Trends Sci. 2025; 22(7): 9930

DM is a global health challenge that has been increasing significantly in recent decades. By the International Diabetes Federation (IDF), over 537 million adults worldwide were living with DM in 2021, a figure projected to rise to 643 million by 2030, and sedentary lifestyles account for approximately 90 - 95 % of all DM cases [1-3]. Uncontrolled DM can lead to chronic hyperglycemia, resulting in severe complications, which may arise in the form of diabetic retinopathy, nephropathy, neuropathy, and heart disease [4]. Moreover, hyperglycemia induces damage to the β-cells in the pancreatic islets of Langerhans, impairing the production of insulin and exacerbating the metabolic state of patients. These impacts not only diminish individual quality of life but also impose a substantial economic burden on global healthcare systems [5].

Conventional diabetes therapies, such as metformin, insulin, and sulfonylureas, have been the primary approaches for managing blood glucose levels [6]. However, these treatments have significant limitations, including risks of side effects such as hypoglycemia, organ toxicity, and a decline in efficacy over time due to drug resistance [7]. Additionally, the high cost of long-term treatment poses a significant challenge, particularly in developing countries. Consequently, exploring alternative therapeutic approaches that are safer, more effective, and affordable, especially those derived from natural sources such as traditional medicinal plants, has become imperative.

One promising traditional medicinal plant is Biwa (E. japonica), widely used in conventional medicine to treat various ailments [8]. This plant is abundant in bioactive substances, including flavonoids, triterpenoids, and polysaccharides, which have been reported to exhibit antidiabetic activity through several mechanisms [9,10]. These include antioxidant activity that protects cells from oxidative damage, glucose regulation through the modulation of glucose-metabolizing enzymes, and protective effects on β-cells in the pancreas, which are often damaged by oxidative stress and inflammation [11]. Although E. japonica holds great potential as an antidiabetic agent, its bioactive compounds often suffer from low bioavailability due to certain chemical properties, such as poor solubility. Nanoherbal technology offers a solution to this limitation by enhancing the stability, bioavailability, and efficacy of active compounds. Nanoformulations enable bioactive compounds to more effectively reach molecular targets, resulting in optimized therapeutic effects [12,13].

In silico research, an essential initial step to evaluate the potential of bioactive compounds through approaches such as molecular docking and pharmacokinetic analysis. This analysis helps identify the interaction of compounds with molecular targets involved in blood glucose regulation, such as specific enzymes or receptors [14]. This study employs molecular docking to investigate potential interactions of inhibitors with protein tyrosine phosphatase 1B (PTP1B) and dipeptidyl peptidase-4 (DPP4), both of which play crucial roles in the pathogenesis of diabetes and obesity [15]. PTP1B is vital in regulating insulin signaling, directly influencing glucose metabolism, while DPP4 impacts incretin peptides essential for maintaining β-cell function in the pancreas [16,17].

Subsequently, in vivo approaches using animal models provide empirical data to confirm antidiabetic activity and protective effects on organs, including pancreatic histology [18]. Integrating of in silico and in vivo data provides a robust scientific foundation for assessing the potential of E. japonica as a diabetes therapy agent.

This study aims to explore the potential of nanoherbal E. japonica as an antidiabetic therapy through an integrative approach involving in silico and in vivo analyses. The primary focus is on blood glucose regulation and histological protection of the pancreas. The findings of this research are expected to bridge knowledge gaps related to the development of nanoherbal-based therapies while providing innovative solutions for managing DM.

Materials and methods

Research material

Biwa (E. japonica) plants were collected from Tangkahan in the Gundaling area, Berastagi District, Karo Regency, North Sumatra Province. The fresh Biwa leaves were dried, ground into a coarse powder, and then processed into a nanoherbal form. The milling process was conducted using a 2 M HCl activator with a Planetary Ball Milling (PBM) machine manufactured by PT Nanotech Indonesia Global Tbk. A mass ratio of 20:1 (balls in the machine: milled powder) was applied. The milling was performed for 2 consecutive 9-hour periods, resulting in a total processing time of 18 h. The milling process was carried out at 350 rpm for a total of 20 h, resulting in the production of nanopowders, which were subsequently analyzed using a Particle Size Analyzer (PSA).

LCMS/MS analysis of nanoherbal Biwa leaves (E. japonica)

Once the Biwa (E. japonica) nanoherbal had been formed, a portion of the sample was extracted using 96 % ethanol for LC-MS/MS analysis. The extraction process was carried out by sonicating the sample for 30 min, followed by centrifugation at 10,000 rpm for 10 min. The supernatant was collected and was filtered through a 0.22 µm membrane before analysis. The analysis was conducted using an AB Sciex 3200 Q-Trap, which was coupled with a Perkin Elmer FX 15 UHPLC system. A pre-packed C18 column (5 μm, 4×250 mm, Phenomenex) was used for separation. The mobile phase was prepared using water (solvent A) and ethanol containing 1 % acetonitrile, 0.1 % formic acid, and 5 mM ammonium formate. Data from the run are processed using MassLynx v4.1 software [19].

In silico analysis compounds nanoherbal Biwa

The bioactive compounds identified from the LC-MS profiling of Biwa leaf (E. japonica) nanoherbal extract include Quinic Acid (PubChem CID: 6508), Caffeic Acid (PubChem CID: 689043), Chrysin (PubChem CID: 5281607), Coumaric Acid (PubChem CID: 637542), Luteolin (PubChem CID: 5280863), Kaempferol (PubChem CID: 5280863), Betulinic Acid (PubChem CID: 64971), Oleanolic Acid (PubChem CID: 10494), Quercetin (PubChem CID: 5280343), Rutin (PubChem CID: 5280805), Epicatechin (PubChem CID: 72276), and Eriodictyl (PubChem CID: 440735). These compounds were subjected to molecular docking analysis against Protein Tyrosine Phosphatase 1B (PTP1B, PDB ID: 5T19) and Dipeptidyl Peptidase-4 (DPP4, PDB ID: 4A5S) to evaluate their potential interactions and therapeutic efficacy in diabetes management.

Drug-likeness evaluation, and biological activity prediction

The drug-likeness of the bioactive compounds identified from the nanoherbal of Biwa leaves (E. japonica) has been assessed using Lipinski’s Rule of Five (Ro5), which is applied to evaluate oral bioavailability based on specific criteria. According to Ro5, good oral bioavailability is likely when compounds meet the following conditions: Log P ≤ 5, no more than 5 hydrogen bond donors, no more than 10 hydrogen bond acceptors, and a molecular weight of ≤500 g/mol [20,21]. Compounds exceeding these thresholds may exhibit limited bioavailability. Additionally, biological activity predictions were carried out using the PASS Online tool. SMILES structures were extracted from PubChem (http://pubchem.ncbi.nlm.nih.gov) and uploaded to the Way2drug server (http://way2drug.com/PassOnline/), where predictions were based on the activation potential (Pa) values. Compounds with a Pa value above 0.7 are considered to have high biological activity, those with Pa values below 0.7 but above 0.5 are considered to have moderate activity, and compounds with Pa values below 0.5 are considered to have low activity potential [22].

ADMET properties and molecular docking analysis

ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) properties of the Biwa nanoherbal compounds (E. japonica Thunb. Lindl.) were evaluated using the pkCSM webserver (http://biosig.lab.uq.edu.au/pkcsm/prediction).

Toxicological risks were analyzed using the ProTox-III server (https://tox.charite.de/protox3/), while the PerMM tool (https://permm.phar.umich.edu/) predicted passive diffusion across lipid bilayers. These analyses provided insights into the pharmacokinetic potential and safety profile of the compounds. Molecular docking simulations were conducted to study the bioactive compounds’ interactions with specific target proteins involved in diabetes management. These include PTP1B (PDB ID:5T19), and DPP4 (4A5S) [15,23]. Ligands were prepared by downloading structural data from PubChem (http://pubchem.ncbi.nlm.nih.gov), followed by energy minimization using Open Babel within PyRx 0.0.8. Protein structures were retrieved from the Protein Data Bank (PDB; https://www.rcsb.org/), and water molecules were removed using PYMOL software. Docking simulations were performed using AutoDock Vina integrated into PyRx, with grid box dimensions optimized to target specific binding sites (Table 1). The results highlighted the optimal binding poses for each protein-ligand complex, providing valuable insights into their potential therapeutic interactions [15,24].

Table 1 Coordinate grid and dimensions of PTP1B and DPP4 proteins for molecular docking.

In vivo analysis

Animals

The in vivo study was approved by the Animal House, Universitas Sumatera Utara, under the ethical committee approval number 0522/KEPH-FMIPA/2019. Male Wistar rats weighing 150 - 200 g were selected to evaluate the antidiabetic effects of nanoherbal Biwa (E. japonica) leaves. The rats were then divided into 5 groups: [C(–)], [C(+)], [T1], [T2], and [T3]. All groups, except [C(–)], were induced with alloxan at a dose of 120 mg/kg BW. The [C(–)] group was administered physiological saline, while the other groups received different treatments after diabetes induction. [T1] was administered nanoherbal Biwa leaves at 250 mg/kg BW, [T2] was administered nanoherbal Biwa leaves at 500 mg/kg BW, and [T3] was administered glibenclamide at 5 mg/kg BW. Nanoherbal and glibenclamide treatments were administered orally for 14 days, and blood glucose levels were measured on days 1, 7, and 14. The results were analyzed to assess the effectiveness of nanoherbal Biwa leaves in reducing blood glucose levels in diabetic rats.

Measurement of blood

Measurement of blood glucose levels in rats (Rattus norvegicus) was performed using the EasyTouch glucometer. The method of taking blood is to clean the tips of the cotton tails that have been given water so that the attached dirt disappears, then clean again with 70 % alcohol. The base of the tail is pricked with a small needle, the blood that comes out is then touched on the glucometer strip. Blood glucose levels will be read on the screen after 10 s and expressed in mg/dL. Measurement of rat blood glucose level was measured before treatment and 4 days after alloxan.

Analysis of decreased blood glucose levels

After treatment the percentage of decrease in blood glucose in the group after being treated using the following formula.

(1)

Analysis of decreased blood glucose levels

Pancreas specimens were fixed in a buffered formalin solution, washed under running water, and subjected to a dehydration process using a graded ethanol series (70, 80, 90 %, and absolute ethanol) for 5 min per concentration, followed by clearing in a xylene-alcohol solution (ratios 3:1, 2:1, and 1:1) and pure xylene for the same duration. The tissue was then infiltrated with molten paraffin at a maintained temperature of 56 - 58 °C for 2 h before being embedded and sectioned into thin slices of 5 - 7 μm thickness. These sections were floated in warm water (56 - 58 °C) and carefully mounted on glass slides to maintain structural integrity and prevent tissue folding. For histological examination, hematoxylin and eosin (H & E) staining was performed, beginning with deparaffinization in xylene for 15 min, followed by rehydration through a descending ethanol series (100, 90, 80, and 70 %) for 5 min at each step. The sections were then stained with hematoxylin for 5 min, rinsed under running water, counterstained with eosin, and subjected to another dehydration cycle using 70, 80, 90 %, and absolute ethanol for 10 min per step. Finally, the sections were cleared in xylene for 5 s, mounted with coverslips, and examined under a light microscope at 400× magnification to analyze histopathological alterations.

Analysis of data

Use SPSS software version 27 to run the ANOVA test at the 5 % level, then move on to the post-hoc Duncan test. If the data are not normally distributed and/or the variance is not homogeneous, use the Kruskal-Wallis test and then proceed to the Mann-Whitney test. Graphing using GraphPad Prism Version 10.2.3 (403).

Results and discussion

Nanoherbal Biwa leaf size analysis

The PSA analysis showed that nanoparticles from Biwa (E. japonica) leaves had a particle diameter of 563.1 nm (Figure 1). Based on these results, Biwa nanoparticles can be categorized as nanoparticles since their size falls within the 100 - 1000 nm range, which has the potential to enhance bioavailability in biological systems [25,26]. A smaller particle size increases surface area, improving solubility, facilitating diffusion, and accelerating the interaction of bioactive compounds with target sites. The PBM process was found to be effective in reducing particle size, resulting in a more homogeneous nanoparticle formulation. The reduction in particle size also plays an important role in the pharmacokinetics of active compounds, potentially improving absorption and distribution. However, further research is required to evaluate the long-term stability of the nanoparticle formulation, the release profile of active compounds, and its overall biological effectiveness.

Figure 1 Nanoparticle size analysis of Biwa (E. japonica) leaves using PSA.

LC-MS analysis of nanoherbal of Biwa leaf (E. japonica)

In recent research conducted with LC-MS on nanoherbal E. japonica leaves, the team successfully identified 12 significant compounds, demonstrating considerable variation in retention times indicative of their diverse physicochemical properties (Table 2). Quinic Acid, showing the shortest retention time at just 6.01 min, suggests its higher solubility or smaller molecular size, which could be beneficial for rapid absorption in therapeutic applications. Conversely, Betulinic Acid, with the longest retention time at 32.4 min, likely has a more complex molecular structure or stronger interactions with the analytical column, suggesting stability or sustained release in pharmaceutical formulations.

Compounds such as Caffeic Acid and Kaempferol, with retention times around 7.26 and 7.7 min, respectively, and others like Chrysin and Luteolin, which exhibit longer retention times at 15.7 and 13.7 min, likely reflect their higher polarity or molecular weight, influencing their bioavailability and efficacy. Additional compounds including Oleanolic Acid, Quercetin, Rutin, Epicatechin, and Eriodictyl, with retention times ranging from 6.9 to 13.5 min, show a range of interactions with the analytical media, which could inform their pharmacokinetic profiles.

This research substantiates the bioactive potential of the compounds in nanoherbal E. japonica leaves, aligning with prior studies that highlight their therapeutic applicability [27]. The detailed retention time profiles facilitate accurate identification and quantification of these compounds and enhance understanding of their biological actions, paving the way for optimized therapeutic formulations.

Table 2 Compounds of nanoherbal Biwa leaf (E. japonica).

In silico analysis of Biwa

Drug-likeness evaluation, and biological activity prediction

Lipinski’s analysis and bioactivity prediction demonstrates the great potential of the nanoherbal compounds from Biwa leaf (E. japonica Thunb. Lindl.) in diabetes therapy, despite some compounds violating certain parameters. Lipinski’s analysis of the Biwa nanoherbal compounds, as listed in Table 3, reveals that several compounds violate specific drug-likeness criteria but still exhibit promising pharmacological properties. Quinic Acid, Caffeic Acid, Chrysin, and Coumaric Acid, although exceeding the limits on the number of oxygen atoms and hydroxyl groups, retain significant pharmaceutical potential, particularly in inhibiting enzymes related to glucose metabolism and exerting antioxidant effects [28]. Betulinic Acid and Oleanolic Acid, despite surpassing the lipophilicity threshold (MlogP), are widely utilized in medicine due to their pronounced anti-inflammatory properties, which play a crucial role in alleviating pancreatic inflammation linked to diabetes [29]. Quercetin, which presents similar violations, remains an essential compound in drug formulations due to its well-established pharmacological benefits. While Rutin violates multiple parameters, it demonstrates noteworthy bioactivity in various therapeutic applications. Furthermore, Epicatechin and Eriodictyl exhibit minor deviations but largely comply with Lipinski’s Ro5, reinforcing their potential in diabetes treatment.

Table 3 Evaluation of nanoherbal Biwa leaf (E. japonica Thunb. Lindl.) compounds based on Lipinski’s Ro5.

Bioactivity prediction analysis using the PASS Online tool (as shown in Table 4) provides further insights into the mechanisms by which these compounds contribute to diabetes therapy. Key compounds such as Quinic Acid, Caffeic Acid, Luteolin, Kaempferol, Betulinic Acid, and Oleanolic Acid exhibit strong activity in modulating diabetes-related pathways, particularly in enhancing insulin secretion, regulating glucose metabolism and exerting anti-inflammatory effects [30-32]. For instance, Quinic Acid demonstrates high insulin-promoting activity alongside potent antioxidant properties. Caffeic Acid functions as a glucose oxidase inhibitor, potentially aiding blood glucose regulation. Luteolin not only promotes insulin secretion but also exerts anti-inflammatory and antioxidative effects. Kaempferol plays a significant role in stimulating insulin release while acting as a robust antioxidant. Betulinic Acid primarily reduces diabetes-induced pancreatic inflammation, while Oleanolic Acid exhibits potent anti-inflammatory effects that could mitigate insulin resistance. Moreover, the active compounds in Biwa leaf nanoherbal predominantly belong to the flavonoid and triterpenoid classes, which are well-documented for their roles in diabetes therapy. Flavonoids such as Luteolin, Kaempferol, and Quercetin exhibit potent antioxidant and anti-inflammatory properties and enhance insulin secretion. Meanwhile, triterpenoids, Betulinic Acid, and Oleanolic Acid are crucial in reducing pancreatic inflammation and improving insulin sensitivity, making them promising candidates for novel diabetes therapeutics [33].

Table 4 Biological activities and criteria of herbal compounds and nanoherbal Biwa leaf.

ADMET and toxicity prediction

The analysis of ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) was considered essential in evaluating the efficacy and safety of nanoherbal Biwa as an antidiabetic agent. Parameters such as intestinal absorption, Caco-2 permeability, and water solubility were assessed to determine how well the nanoherbal formulation was absorbed and distributed in the body, while BBB permeability was analyzed to indicate whether the compounds could cross the blood-brain barrier for potential neurological effects. Additionally, interactions with metabolic enzymes such as CYP2D6 substrates and inhibitors and OCT2 substrates were evaluated, as they influenced drug metabolism and excretion, potentially affecting efficacy and the risk of drug interactions in diabetes therapy [34,35]. The ADMET analysis in Table 5 highlights significant variations in parameters such as intestinal absorption, blood-brain barrier (BBB) permeability, Caco-2 permeability, and water solubility among the tested compounds. High intestinal absorption (>70 %) was observed for chrysin (93.761 %), Coumaric Acid (93.494 %), and Betulinic Acid (99.763 %), indicating good potential for oral bioavailability. Conversely, Quinic Acid (32.274 %) and rutin (23.446 %) exhibited low absorption levels, which may hinder their therapeutic application via oral administration without specialized formulations. Caco-2 permeability, representing a compound’s ability to cross intestinal epithelial membranes, showed high values for Betulinic Acid (1.175) and Oleanolic Acid (1.168), supporting their potential for systemic absorption. However, quercetin (–2.925) displayed low permeability, potentially limiting its absorption through the intestine. Regarding BBB permeability, most compounds exhibited negative values, such as Quinic Acid (–0.894) and Luteolin (–0.907), indicating a restricted capacity to traverse the blood-brain barrier. Nonetheless, caffeic acid (69.407) demonstrated high permeability into the central nervous system, making it a promising candidate for brain-related therapies. None of the compounds were identified as substrates or inhibitors of the CYP2D6 enzyme, suggesting a low risk of drug-drug interactions involving this metabolic pathway. Additionally, most compounds were not substrates for the OCT2 transporter, supporting their potential for systemic distribution. In terms of water solubility, Quinic Acid (–1.119) exhibited relatively high solubility, whereas chrysin (–3.538) and Kaempferol (–3.04) had low solubility values, which could impact their absorption process, necessitating specialized formulations to enhance solubility.

Table 5 ADMET prediction of the compound from nanoherbal Biwa leaf.

Table 6 Toxicity prediction of nanoherbal Biwa leaf compounds.

The analysis conducted in this study presents the rat oral acute toxicity (LD₅₀) values in mg/kg, predicted toxicity classes (I - VI), and prediction accuracy percentages for various compounds, as shown in Table 6. Based on the classification, Class I is defined as fatal toxicity if swallowed (LD₅₀ ≤ 5), Class II is categorized as fatal toxicity (5 < LD₅₀ ≤ 50), Class III is described as toxic if ingested (50 < LD₅₀ ≤ 300), Class IV is considered harmful if swallowed (300 < LD₅₀ ≤ 2000), Class V is classified as potentially harmful if swallowed (2000 < LD₅₀ ≤ 5000), and Class VI is regarded as non-toxic (LD₅₀ > 5000) [36]. The findings indicate that Quinic Acid and Epicatechin exhibit the most favorable pharmacokinetic and toxicity profiles, positioning them as potential candidates for diabetes treatment. Both compounds are classified under Class VI, indicating very low toxicity. Conversely, quercetin is classified under Class III (50 < LD₅₀ ≤ 300), signifying high toxicity, which may limit its clinical application. Furthermore, most other compounds, including caffeic acid, Luteolin, and Kaempferol, are categorized under Class IV and V, indicating moderate to low toxicity. Compounds with high average similarity and prediction accuracy, such as Coumaric Acid and Rutin, are considered more reliable for further development. The heatmap shown in Figure 2 illustrates the predicted organ toxicity, where values approaching –0.5 indicate active toxic effects. The results demonstrate that quercetin exhibits high toxicity, particularly in terms of hepatotoxicity and cardiotoxicity, whereas Betulinic Acid and Oleanolic Acid are shown to induce moderate nephrotoxicity and hepatotoxicity. In contrast, Quinic Acid, Epicatechin, and Rutin exhibit low or no toxicity across different organs, making them safer options. Additionally, chrysin and Kaempferol are found to cause mild hepatotoxicity and nephrotoxicity, while eriodictyol is predicted to exhibit potential neurotoxicity but remains relatively safe for other organs.

Figure 2 Headmap organ toxicity of components in nanoherbal from Biwa leaves.

In diabetes therapy, Quinic Acid is particularly promising due to its high solubility, low toxicity, and well-documented anti-inflammatory and antioxidant properties, which contribute to protecting pancreatic tissue from oxidative stress, a key mechanism in the pathogenesis of diabetes [37]. Similarly, Epicatechin, which is also safe and exhibits reasonable bioavailability, has been reported to enhance insulin sensitivity and reduce insulin resistance through the modulation of oxidative stress and inflammation [38]. This compound also offers protection to β-pancreatic cells, supporting glycemic control. Conversely, compounds such as quercetin, despite their anti-diabetic activity through α-glucosidase inhibition and protective effects on the pancreas, exhibit high organ toxicity, limiting their therapeutic application. Nanotechnology-based formulations may provide a viable solution to improve the safety and efficacy of such compounds.

Additionally, caffeic acid, which demonstrates high BBB permeability, shows potential in addressing diabetes-related neurological complications such as diabetic neuropathy through its antioxidant and anti-inflammatory mechanisms [39]. In conclusion, Quinic Acid and Epicatechin are promising candidates for diabetes therapy due to their favorable safety profiles and biological activities supporting glycemic regulation. However, specialized formulations, such as quercetin, are essential for compounds with pharmacokinetic or toxicological limitations to optimize their clinical potential.

PerMM prediction

This study evaluated the passive permeability potential of 11 phytochemicals identified through LC-MS analysis of Biwa (E. japonica) leaf nanoherbal compounds using the PerMM method. PerMM is a web server that predicts membrane partitioning and translocation of small molecules based on free energy calculations [40]. This analysis is essential for determining whether a compound crosses membrane passively or requires specific transport mechanisms [41]. As illustrated in Figure 3, the energy profiles of the analyzed compounds exhibited distinct interaction patterns with lipid membranes, influencing their pharmacokinetic potential.

Figure 3 The PerMM analysis identified 11 compounds: (a) Quinic Acid, (b) Caffeic Acid, (c) Chrysin, (d) Coumaric Acid, (e) Luteolin, (f) Kaempferol, (g) Betulinic Acid, (h) Oleanolic Acid, (i) Quercetin, (j) Epicatechin, and (k) Eriodictyl.

To further examine permeability trends, individual compound energy profiles were analyzed. Assessing the

membrane permeability of Biwa (E. japonica) leaf nanoherbal compounds is essential for optimizing their pharmacokinetic properties in diabetes therapy. Enhanced permeability facilitates improved bioavailability, promoting glucose homeostasis, insulin signaling modulation, and anti-inflammatory activity. PerMM provides valuable insights into the interaction of these nanoherbal compounds with lipid membranes, enabling predictions regarding their cellular uptake and pharmacological efficacy.

Quinic Acid exhibited moderate permeability, with maximum stability at –1.62 kcal/mol at Z = ±22 Å and the highest resistance at Z = 0 Å. Caffeic Acid demonstrated low permeability, peaking at –1.7 kcal/mol at Z = ±17 Å, while Chrysin exhibited enhanced membrane penetration efficiency, stabilizing at –3.61 kcal/mol at Z = ±16 Å. Coumaric Acid required additional energy to traverse hydrophobic regions, with a peak of +3.54 kcal/mol at Z = 4 Å and a minimum of –2.02 kcal/mol at Z = ±17 Å. Luteolin encountered substantial energy barriers within the hydrophobic membrane core, with a minimum energy of –2.21 kcal/mol at Z = ±16 Å. In contrast, Kaempferol exhibited strong surface interactions, similar to Luteolin, with a minimum energy of –2.04 kcal/mol at Z = ±17 Å. Betulinic Acid and Oleanolic Acid demonstrated high stability and efficient membrane traversal, with Betulinic Acid reaching –6.27 kcal/mol at Z = ±16 Å and Oleanolic Acid maintaining consistently negative energy values. Compounds with low energy transfer values can passively permeate membranes, reducing the need for specialized drug delivery systems such as liposomes or nanoparticles [42].

Most compounds exhibited stability at the membrane’s peripheral regions (Z = ±16 to ±22 Å), with Betulinic Acid and Oleanolic Acid displaying the highest permeability. In contrast, Caffeine Acid and Luteolin faced significant energy barriers. Eriodictyol demonstrated a flat energy profile, suggesting computational limitations due to incomplete molecular data. These findings reinforce the importance of free energy calculations in predicting molecular transport across lipid bilayers, which is critical for developing nanoherbal therapeutics for diabetes management.

Molecular docking and visualization of interactions with PTP1B and DPP4

This comprehensive study explores the antidiabetic potential of natural compounds extracted from the nanoherbal leaves of Biwa (E. japonica), emphasizing their role in inhibiting key therapeutic targets, specifically PTP1B and DPP4. These enzymes are instrumental in regulating blood glucose levels and enhancing insulin sensitivity, which are pivotal factors in diabetes management. The study focuses on flavonoids such as Luteolin and quercetin, as well as terpenoids like Oleanolic Acid, which are known for their effectiveness in improving insulin sensitivity and reducing blood glucose levels by targeting these enzymes. Notably, Luteolin and quercetin enhance insulin sensitivity by binding to critical residues within PTP1B’s active site, corroborating the molecular interactions detailed in this research [43]. Additionally, research by Zhao et al. [44] highlights Oleanolic Acid’s capability to significantly decrease insulin resistance in type 2 diabetes models.

Table 7 Interaction of nanoherbal leaf E. japonica with PTP1B and binding affinity.

Table 8 Interaction of nanoherbal leaf Eriobotrya japonica with DPP4 and binding affinity.