 (van der Waals); Thr260 and Gln257 (conventional hydrogen bonds); Leu112 (pi–sigma); Phe204 (pi–pi stacked); Lys199 and Val264 (alkyl and pi–alkyl).

3.11.2.4 Interaction of quercetin and ramipril with AT2R

The binding affinities of quercetin and ramipril toward AT2R were −6.87 and −7.32 kcal/mol, respectively, indicating that quercetin has a strong affinity for AT2R, though slightly weaker than that of ramipril. Figure 7 shows the 3D and 2D ways that quercetin and ramipril interact with the AT2R (PDB ID: 7JNI). Quercetin was found to form a complex with AT2R through conventional hydrogen bonds, pi–sigma, pi–donor hydrogen bonds, and van der Waals interactions. The amino acids in AT2R that interacted with quercetin include Arg142, Gln326, Gly322, Lys77, Asn323, and Val65 (for conventional hydrogen bonds); Ile83 (for pi–sigma); Met138, Ser79, Tyr318, Asp141, Phe69, Phe320, and Leu68 (for van der Waals interactions); and Ser80 (for pi–donor hydrogen bonds). In contrast, ramipril formed interactions via conventional hydrogen bonds, pi–sulfur, pi–pi T-shaped, alkyl, pi–alkyl, pi–sigma, and unfavorable positive–positive interactions. The amino acid residues of renin interacting with ramipril were Thr178, Thr125, Gly121, Phe308, Tyr103, Trp100, Ilr47, and Trp269 (van der Waals); Met128 (pi–sulfur); Phe272 (pi–pi T-shaped); Leu124, Tyr108, Tyr104, and Pro301 (alkyl, pi–alkyl), and Ile304 (pi–sigma); Arg182 (unfavorable positive); and Tyr51 (conventional hydrogen bonds).

3.11.2.5 Interaction of quercetin and ramipril with ACE-2

The binding affinities of quercetin and ramipril for ACE2 were found to be −5.77 and −6.1 kcal/mol, respectively. This indicates that quercetin exhibits strong affinity for AT2R2, although slightly weaker than that of ramipril. Figure 6i and j shows how quercetin and ramipril interact with the ACE2 (PDB ID: 8B9P) in both 3D and 2D views. Quercetin was found to form a complex with ACE2 through conventional hydrogen bonds, pi–alkyl interactions, carbon–hydrogen bonds, and van der Waals forces. The specific ACE2 amino acid residues involved in interactions with quercetin were Asp543, His535, Glu536, and Glu549 (conventional hydrogen bonds); Thr548, Asn546, Ser545, Ile544, Lys416, Cys542, Gly537, and Lys534 (van der Waals interactions); Ala533 (pi–alkyl interaction); and Ser547 (carbon–hydrogen bond). In contrast, ramipril formed interactions with ACE2 via conventional hydrogen bonds, van der Waals forces, pi–alkyl interactions, pi–cation interactions, unfavorable positive–positive interactions, attractive charge interactions, and salt bridges. The ACE2 residues involved in ramipril interactions included Thr 294, Thr 276, TyrR 279, Asn 277, Asn 290, and Phe 438 (van der Waals); Met 366, Lys 441, and Asp 367 (conventional hydrogen bonds); Ile 291 (pi–alkyl); Asp 292, Asp 367, and Lys 441 (pi–cation, attractive charge, and salt bridge interactions); and Ile 291 (pi–alkyl).

Molecular docking studies revealed distinct binding profiles for quercetin and ramipril across various targets in the RAS. Quercetin showed stronger ability to attach to ACE and renin than ramipril, indicating it might work better to block these enzymes. However, ramipril, as a standard drug, showed slightly higher affinity for ACE2 and AT2R, where both compounds exhibited comparable binding. While quercetin created a greater range of connections, including π-based interactions, ramipril had more alkyl, salt bridge, and negative interactions between positive charges. These findings suggest that quercetin has potential as an alternative inhibitor, while ramipril, due to its targeted affinity for ACE2 and AT2R, remains a strong therapeutic agent.

4 Discussion

Metabolic disorders are metabolic syndromes associated with insulin resistance and cardiovascular risk factors, including myocardial infarction, heart failure, and hypertension [33]. Excess glucose in the bloodstream leads to hyperglycemia. Glucocorticoids can cause insulin resistance by blocking the liver’s ability to use glucose, which may lead to problems in making glycogen and higher amounts of free fatty acids. While the precise mechanisms at play remain elusive, insulin resistance potentially plays a role in the pathogenesis of hypertension, impaired glucose tolerance, and lipids [34]. ACE inhibitors stop the ACE in the RAS and kallikrein system, and they are known to be very effective for treating high BP [35]. Patients afflicted with diabetic nephropathy now benefit more from ACE inhibitors as a result of their well-documented safety. Nonetheless, certain extremely hazardous side effects are present in all allopathic medications and can be mitigated through the use of herbs [36].

T. graecum L., a traditional Indian herb, belongs to the Fabaceae family. This study explores T. graecum L. leaves for managing the symptoms of diabetes-induced hypertension [37]. In our investigation, both extracts confirmed the presence of flavonoids and alkaloids; however, glycosides, carbohydrates, proteins, and amino acids were only found in the chloroform extract. The chloroform extract also demonstrated antioxidant capability with an inhibitory concentration (IC₅₀) value of 1.434 μg/ml, which is similar to ascorbic acid’s value of 1.686 μg/ml. Quercetin is an important flavonoid molecule that is present in T. graecum L. HPLC analysis detected the small concentration of quercetin in both the extracts, which is compared with the standard quercetin. The retention of quercetin was similar to the reported studies [38]. Antioxidant or free radical scavenging properties of T. graecum L. can be ascribed to the plant’s flavonoid (quercetin) and saponin contents. The protective effects of flavonoids in living systems are thought to come from their ability to move electrons, bind to metal catalysts, boost antioxidant enzymes, lower alpha-tocopherol radicals, and blocks the oxidation tadical system [39]. Our study revealed that both extracts led to a statistically significant (p < 0.0001) decrease in blood glucose levels persistently for 45 days. Furthermore, extracts at 400 mg/kg exhibited a reduction in LDL and triglyceride levels. Chloroform and hydroethanolic extracts at 400 mg/kg had HDL levels that were about the same as those in the group treated with ramipril. The insulin level returned to near normal after 45 days of treatment.

At doses of 200 and 400 mg/kg chloroform and hydroethanolic extracts, the systolic BP levels were measured on the 15th and 30th days, while the diastolic BP levels did not show any significant reduction at either interval. Another study reported that the methanolic extract showed similar reductions in BP levels. Histological exams showed that the shape and structure of kidneys in diabetic patients improved significantly in both groups that received the extract after treatment. Pharmacokinetic evaluation revealed traces of quercetin in the serum samples of the treated groups. This plant has a lot of important compounds, including quercetin, fenugreekine, nicotinic acid, sapogenins, phytic acid, scopoletin, trigonelline, diosgenin, gitogenin, neogitogenin, homoorientin, and saponaretin [40]. The likely process might turn on a specific receptor that works with tyrosine, which is found on the inside of the cell membrane. With the aid of ATP, it phosphorylates itself, modifies its conformation, and becomes activated via G-proteins. These processes result in the secretion of multiple second messengers, which subsequently stimulate protein kinases, allowing for Ca²⁺ influx and generating an effect comparable to insulin. It might also lower the production of phosphoenolpyruvate carboxykinase by depending on phosphoinositide 3-kinase (PI3K) and working like insulin by increasing PI3K and mitogen-activated protein kinase (MAP kinase). Trigonelline may enhance glucose uptake and insulin sensitivity via peroxisome proliferator activated receptor gamma (PPAR-γ) and GLUT-4 proteins. This upregulation of protein expressions has been linked to protective activity in diabetic nephropathy [13,41].

We investigated molecular receptor targets using an in silico molecular docking approach. PPARγ ligands have been studied for their potential effects on BP, and some research suggested that they may have a beneficial impact, particularly in models of hypertension [42]. PPARγ is a nuclear receptor that plays a key role in the regulation of various physiological processes, including glucose and lipid metabolism, inflammation, and vascular functions. Activation of PPARγ by its ligands can have systemic effects on these processes. Some proposed mechanisms by which PPARγ ligands influence BP include: (1) improvement of insulin sensitivity: PPARγ activation improves insulin sensitivity, and insulin resistance is often linked to hypertension and (2) anti-inflammatory activity [43].

Targeting PPARγ activation along with inhibiting the renin angiotensin-aldosterone system (RAAS) pathway may manage diabetes-associated hypertension [44]. Computational methods revealed that quercetin exhibited noteworthy high absorption, low BBB permeability, and a favorable toxicity profile. Docking studies unraveled high affinity of quercetin toward RAAS proteins, i.e., ACE, renin, AT1R, AT2R, and ACE-2. The RAAS pathway commences with the conversion of angiotensinogen to angiotensin I by renin, a rate-limiting step. Renin is a central hormone for modulation of BP and is the rate-limiting step in the RAAS. It cleaves angiotensinogen and releases angiotensin I, thus representing a viable target for managing hypertension [45]. This is evident by several effective clinically used renin inhibitors like enalkiren, remikiren, and aliskiren (Drug Bank n.d.). In the current study, ramipril was observed to bind with the S2 pocket and S3sp subpocket of renin [46]. Quercetin was observed to have high affinity toward renin and formed interactions with different amino acids. The interaction with Thr298 shows that quercetin attaches in the S1 pocket of renin, indicating that quercetin could influence how renin works [47]. Notably, ACE inhibitors like ramipril, lisinopril, captopril, etc., are effective in the clinical management of diabetic hypertension [48]. The S1 and S2 active sites of ACE are crucial for interaction with its inhibitors. These sites contain Glu162 and His353 amino acid residues. In fact, lisinopril, a well-established clinically used ACE inhibitor, binds with the S1 (Ala354 and Glu384) and S2 (His353) active sites of ACE. The analysis from the current study revealed that quercetin developed van der Waals interactions with Glu162 and His353 amino acid residues, suggesting that quercetin binds at the active site of ACE and engages the crucial amino acids required for inhibition of ACE activity. Moreover, the binding of quercetin specifically with His353, an amino acid that is involved in lisinopril-mediated ACE inhibition, confirms that quercetin has inhibitory activity on ACE [35]. Similarly, quercetin was identified to interact with ACE2; however, interactions of quercetin with those amino acids of ACE2 were not shown to interact with other ACE2 modulators in the literature. From this study we concluded that quercetin has more affinity and selectivity toward ACE than ACE2. Angiotensin II is a bioactive peptide that regulates the RAAS pathway. It binds to the AT-1 and AT-II receptors and creates vasoconstriction. AT-II connects with the Lys102 and Trp94–Gly97 parts of the AT-1 receptor, leading to effects on the heart and blood vessels, such as narrowing of the blood vessels. Lys102 and Trp94–Gly97 are present in the TMD3, ECL1, and WPFG motif regions of the AT-1R, respectively. Lys102 is crucial for the binding of AT-II with its AT1R, while the Trp94–Gly97 amino acids are crucial for AT1R receptor activation. In this study, quercetin interacts with Lys102 and Gly97, indicating that it can attach to important amino acids of AT-1R needed for activating the receptor. It also suggests that it can interfere with the binding of AT-II with AT1R, causing interruption in the RAAS pathway [49]. Quercetin was also observed to bind and form interactions with the amino acid residues of AT2R, suggesting it might disrupt AT-II binding and AT2-receptor activation. Moreover, the observations made in the current study state that quercetin interacts with AT2R amino acids; however, its interaction with the crucial amino acids of AT1R indicates its selective affinity toward AT1R. We observed that quercetin has the potential to modulate hypertension by interacting with the RAAS cascade through antagonism of renin, ACE, and AT1R, offering promising avenues for future therapeutic interventions. Additional research is required to gain a more profound understanding of the mechanisms underlying the hypoglycemic effects of this plant.

5 Conclusions

The study provides the usefulness of T. graecum L. in the treatment of diabetes-associated hypertension via pre-clinical studies. The chemical constituents present in the plant might be responsible for therapeutic effects of T. graecum L. in the treatment of diabetes-associated complications via multiple molecular mechanistic pathways by acting on distinct receptors, and an in silico study supported these results. A molecular mechanistic study of the plant extract may provide deeper understanding of the mechanisms related to diabetes-associated disorders. Further investigation is needed using clinical studies to validate its usefulness in treatment of this disorder.