Green approach for the synthesis of zinc oxide nanoparticles from methanolic stem extract of Andrographis paniculata and evaluation of antidiabetic activity: In silico GSK-3β analysis

2.1 Materials

Metformin, acarbose, dimethyl sulfoxide (DMSO), and methanol were procured from Haymankimia (Witham, UK). Streptozotocin (STZ) was procured from Hi-media (Mumbai, India). Ascorbic acid was procured from Scharlab (Barcelona, Spain). Zinc sulfate (ZnSO₄) was procured from Sigma Aldrich (Mumbai, India). The plant components were extracted using methanol and distilled water. The other substances were all of analytical grade.

2.2 Preparation of methanolic stem extract of A. paniculata (MEAP)

The traditional zone for the collection of A. paniculata was Udupi district, Karnataka, South India. The plant collection was done in September 2023. The Indian Institute of Horticultural Research successfully identified the plant material. The collected stems were then cleaned twice, once with sterile distilled water and once with tap water. The stem portion was then allowed to air dry at room temperature and shielded from the sun. The dried part of the stem was ground into fine powder using an electric grinding machine and sieved using mesh no. 40. After sieving, 100 g of the sample (fresh sample) was soaked in 1,000 mL of solvent in the Soxhlet apparatus as the first extraction solvent [8]. After raising the temperature to 40°C, the procedure lasted for 24 h. The following day, the extract was emptied, 1,000 mL of solvent was added back, and the procedure was repeated for 24 h. At the end of the extraction period, the extract was distilled at 40°C under reduced pressure in a rotary evaporator. Once concentrated to a small volume, the extract was dried completely in an oven. In preparation for later use, the obtained extracts (9 g) were stored at 4°C. The ratio between the fresh sample and the obtained extract powder was 100:9. The obtained yield for the extract was 9%.

2.3 Phytochemical screening

In accordance with a previously described procedure [9], the MEAP was phytochemically screened and analyzed to ascertain the presence of various phytoconstituents. The presence of various phytoconstituents was determined qualitatively by following standard procedures, such as Mayer’s test, Dragendroff’s test, and Wager’s test for alkaloids; Molisch’s test, Benedict’s test, and Fehling’s test for carbohydrates; Shinoda test and alkaline test for flavonoids, tannins, and terpenoids [9].

2.4 Green formulation of ZnO-NPs

A 0.01 M ZnSO₄ solution was used to accomplish the biosynthesis of ZnO-NPs using MEAP. Essentially, 95 mL of ZnSO₄ solution (0.01 M) and 5 mL of MEAP (100 μg·mL⁻¹) were mixed and stirred for an hour at 40°C (550 rpm) using a magnetic stirrer. By forming reduced precipitates, the bioformulation of ZnO-NPs was identified. After centrifugation for 30 min at 8,000 rpm, the bioreduced precipitates of ZnO-NPs were finally collected. The pH of the synthesized NPs was 6.8. The recovered precipitates were rinsed twice with sterile distilled water to remove any contaminants after the supernatants were disposed of [10].

2.5 Physicochemical evaluation of the biosynthesized ZnO-NPs

2.6 Screening of the free radical scavenging activity assay

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was used to estimate the sample using the previously published methodology [13]. Initially, 2 mL of DPPH˙ radical methanol solution (6 × 10⁻⁵ M in 80% methanol) was combined with 50 µL of sample solutions at various concentrations. After 16 min, the absorbance reading at 515 nm was obtained for each sample. All samples were evaluated in triplicate. The proportion of inhibition of the DPPH˙ radical [14] was ascertained using Eq. 1:

where AS(t) is the sample’s absorbance (t = 16 min) and AC(0) is the control’s absorbance (t = 0 min). Ascorbic acid was used as a reference, and the graph was used to calculate the sample concentration that exhibited 50% inhibition (IC₅₀).

2.7 In vitro α-amylase inhibition assay

A total of 600 µL of the test sample (10, 20, 40, 60, 80, and 100 µg·mL⁻¹) and 1.2 mL of starch in phosphate buffer (pH 6.9) with 6.7 mM sodium chloride were added for the in vitro antidiabetic activity assessment of amylase inhibition. Then, 600 µL of porcine pancreatic amylase was added to start the reaction, which was then incubated for 1 h at 37°C. After extracting 600 µL of the above combination, 300 µL of DNSA (1 g of DNSA, 30 g of sodium potassium tartrate, and 20 mL of 2 N sodium hydroxide were added and built up to a final volume of 100 mL with distilled water) were added, and the mixture was heated to a boil for 15 min. The absorbance was measured at 540 nm after diluting the reaction mixture with 2.7 mL of water. Blank tubes were made for each concentration by substituting the enzyme solution with 600 μL of distilled water. Without a test sample, the control represents 100% of the enzyme activity created identically. An identical methodology was used three times to repeat the experiment. Using Eq. 2, the α-amylase inhibitory activity was determined:

A graph of the concentration of the sample and the reference against the percentage of inhibition was plotted to determine the IC₅₀ value.

2.8 In vitro α-glucosidase inhibition assay

To create the enzyme solution, 0.5 mg of α-glycosidase was dissolved in 10 mL of phosphate buffer (pH 7.0) that contained 20 mg of bovine serum albumin. Phosphate buffer was added to the solution to further dilute it at a 1:10 ratio. The sample blank was created by dissolving 4 mg of material in 400 µL of DMSO to create the sample solution. Five concentrations were prepared: 50, 100, 150, 200, and 250 µg·mL⁻¹. Phosphate buffer (pH 7.0) containing p-nitrophenyl α-d-glucopyranoside was added to 5 µL of the sample solution and DMSO. The solutions were incubated at 37°C for 15 min. Then, 0.1 N Na₂CO₃ (1,000 µL) solution was added after 15 min. The sample’s absorbance against the sample blank was measured at 400 nm using a UV-visible spectrophotometer [15].

The α-glucosidase inhibitory activity was calculated using Eq. 3:

A graph of the sample and reference values against the percentage of inhibition was plotted to obtain the respective IC₅₀ values.

2.9 In silico studies

2.10 Experimental animals

Wistar Albino rats, with an average body weight of 180 g(weighing between 160 and 200 g; 6–8 weeks old), were obtained from Varunya Biolabs Pvt. Ltd. (CPCSEA Registration No. 2076/P O/RcBi Bt/S/19/C PCSEA; Bengaluru, Karnataka, India). The animals were kept in groups of eight, each at a temperature of 24–28°C, a relative humidity of 60–70%, and a 12-h cycle of light and dark. As per CCSEA guidelines, the animals were provided with commercially available palatable, non-contaminated, and nutritionally adequate food pellets (10–14 mm) containing moisture, crude fiber, crude protein, essential vitamins, minerals, crude fat, carbohydrate, and fresh, potable, uncontaminated drinking water ad libitum. Sri Adichunchanagiri College of Pharmacy in India’s Institutional Animal Ethical Committee (IAEC) approved the experimental protocols (IAEC approval No.: SACCP-IAEC/2023-02/85).

2.11 Antidiabetic effects of MEAP and bioformulated ZnO-NPs

2.12 Histopathological examination

Following their removal, the kidney and pancreas were preserved by being submerged in a 10% formalin solution, often washed with phosphate-buffered saline (1× PBS, pH 7.4), and embedded in paraffin blocks. Using a semi-automated rotator microtome, 5 μm-thick slices were created and then stained with hematoxylin and eosin. A biological inverted microscope was used to image the sections [23].

2.13 Statistical evaluation

The data are presented as mean ± SEM, and GraphPad Prism (version 8.0.2, San Diego, CA, USA) was utilized to analyze variance (ANOVA) to assess the statistical differences between the groups. A p-value of less than 0.05 was deemed statistically significant.

3.1 Preliminary phytochemical screening

The phytochemical analysis of the MEAP revealed the presence of alkaloids, carbohydrates, flavonoids, tannins, terpenoids, and quinone. The related data are summarized in Table S1.

3.2 Green biosynthesis of ZnO-NPs

MEAP was used in the biosynthesis of biogenic ZnO-NPs, as shown in Figure 1. Thus, the first indication of ZnO-NP biosynthesis was the formation of a reddish-brown precipitate (Figure S1) after MEAP was added to the colorless ZnSO₄ solution. A previous study [24] reported that the first indication of ZnO-NP biofabrication was the formation of a reddish-brown precipitate. It is well known that phytochemical components such as tannins, alkaloids, flavonoids, and quinone are abundant in plant extracts. Because of their potential to neutralize chelate metals, free radicals, and ROS, these ingredients are referred to as antioxidants [25]. According to reports, MEAP has a high antioxidant content that supports its antioxidant effects [26]. These phytoconstituents, which are found in the stem of A. paniculata as phenolic compounds, flavonoids, quinone, alkenes, and proteins, are thought to be responsible for the biofabrication of ZnO-NPs because they have the ability to reduce or chelate metal ions and function as stabilizing and capping agents for the biogenic ZnO-NPs [27]. According to a different study, phytochemical components assigned their electrons to the biostabilization of Zn²⁺ complex ions and were then thermally annealed to form ZnO-NPs [28].

Figure 1

Diagrammatic representation of the ZnO-NP synthesis process.

3.3 Characterization of ZnO-NPs from A. paniculata

3.3.1 Particle size and ZP analysis

Figure S2 shows the acceptable uniformity in the particle size distribution of ZnO-NPs in an aqueous medium. It was derived using dynamic light scattering and based on the reasonably low polydispersity of 0.286, which fits the 0.2–0.7 range. The average particle size of the NP obtained is 93.61 nm, which demonstrated relatively small NPs and uniform particle size distribution [29].

ZP levels must be analyzed in order to forecast and understand the electrostatic potential at NP surfaces. The ZP level represents the NPs’ colloidal stability. NP aggregation does not occur when particles with large positive and negative ZP values reject one another. NPs with ZP values less than −25 mV or greater than +25 mV frequently exhibit strong stability. van der Waals interparticle attraction is the reason for aggregation at lower ZP values [30]. The ZP of the biogenic ZnO-NPs was determined to be −32.2 mV (Figure 2a), indicating a possible long-term stability and uniform distribution. In actuality, our findings show that NPs are reasonably stable and may be protected from self-aggregation.

Figure 2

(a) ZP of the biosynthesized ZnO-NPs. (b) SEM image of biosynthesized ZnO-NPs.

3.3.4 FTIR spectroscopy studies

FTIR spectroscopy provides information on the rotational and vibrational modes of a molecule, making it a reliable method for chemical identification and characterization [32]. The FTIR spectra of ZnO-NPs obtained using the biological process are displayed in Figure 3. Potential biomolecules that could be in charge of ZnO reduction and capping agent peaks at limited wave numbers were found. The O–H stretching, intermolecular hydrogen bonding, and the presence of primary and secondary amines of alcohols are all indicated by the bands at 3,500 cm⁻¹ [32,33]. Asymmetric and symmetric stretching vibrations of C–H were attributed to the bands detected at 3,000 and 2,852 cm⁻¹ [33] (Table S2). The amide I and amide II regions, which are features of proteins and enzymes, are responsible for the bands at around 1,593 and 1,340 cm⁻¹ [32]. A ZnO absorption band is clearly visible in the spectra at 675.22 cm⁻¹ may be explained by the metal oxide’s stretching vibration, which is a member of the ZnO metal group [33]. Furthermore, C–H bending, C–N stretching vibration of aliphatic and aromatic amines, and C–O stretching of polysaccharides, alcohols, and phenolic groups were linked to the strong bands detected at 1,593, 1,408, 1,340, and 1,340 cm⁻¹, respectively [34].

Figure 3

FTIR spectra of ZnO-NPs from MEAP.

3.3.5 XRD spectra of the biogenic ZnO-NPs

The crystalline character of the biogenic ZnO-NPs was examined using the XRD spectra. Eighteen diffraction peaks were detected at 2θ degrees using the XRD configuration, as shown in Figure 4: 4.20°, 27.66°, 31.99°, 33.38°, 34.65°, 36.48°, 45.69°, 56.79°, 63.04°, 66.55°, 68.12°, 72.77°, 75.47°, 77.14°, 81.55°, 84.05°, and 89.75°. All of these findings supported the hexagonal wurtzite structure of biogenic ZnO-NPs. Our findings were in agreement with an earlier report that detailed the environmentally friendly biosynthesis of ZnO-NPs using MEAP, and they confirmed that the hexagonal wurtzite structure formed as indicated by diffraction peaks detected at 2θ for 4°, 27°, 31°, 33°, 34°, 36°, 45°, 56°, 63°, 66°, 68°, 72°, 77°, 81°, 84°, and 89°, respectively, in the XRD data. Using Scherrer’s formula, the crystalline size of the biogenic ZnO-NPs was calculated as follows: D = (kλ/β cos θ), where D is nanosize of biogenic ZnO-NPs, λ is the wavelength of X-ray (2.46065 Å), K is Scherer’s constant, which is found to be the full width at half-maximum (FWHM) of the most intense diffraction peak (0.2230), and θ is the diffraction angle (36.4860). Accordingly, the crystalline size was determined to be 12.90 nm [35] by using the peak with the maximum intensity, which relates to the plane at 36.4860.

Figure 4

XRD spectra of the biogenic ZnO-NPs utilizing MEAP.

3.4 DPPH radical scavenging activity

Using the DPPH free radical, the antioxidant activity of A. paniculata stem extract was examined. Figure S4 illustrates the antioxidant activity of the extracts. The DPPH free radical scavenging activities of the A. paniculata extracts depend on IC₅₀ values. The highest antioxidant activity of A. paniculata was observed with an IC₅₀ value of 45.44 μg·mL⁻¹ and the IC₅₀ value of ascorbic acid was found to be 66.42 μg·mL⁻¹.

3.5 In vitro α-amylase and α-glucosidase inhibitory activity of stem extract of A. paniculata

The inhibitory potentials of A. paniculata against α-amylase and α-glucosidase were analyzed, and it was found that the inhibition related to the commercial drug acarbose. The IC₅₀ values of the A. paniculata and acarbose were determined graphically. The IC₅₀ values of A. paniculata and acarbose on the activity of α-amylase were estimated to be 17.76 and 20.97 μg·mL⁻¹, respectively, as shown in Figure 5. In the case of α-glucosidase activity, the IC₅₀ values were calculated to be 176.85 and 163.44 μg·mL⁻¹ for A. paniculata and acarbose, respectively, as shown in Figure 6. Though acarbose inhibited α-amylase at a higher concentration, A. paniculata reduced its activity by half at a lower concentration and inferred that A. paniculata could act as an α-amylase inhibitor. When the diabetes-associated α-glucosidase inhibition of acarbose and A. paniculata was compared, it was found that A. paniculata had better inhibition and a lower IC₅₀ value [36].

Figure 5

Inhibitory potential of A. paniculata and acarbose against α-amylase enzyme.

Figure 6

Inhibitory potential of A. paniculata and acarbose against α-glucosidase enzyme.

3.6 Molecular docking

Molecular docking analysis was used to ascertain the molecular interactions between the target protein and seven different ligands. The findings are shown in Figures 7–10. ADT 1.5.7 was utilized to dock these phytochemicals with the GSK-3β protein. 1Q41 was observed to exhibit strong binding interactions, with negative free energy values in the grid box (ranging from 4.90 to 10.19). Given that binding energy is known to significantly affect ligand involvement and even protein flexibility [37], it suggests a high affinity between the two components. It has also been shown that a few additional substances, including andrographolide, apigenin, and neoandrographolide, have substantial binding affinities with the GSK-3β protein. Table 1 displays the active compounds together with their corresponding free energies from docking experiments, with metformin serving as the control medication. Six different compounds were discovered to have binding energies lower than that of the medication used as a reference, metformin, which has a docking score of −4.90 kcal·mol⁻¹. This indicates that bioactive molecules of A. paniculata are more active than those of the typical medication and have a propensity to interact with and disrupt the function of GSK-3β. The four best ligands – isoandrographolide, apigenin, andrographolide, and neoandrographolide – were selected based on their binding affinities of ≤7.22 kcal·mol⁻¹, and their molecular interactions and pharmacokinetic characteristics were examined.

Figure 7

Molecular docking results of isomandrographolide in the GSK-3β protein domain. (a) 3D binding mechanism of isomandrographolide in the protein active site. (b) Two-dimensional (2D) interaction displaying the hydrophobic and hydrogen-interacting amino acid residues.

Figure 8

Molecular docking results of apigenin on the GSK-3β protein domain. (a) 3D binding mode of apigenin in the protein active site. (b) 2D interaction displaying the hydrophobic and hydrogen-interacting amino acid residues.

Figure 9

Molecular docking results of andrographolide in the GSK-3β protein domain. (a) 3D binding mechanism of andrographolide in the protein active site. (b) 2D interaction displaying the hydrophobic and hydrogen-interacting amino acid residues.

Figure 10

Molecular docking of neoandrographolide in the GSK-3β protein domain. (a) Neoandrographolide’s 3D binding mechanism in the protein active site. (b) 2D interaction displaying the hydrophobic and hydrogen-interacting amino acid residues.

3.7 Molecular interaction

Using a Discovery Studio visualizer, the ligand−receptor molecular interaction was studied. Hydrogen bonding, hydrophobic interactions, and polar interactions mostly mediated the binding of the chemical in the GSK-3β protein’s active region. A thorough analysis was conducted on the binding orientations of the top four phytochemical analogues: isoandrographolide, apigenin, andrographolide, and neoandrographolide. The ideal positions were identified, providing a detailed description of the various amino acid residues engaged in the interaction (Table 2). The 3D structure of andrographolide and neoandrographolide (Figure S5) demonstrated that hydrogen bonds were more common than hydrophobic interactions in the protein’s binding pocket. This most likely illustrates how these bonds help the molecule function as an effective ligand [38]. Hydrophobic contact is more prevalent in biological complexes, such as those containing apigenin. Since none of the ligands exhibited a salt bridge interaction, it is possible that these forces contributed less to the stability of the protein. Based on its binding energy of −10.19 kcal·mol⁻¹, neoandrographolide had the strongest interaction of all the phytocompounds with the GSK-3β protein. Alanine (Ala) (3.98), leucine (Leu) (4.97), tyrosine (Tyr) (5.39), valine (Val) (4.41), and isoleucine (Ile) (4.50) were involved in hydrophobic contact, and three polar residues, Val (1.91), lysine (Lys) (2.05), and glycine (Gly) (2.54) were involved in hydrogen bonding between the receptor and ligand.

The second-best medication is andrographolide, whose binding energy is −8.69 kcal·mol⁻¹. After a thorough analysis, it was determined that this molecule fits into the receptor’s active binding site steadily by interacting with key amino acid residues, such as Lys (5.26) and proline (Pro) (4.13), which are important for hydrogen bonding with the receptor, and Leu (1.89), aspartic acid (Asp) (1.97), asparagine (Asn) (2.86), and arginine (Arg) (2.31), which are involved in hydrophobic interaction. Additionally, the third-best analogue, apigenin, has a binding affinity of −8.51 kcal·mol⁻¹ and is primarily composed of Arg (5.20), Arg (2.06), insulin-link growth factor (Ilg) (2.84), Tyr (3.16), cysteine (Cys) (2.57), Arg (2.27), Tyr (3.39), Asp (3.33), and Asp (1.33). The fourth-best compound, isoandrographolide, forms two hydrogen bonds and six hydrophobic interactions with the GSK-3β protein. The following amino acids are the main players in these interactions: Asp (2.18), Asn (3.03), Cys (4.97), Leu (5.19), Ala (3.34), Leu (4.20), Val (4.44), and Val (5.22). The studied compounds of MEAP were found to be orally bioavailable as they followed the statement of Lipinkski’s rule of five [39].

3.8 Effect of MEAP and bioformulation on blood glucose levels

STZ is frequently used to induce type 2 DM in experimental animals. By targeting biological components and membranes, ROS mediates the cytotoxic action of the STZ-diabetogenic agent, resulting in diabetes complications [40]. The anti-hyperglycemic effects of MEAP and bioformulation were investigated in STZ-induced diabetic rats. Figure 11 shows the hypoglycemic effect of MEAP and bioformulation in an STZ-diabetic rat. After receiving an injection of 40 mg·kg⁻¹ of STZ intraperitoneally, diabetic rats developed blood glucose levels above 300 mg·dL⁻¹ on day 7. Comparing the MEAP and bioformulated-treated group with the STZ-treated diabetic control group from day 1 to day 30 following STZ treatment revealed heterogeneous significance (p < 0.01). In STZ-induced diabetic rats, Groups IV, V, and VI receiving MEAP and Groups VII, VIII, and IX receiving bioformulation at different doses showed a substantial effect on the blood glucose levels. Group III received metformin 250 mg·kg⁻¹. Groups IV, V, and VI showed dose-dependent effects on blood glucose regulation, indicating its potential as a treatment for lowering blood glucose in diabetic conditions. Groups VII, VIII, and IX, potentially combining MEAP with ZnO, showed similar trends to MEAP alone, suggesting that the addition of ZnO may enhance the anti-diabetic properties of MEAP. Metformin consistently showed effectiveness in reducing blood glucose levels. MEAP and MEAP-mediated ZnO-NP treatments exhibited dose-dependent effects, with higher doses generally leading to more substantial reductions in blood glucose levels.

Figure 11

Graphical representation of the MEAP and MEAP-derived ZnO-NPs on glycemia (mg·mL⁻¹) in STZ-induced diabetic rats. Group 1: normal control (0.1% saline); Group 2: diabetic control (received STZ 40 mg·kg⁻¹ body weight); Group 3: metformin (250 mg·kg⁻¹); Group 4: MEAP (300 mg·kg⁻¹); Group 5: MEAP (600 mg·kg⁻¹); Group 6: MEAP (1,200 mg·kg⁻¹); Group 7: MEAP-ZnO-NPs (100 mg·kg⁻¹); Group 8: MEAP-ZnO-NPs (200 mg·kg⁻¹); and Group 9: MEAP-ZnO-NPs (400 mg·kg⁻¹). Values are expressed as mean ± SEM, n = 8; ****p < 0.0001, ***p < 0.001, **p < 0.004, and *p < 0.0102 compared to diabetic animal. P-values <0.05 were considered statistically significant.

3.9 Effect on body weight

Figure 12 shows the impact of extract and bioformulation on the body weight of rats under normal and diabetes conditions. Rats in the normal control group were found to have stable body weights; however, throughout a 30-day period, the body weight of the diabetic rats significantly decreased. The body weight reduction caused by STZ was effectively counteracted by the formulation and extract.

Figure 12

Graphical representation of MEAP and MEAP-mediated ZnO-NP effects on body weight (g) in STZ-induced diabetic rats for different group treatments. Values are expressed as mean ± SEM (n = 8); p < 0.36 (ns), **p < 0.01 ***p < 0.001 and ****p < 0.0001 compared to diabetic animals/Group II (two-way ANOVA followed by a Dunnett’s t-test). P-values <0.05 were considered statistically significant; ns: non-significant.

3.10 Serum biochemical parameters

Compared to normal control rats, diabetic control rats had lower levels of HDL and higher levels of TG, TC, LDL, and VLDL after 30 days of therapy (Figure 13). Metformin (250 mg·kg⁻¹), MEAP, and MEAP-mediated ZnO-NP treatment group had these parameters reversed; the effect of MEAP-derived ZnO-NPs was greater than that of MEAP. This indicates that the lipid profile of MEAP-mediated ZnO-NPs was improved in a dose-dependent manner.

Figure 13

Graphical representation of the effects of MEAP and MEAP-ZnO NPs on the lipid profile, i.e., TG, TC, LDL, VLDL, and HDL (mg·dL⁻¹) in STZ-induced diabetic rats. Values are expressed as mean ± SEM (n = 8) and were analyzed by one-way ANOVA followed by a Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ^& p < 0.05, ^&& p < 0.01, ^&&& p < 0.001, and ^### p < 0.001.

3.11 Diabetic nephropathy analysis

The study investigated the therapeutic effects of MEAP and MEAP-mediated ZnO-NPs on diabetic nephropathy parameters in diabetic rats. The main parameters assessed were BUN, creatinine, and uric acid levels over 30 days of therapy. Diabetic control rats exhibited elevated levels of these parameters, indicating impaired kidney function associated with diabetic nephropathy. The results demonstrate that both MEAP and MEAP-derived ZnO-NPs can effectively mitigate the adverse effects of diabetic nephropathy, with MEAP-derived ZnO-NPs showing superior efficacy (Figure 14). This suggests a potential for MEAP-derived ZnO-NPs as a promising therapeutic strategy for improving kidney function under diabetic conditions.

Figure 14

MEAP and MEAP-mediated ZnO-NPs mediated diabetic nephropathy in diabetic rats: (a) BUN, (b) creatine, and (c) uric acid for different groups. Values are expressed as mean ± SEM (n = 8); **p < 0.01 and ***p < 0.001 compared to diabetic animals/Group II (one-way ANOVA followed by a Tukey’s test). P-values *p < 0.05 were considered statistically significant; ns: non-significant.

3.12 Effect of MEAP and bioformulation on histopathology of pancreas and kidney

3.12.1 Pancreatic cells

The pancreatic histology architecture in the normal control rats was predicted (Figure 15a). It took the shape of an acinar structure containing typical Langerhans (IL) islets. The pancreas of the diabetic control group showed necroptosis, vacuolar degeneration, and acini shrinkage, in addition to a notable decrease in the islets of Langerhans (Figure 15b). With normal acinar cells and minimal necrotic alterations, IL recovered to a regular size in the metformin-treated group (Figure 15c). The pancreas of the MEAP-treated groups showed minimal necrotic changes and a slight reconstitution of IL cells (Figure 15d–f). With the restoration of IL and minimal necrotic alterations, the pancreas of the MEAP-mediated ZnO-NP-treated groups appeared normal (Figure 15g–i). Both the standard and test drug-treated groups showed improvements in pancreatic cells (Figure 15d–i).

Figure 15

The pancreatic section of histology was stained with hematoxylin and eosin (40× magnification). (a) Group I: normal control (0.1% saline); (b) Group II: diabetic control (received STZ 40 mg·kg⁻¹; (c) Group III: metformin (250 mg·kg⁻¹); (d) Group IV: MEAP (300 mg·kg⁻¹); (e) Group V: MEAP (600 mg·kg⁻¹); (f) Group VI: MEAP (1,200 mg·kg⁻¹); (g) Group VII: MEAP-ZnO NPs (100 mg·kg⁻¹); (h) MEAP-ZnO NPs (200 mg·kg⁻¹); and (i) MEAP-ZnO NPs (400 mg·kg⁻¹). NIL – normal islet of Langerhans, RSIL – reduction of the size of IL, DBC – degeneration of beta cells, N – necroptosis, VD – vacuolar degeneration, NAC – normal acinar cell, MNC – mild necrotic cells, RDBC – recovering degeneration of beta cells, RAC – recovering acinar cells, RCIL – reconstitution of IL, MNIL – minimal necrotic IL, NBC – normal Bowmen’s capsule, MRIL – modest reconstitution of IL, MAA – mild acini atrophy, NG – normal glomerulus, MN – minimal necroptosis, MCIL – modest reconstitution of IL, NOBC – normal ovoid beta cells, RILN – reconstituted IL cells with minimal necrotic alterations, RAA – recovered acini atrophy, and VR – vacuolar regeneration.

3.12.2 Kidney cells

Microscopically, the histopathological examination of the kidney of the normal control group showed normal, well-defined glomeruli and tubular cells had a uniform size and shape with distinct nuclei. There were no signs of pathological changes or abnormalities (Figure 16a). Examination of kidney tissues of rats in the disease group, which suffer from diabetes, showed pathological changes in the kidney structure compared to that of the normal control group. They showed signs of hypertrophy, thickening of the glomerular basement membrane, tubular atrophy, and interstitial fibrosis. These changes are indicative of the progressive nature of diabetic kidney disease (Figure 16b). Meanwhile, the kidney section of diabetic rats in Groups IV–VI treated with MEAP for 30 days seemed to reduce damage to glomeruli, tubules, and interstitial space compared to untreated diabetic rats. This suggests a potential therapeutic benefit in preserving kidney function. On the other hand, the kidney section of diabetic rats in Groups VII–IX treated with the MEAP-mediated ZnO-NPs seemed to preserve glomerular and tubular structures, reduce fibrosis, and less inflammation compared to untreated diabetic rats.

Figure 16

The kidney section of histology stained with hematoxylin and eosin (40× magnification). (a) Group I: normal control (0.1% saline); (b) Group II: diabetic control (received STZ 40 mg·kg⁻¹); (c) Group III: metformin (250 mg·kg⁻¹); (d) Group IV: MEAP (300 mg·kg⁻¹); (e) Group V: MEAP (600 mg·kg⁻¹); (f) Group VI: MEAP (1,200 mg·kg⁻¹); (g) Group VII: MEAP-ZnO NPs (100 mg·kg⁻¹); (h) MEAP-ZnO NPs (200 mg·kg⁻¹); and (i) MEAP-ZnO NPs (400 mg·kg⁻¹). NBC – normal bowmen’s capsule, NG – normal glomerulus, NDT – normal distal tubule, NT – normal tubule, SPT – swollen proximal tubule, CSRT – congested and swelling renal tubules, HGT – hemorrhage glomerular tuft, NPT – normal proximal tubules, NBC – normal Bowmen’s capsule, NG – normal glomerulus, NRT – normal renal tubule, LCG – low congested glomerulus, MSPT – mild swollen proximal tubule, NPT – normal proximal tubule, MCT – mild congested tubule, LH – low hemorrhage, PT – proximal tubule, MST – mild swollen tubule, LGH – low glomerular hemorrhage, NMC – normal macula dense cells, NDT – normal distal tubule, RSPT – recovered swollen proximal tubule, MHG – mild hemorrhagic glomerulus, and RST – recovered swollen tubule.