Assessment of In Vitro Antioxidant Activity and Subacute Toxicity and Safety of Tetrahydroserpentine in Hyperglycemic Mice

ABSTRACT

Tetrahydroserpentine (ThS), an indole alkaloid and α₁‑adrenergic receptor blocker, is used for treating hypertension, a condition often linked with diabetic pathophysiology. However, its toxicological profile and safety under hyperglycemic conditions remain unclear. This study aimed to assess the antioxidant activity, subacute toxicity, and effect of ThS under hyperglycemic conditions using in silico, in vitro, and in vivo approaches. In vitro toxicity analysis demonstrated that ThS exhibited dose‐dependent cytotoxicity in both cancerous and noncancerous cells at ≥ 25 µM. It showed moderate antioxidant activity by reducing H₂O₂‐induced ROS, although its antioxidant effect declined with time. In silico network pharmacology predicted 110 targets involved in metabolic, inflammatory, and proliferative pathways. In vivo subacute toxicity studies in mice showed that oral doses ≥ 5.0 mg/kg/day led to liver and kidney damage, as evidenced by altered hematological, biochemical, and histological parameters. A lower dose (2.5 mg/kg/day) was identified as non‐toxic and used in streptozotocin‐induced hyperglycemic mice. At this dose, ThS did not improve hyperglycemia‐induced liver and kidney damage and, notably, worsened serum SGOT and urea levels. These findings highlight that while ThS possesses limited antioxidant capacity, it can be toxic at higher doses and harmful under hyperglycemic conditions. Further studies are required to ensure its safe therapeutic use in hyperglycemic conditions.

Integrated in vitro, in silico, and in vivo analyses show that tetrahydroserpentine (ThS) exhibits dose‐dependent cytotoxicity and limited, time‐dependent antioxidant activity. Network pharmacology identified 110 protein targets. Subacute dosing (≥5.0 mg/kg/day) caused liver and kidney damage, while a lower dose (2.5 mg/kg/day) failed to improve hyperglycemia and worsened organ injury, indicating potential risk under hyperglycemic conditions.

1. Introduction

Plants are the greatest source of certain types of secondary metabolites, including alkaloids (e.g., caffeine, nicotine), terpenoids (e.g., essential oils, carotenoids), and phenolics (e.g., flavonoids, lignins), having various functions, including defense against herbivores and pathogens, attracting pollinators, and adaptation to environmental stress [1]. Among them, alkaloids (20% of all secondary metabolites) are nitrogen‐containing compounds derived from amino acids and have been well studied for their numerous therapeutic potentials, such as anesthetic, cardioprotective, antioxidant, and anti‐inflammatory activities [2, 3, 4]. Tetrahydroserpentine (ThS) (also known as ajmalicine or raubasine) is one of the indole alkaloids found in Catharanthus roseus and Rauwolfia serpentina. Due to its antihypertensive effect [5, 6], an estimated 3500 kg of ThS is being isolated annually by pharmaceutical companies either from Rauwolfia or Catharanthus spp. [7]. ThS showcases its antihypertensive effect by blocking the α₁‑adrenergic receptor [6, 8, 9]. In combination with almitrine, ThS was reported to improve cognitive disorders and cerebral impairments by enhancing arterial oxygen level, cerebral oxygen and glucose uptake, mitochondrial function, redox homeostasis, and by modulating noradrenergic activity [10, 11]. The oxidized form of ThS, serpentine, has been reported to reduce reactive oxygen species (ROS) generation and to inhibit NF‐κB activation by interfering with p65 translocation to the nucleus in HABP1‐overexpressing murine fibroblast F111 cells [12]. Furthermore, ThS was reported to induce pyroptosis by inducing oxidative stress in hepatoma H22 cells [13], indicating its potential in anticancer therapy.

Apart from the hypotensive activity, α₁‐adrenergic receptor blockers are now being studied for metabolic benefits, including reductions in blood glucose and lipid levels, by improving insulin sensitivity and glucose tolerance [14]. This is particularly relevant in hyperglycemic patients, where 80% of Type 1 diabetic patients develop hypertension due to diabetic nephropathy and microalbuminuria. In contrast, Type 2 diabetic patients often develop hypertension even in the absence of kidney disease and are at a higher risk of chronic cardiovascular complications [15]. Apart from this, diabetes is associated with liver damage [16]. A major concern regarding ThS is its ability to inhibit CYP2D6, a key liver enzyme that metabolizes ∼20% of drugs, raising the possibility of significant drug interactions and systemic toxicity [17, 18, 19]. However, the toxicological profile of ThS is not well explored [20]. Only a single study reported that ThS up to 40 mg/kg body weight (BW)‐day oral doses to rats (the strain was not specified) for 24 weeks did not show any sign of intoxication [21]. Considering these facts, the main objective of this study is to investigate the effects of ThS under hyperglycemic conditions, specifically its impact on the liver and kidney, an area that has not been explored.

Taking all of these into account, the present study investigated the antioxidant potential, toxicity, and effects of ThS under hyperglycemic conditions. Antioxidant activity was evaluated in F111 murine fibroblast cells exposed to H₂O₂, while in vivo toxicity and the effects of ThS in hyperglycemic conditions were evaluated in Swiss albino mice. In addition, in silico network pharmacology approaches were employed to predict its toxicity before in vivo experimentation. This research may help to determine whether ThS should be taken (as a hypotensive agent) in hyperglycemic conditions.

2. Results

2.1. Cytotoxicity Assessment of ThS

Cytotoxicity of ThS was investigated in murine fibroblast (F111), human liver (HepG2 and WRL‐68), and human embryonic kidney (HEK293) cells. Results indicated that ThS induced a dose‐dependent cytotoxicity in all cells. Significant cell death was observed at ≥ 50 µM concentration in F111 (Figure 1A), HepG2 (Figure 1B), and HEK293 cells (Figure 1D), whereas it was observed at ≥ 250 µM in WRL‐68 cells (Figure 1C). The IC₅₀ values were found to be 741.37 µM for F111 (Figure S1A), 316.22 µM for HepG2 (Figure S1B), 338.47 µM for WRL‐68 (Figure S1C), and 299.58 µM for HEK293 (Figure S1D). As with ThS ≤ 25 µM, no cytotoxicity was observed in any of the cells. Further in vitro experiments were conducted at this concentration.

FIGURE 1.

Effect of tetrahydroserpentine on cell viability. Bar graphs display changes in cell viability after 24 h exposure to varying concentrations of ThS in (A) F111, (B) HepG2, (C) WRL‐68, and (D) HEK293 cells. VC_max = vehicle control (DMSO) concentration equal to its concentration in the treatment group having maximum selected ThS concentration (750 µM). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 versus control or between the indicated groups.

2.2. ThS Had In Vitro Antioxidant Capacity

The antioxidant potential of ThS was assessed through two methods. First, its capacity to scavenge radicals was evaluated by reacting with DPPH radicals [22]. ThS exhibited concentration‐dependent DPPH free radical scavenging, with an IC₅₀ value of 720 µM (Figure 2A). As ThS showed the least cytotoxicity in F111 and HepG2 cells, further cellular antioxidant assays were performed in these cells. In F111 cells, 50 and 100 µM H₂O₂ increased intracellular ROS levels by 2.93‐ and 3.57‐fold, respectively, which was reduced by 1.70‐ and 1.48‐fold (Figure 2B) by 25 µM ThS (2 h pretreatment). Similarly, in HepG2 cells, 50 and 100 µM H₂O₂ led to an increase in ROS by 1.40‐fold and 2.22‐fold, respectively, while ThS significantly reduced this effect by 1.44‐fold and 2.01‐fold (Figure 2C). These observations indicated that 25 µM ThS exhibited antioxidant activity against H₂O₂‐induced ROS.

FIGURE 2.

Antioxidant activity and effect of tetrahydroserpentine on H₂O₂‐induced ROS production and cell death. (A) Bar graph displays the DPPH free radical scavenging capacity of ThS across 100–1000 µM; positive control = Ascorbic acid (Asc. A). (B, C) Detection of ROS levels in (B) F111 cells and (C) HepG2 cells after 2 h of pretreatment with tetrahydroserpentine, followed by treatment with H₂O₂ for 15 min. (D–F) Cell viability in F111 cells after 2 h of pretreatment with ThS, followed by (D) 3 h, (E) 6 h, and (F) 12 h of treatment with H₂O₂. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 versus control or between the indicated groups.

2.3. ThS Reduced H₂O₂‐Induced Cell Death in F111 Cells

Since ThS exhibited antioxidant activity, its protective effect against H₂O₂‐induced cytotoxicity was further evaluated in noncancer‐origin F111 cells. No significant change in cell viability was observed across any treatment group at 3 h (Figure 2D). After 6 h, 100 µM H₂O₂ significantly reduced cell viability to 71.25%, whereas 25 µM ThS pretreatment improved viability to 94% (Figure 2E). At 12 h, H₂O₂ further decreased cell viability to 37.25%, and ThS partially restored viability to 56% (p < 0.01). Despite this partial protection, cell viability remained significantly lower (p < 0.0001) in the ThS + H₂O₂‐treated group compared to the control (Figure 2F), indicating that ThS failed to completely prevent H₂O₂‐induced cytotoxicity during prolonged exposure. Collectively, these findings suggest that although ThS exerts early antioxidant protection, its efficacy diminishes over time under sustained oxidative stress.

2.4. Effect of ThS on H₂O₂‐Induced Cell Cycle Arrest

H₂O₂ has been reported to alter the cell cycle progression [23]. Furthermore, we decided to investigate how ThS affects the H₂O₂‐induced cell cycle arrest. It was observed that H₂O₂ (100 µM) induced a G₂/M and S phase arrest. However, ThS significantly (p < 0.05) decreased the G₂/M phase arrest in H₂O₂‐treated cells but was found to increase (p < 0.05) the percentage of S phase arrest significantly (Figure 3A,B). This indicated that ThS might interfere with the transition from the S phase to the G₂/M phase of the cell cycle. N‐acetyl cysteine (NAC) 50 µM, used as a positive control, significantly reduced G₂/M arrest but did not alter H₂O₂‐induced S‐phase arrest.

FIGURE 3.

Effect of tetrahydroserpentine on cell cycle profile of F111 cells exposed to H₂O₂. (A) Flow cytometry histograms illustrate DNA content variations across cell cycle phases in different treatment groups after 12 h. (B) Statistical analysis demonstrates the significance level of the alterations in the distribution of cell cycle phase and sub‐G₀/G₁ cell populations. N‐Acetyl cysteine (NAC), 50 µM used as a positive control. Significance is denoted as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 versus control or between the indicated groups; n = 3.

2.5. Network Pharmacology Revealed the Top Target for ThS

The probable gene targets for ThS were identified using SwissTargetPrediction (swisstargetprediction.ch) and STITCH (stitch.embl.de) databases. A number of total 110 potential targets were predicted by these databases, which were, ADRA2C, ADRA2B, CYP2D10, CYP2D26, CYP2D22, ADRA2A, CYP2D12, CYP2D11, CYP2D9, CYP2D34, BCHE, OPRD1, DRD2, HTR1A, SLC6A1, NPY5R, HCRTR2, HCRTR1, PREP, PDE10A, MAPK14, ACHE, JAK3, HRH4, TNKS, SLC6A9, CTSG, JAK2, TNKS2, P2RX7, WNT3A, CTSL, NOS3, EGFR, GSK3B, GCK, MAOB, SCD1, PDE7A, SLC27A1, DYRK1A, IMPDH2, AKT1, MMP13, BDKRB1, LCK, CES1D, MCL1, FAAH, CSF1R, F2, AURKB, CDK1, MAPK9, LTA4H, MGAT2, TYMS, ADORA2A, AOC3, CDC25B, GSK3A, MDM2, OPRM1, CTSS, HIF1A, FLT1, NAT2, ADORA1, PORCN, TGFBR1, ALK, KCNA3, PIK3CA, NAMPT, HTR4, EPHX2, DHODH, STAT3, DRD1, HSD11B2, mTOR, AR, KDR, TAAR1, ALOX5, DAGLA, PARP1, ABL1, SLC5A2, CA13, MAPK11, CA7, AVPR1A, RAPGEF4, HTR7, ALPL, INSR, ALOX12, PTAFR, ADORA2B, S1PR1, TRHR, TRH‐R2, SCN10A, SHH, SMO, HCAR2, OPRK1, PPARA, TGM2. A protein–protein interaction (PPI) network between these genes was analyzed using the String database (string‐db.org) and visualized by Cytoscape software (Figure 4A). Furthermore, using the CytoHubba plug‐in, top hub proteins were identified in the Cytoscape platform, which were AKT1, STAT3, GSK3B, EGFR, mTOR, HIF1A, MAPK14, PARP1, PIK3CA, and MCL1 (Figure 4B). Furthermore, using the Metascape platform (metascape.org) association analysis was conducted using PaGenBase, TRRUST ontology (criteria‐terms with a p < 0.01, a minimum count of 3, and an enrichment factor > 1.5) and found that genes were liver tissue and mast cell‐associated (Figure 4C); and linked with several transcription factors including SP1, NF‐κB1, NR2F2, and STAT3 (Figure 4D).

FIGURE 4.

Protein–protein interaction (PPI) network and association analysis of tetrahydroserpentine targeted genes (A, B) PPI network shows interactions among (A) all potential protein targets, and (B) hub (key) protein targets of ThS. (C) Gene‐target tissue association analysis (D) ThS target genes‐transcription factor association.

2.6. Function Analysis of the ThS Targeted Genes

All the ThS targeted genes were subjected to functional analysis using the Metascape platform (metascape.org). Enrichment analysis was conducted using GO Biological Processes, KEGG Pathway, Reactome Gene Sets, CORUM, WikiPathways, and PANTHER Pathway. Terms (pathways and processes) meeting certain criteria, including a p value below 0.01, a minimum count of 3, and an enrichment factor greater than 1.5, were collected. These terms were then grouped into clusters based on their similarities in membership. To calculate p values, the cumulative hypergeometric distribution [24] was employed, while q values were determined using the Benjamini–Hochberg procedure [24] to correct for multiple testing. Hierarchical clustering of enriched terms was performed using kappa scores [24] as the similarity metric. Sub‐trees with a similarity exceeding 0.3 were considered as clusters. Within each cluster, the most statistically significant term was selected to represent the cluster. The top 20 clusters were as follows: GPCRs non‐odorant, circulatory system process, response to the hormone, positive regulation of smooth muscle cell proliferation, response to organic cyclic compound, positive regulation of phosphorylation, behavior, regulation of secretion, regulation of monoatomic ion transport, phospholipase C‐activating G protein‐coupled receptor (GPCR) signaling pathway, regulation of growth, Serotonergic synapse—Mus musculus, regulation of muscle system process, Pathways in cancer—M. musculus, regulation of blood pressure, PD‐L1 expression and PD‐1 checkpoint pathway in cancer—M. musculus, rhythmic process, regulation of body fluid levels, proteoglycans in cancer—M. musculus, multicellular organismal‐level homeostasis. Figure 5A describes the p values along with all of the terms. In addition, a subset of enriched terms (referring to pathways and processes) was meticulously chosen based on specific criteria to enhance our understanding of the relationships between pathways and processes. The best p values were chosen from each of the 20 clusters, ensuring that no cluster contained more than 15 terms and the total number of terms did not surpass 250. Thereafter, these terms were rendered as a network plot where terms with a similarity exceeding 0.3 were connected by edges (Figure 5B).

FIGURE 5.

Analysis of the functionality of tetrahydroserpentine targeted genes using GO biological processes, KEGG pathways, Reactome gene sets, CORUM, WikiPathways, and PANTHER pathways via the Metascape platform. (A) Bar graph illustrates the top 20 clusters of enriched pathways and processes among the genes. (B) Network analysis of the top biological processes and pathways in each of the clusters mentioned in Figure A, with nodes colored by cluster ID, indicating nodes with the same cluster ID are usually in close proximity.

2.7. Subacute Administration of ThS Altered BW and Daily Food and Water Uptake in Mice

Daily BW and food, and water uptake were monitored throughout the experiment. Results revealed an increase in BW percentage (relative to Day 0) in a dose‐dependent manner. Notably, the maximum BW percentage increase was recorded with the 10 mg/kg BW‐day dose (Figure 6B and Figure S1E). In terms of food uptake, the group receiving a 2.5 mg/kg BW‐day dose exhibited an increase in their daily food intake. Conversely, the groups administered with 5.0 and 10.0 mg/kg BW‐day showed a decrease in their daily food consumption (Figure 6C and Figure S1F). Simultaneously, an elevated daily water intake was observed in all ThS doses, with a maximum in the 2.5 mg/kg BW‐day‐dosed group compared to those mice receiving 5.0 and 10.0 mg/kg BW‐day ThS (Figure 6D and Figure S1G). These findings underscore that ThS affected the food and water uptake patterns in mice.

FIGURE 6.

Toxicity assessment of tetrahydroserpentine in mice. (A) The schematic diagram shows the plan of the ThS administration and selected parameters for toxicity assessment. (B) The bar graph shows the change in body weight at the end of the treatment period. (C, D) Line graphs illustrate day‐wise variation in (C) food, and (D) water intake after treatment, relative to the control group (control group's body weight normalized to zero for each day). (E) The bar graph shows variation in fasting blood glucose levels after 14 days of treatment (F). The bar graph shows blood glucose levels in different groups subjected to an oral glucose tolerance test (G). Change in liver and kidney weight, relative to body weight, in control and treated groups. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 versus control or between the indicated groups; n = 5.

2.8. Higher Dose of ThS Altered Fasting Blood Glucose in Mice

After 14 days of ThS treatment, a significantly elevated blood glucose level was observed in the mice receiving 10.0 mg/kg BW‐day ThS (Figure 6E). Furthermore, the oral glucose tolerance test (OGTT) revealed that blood glucose levels at this dose were significantly elevated at both 1 and 2 h post‐glucose administration, indicating slower glucose metabolism (Figure 6F).

2.9. Higher Dose of ThS Decreased Liver and Kidney Weight

At 10 mg/kg BW‐day ThS, a significant decrease in the relative liver and kidney weight (%) was observed (Figure 6G). Conversely, no significant changes were observed in the relative spleen, pancreas, and testis weight across any of the administered doses (Figure S1H).

2.10. Hematological Analysis Indicated Blood Parameter Alteration at a Higher ThS Dose

Notable alterations in serum parameters were observed in ThS‐dosed mice compared to the control mice. Specifically, there was a significant decrease in total leukocyte count observed in the 2.5 mg/kg BW‐day group but a significant increase in 10.0 mg/kg BW‐day‐dosed mice. Neutrophil counts, including absolute neutrophil numbers, were significantly elevated in the 5.0 and 10.0 mg/kg BW‐day groups, while lymphocyte percentages were significantly reduced at these doses. A significant decrease in absolute lymphocyte count was also observed in the 2.5 mg/kg BW‐day group. Furthermore, hematocrit % was found notably higher (with a p < 0.05) in the 10.0 mg/kg BW‐day group (Table 1).

TABLE 1.

Hematological parameters of control and tetrahydroserpentine (2.5–10.0 mg/kg) orally administered mice.

Hematological parameters	Control	2.5 mg/kg BW‐day	5.0 mg/kg BW‐day	10.0 mg/kg BW‐day
Hemoglobin (g/dL)	14.67 ± 0.78	15.4 ± 0.14	15.23 ± .0.38	16.13 ± 0.67
Total leucocyte count (cumm)	5300 ± 600	4600 ± 424.26*	5500 ± 435.89	6300 ± 1374.77***
Neutrophil (%)	11 ± 1	20 ± 7.07	29 ± 6.56**	31.67 ± 2.89***
Lymphocytes (%)	68.33 ± .3.06	63.00 ± 9.89	58.67 ± 3.21*	53.33 ± 2.89**
Eosinophil (%)	9.33 ± 0.58	8 ± 8.48	5.0 ± 3.0	6.33 ± 3.21
Monocytes (%)	11.33 ± 2.31	9 ± 8.48	7.33 ± 4.62	8.67 ± 2.31
Basophils (%)	0	0	0	0
RBC (millions/cumm)	9.07 ± 0.53	9.54 ± 0.13	9.5 ± 0.40	10.11 ± 0.36
Haematocrit (%)	48.4 ± 2.87	53.5 ± 2.12	53.23 ± 1.24	54.9 ± 2.34*
Mean corpuscular volume (fL)	53.35 ± 0.96	57.07 ± 3.01	56.10 ± 2.80	54.32 ± 2.07
Mean corpuscular haemoglobin (pg)	16.17 ± 0.43	16.13 ± 0.77	16.04 ± 0.33	15.96 ± 0.55
Mean corpuscular haemoglobin concentration (g/dL)	30.31 ± 0.35	28.81 ± 1.41	28.63 ± 1.17	29.39 ± 0.13
Platelet count (103/cumm)	848 ± 424.43	934.5 ± 27.58	1131.67 ± 89.54	729.33 ± 394.51
Absolute neutrophil/cumm	581 ± 60.00	905 ± 240.42	1581.33 ± 283.52***	2020 ± 629.52****
Absolute lymphocyte/cumm	3633.67 ± 567.36	2877 ± 188.09*	3320 ± 186.82	3380 ± 872.75
Absolute eosinophil/cumm	492.67 ± 33.38	388 ± 424.26	282 ± 178.13	374 ± 117.93
Absolute monocyte/cumm	592.67 ± 64.04	432 ± 428.51	416.67 ± 274.29	526 ± 67.44

Note: Statistical significance was assessed utilizing a two‐way analysis of variance (ANOVA) coupled with Tukey's multiple comparison test.

*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared to control group (n = 5 mice per group).

2.11. Serum Biochemical Analysis Revealed an Alteration in Liver and Kidney Parameters at Higher ThS Dose

Serum biochemical analysis of liver function parameters revealed increased total bilirubin, direct bilirubin, serum glutamic oxaloacetic transaminase (SGOT), alkaline phosphatase (ALP), total protein, albumin, and globulin levels in 5.0 and 10.0 mg/kg BW‐day ThS‐dosed groups. In addition, these two groups exhibited a significant increase in urea, blood urea nitrogen (BUN), and the BUN:creatinine ratio. Moreover, a significantly higher uric acid level was noted in mice treated with 10.0 mg/kg BW‐day ThS (Table 2). These observations indicated liver and kidney impaired liver and kidney function [25]. Conversely, in the 2.5 mg/kg BW‐day ThS group, only the BUN level was found to be significantly higher, with no significant alteration observed in the BUN:creatinine ratio.

TABLE 2.

Serum parameters of control and tetrahydroserpentine (2.5–10.0 mg/kg) orally administered mice.

Serum parameters	Control	2.5 mg/kg BW‐day	5.0 mg/kg BW‐day	10 mg/kg BW‐day
Bilirubin total (mg/dL)	1.07 ± 0.32	1.20 ± 0.20	5.30 ± 0.70**	18.0 ± 1.85***
Bilirubin direct (mg/dL)	0.8 ± 0.36	0.70 ± 0.40	5.00 ± 0.70**	16.90 ± 2.26***
Bilirubin indirect (mg/dL)	0.27 ± 0.07	0.5 ± 0.20	0.3	1.10 ± 0.50
SGOT (U/L)	147.33 ± 14.66	192.0 ± 15.00	248.0 ± 36.0***	362.00 ± 46.36.00****
SGPT (U/L)	57.67 ± 12.33	57	64.5 ± 7.50	69.67 ± 5.80
SGOT: SGPT	2.72 ± 0.50	3.37 ± 0.80	3.83 ± 0.11	5.16 ± 0.24*
ALP (U/L)	74.33 ± 4.26	56.00 ± 11.00	26.5 ± 6.50**	41.0 ± 0.60**
Total protein (g/dL)	5.2 ± 0.20	5.05 ± 0.15	7.65 ± 0.05****	11.00****
Albumin (g/dL)	2.63 ± 0.19	2.55 ± 0.05	4.30 ± 0.20***	6.00****
Globulin (g/dL)	2.57 ± 0.03	2.5 ± 0.10	3.35 ± 0.15***	5.00****
Albumin: Globulin	1.02 ± 0.07	1.02 ± 0.02	1.29 ± 0.12	1.2
Urea (mg/dL)	33.17 ± 5.35	48.85 ± 13.91	67.41 ± 7.50**	56.35 ± 2.60**
Creatinine (mg/dL)	0.16 ± 0.02	0.25 ± 0.05	0.2	0.20 ± 0.01
Uric acid (mg/dL)	3.4 ± 0.17	4.75 ± 0.75	4.9	6.10 ± 0.70*
BUN (mg/dL)	15.5 ± 2.50	27.5 ± 6.50**	31.50 ± 3.50**	26.33 ± 1.20**
BUN: Creatinine	85.44 ± 10.44	120.0 ± 30.0	157.50 ± 17.50***	127.42 ± 4.33**

*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared to control group (n = 5 mice per group).

2.12. Histomorphology Examination of Liver and Kidney

Hematoxylin and eosin (H&E) staining of liver sections revealed no significant alteration of morphology in the 2.5 mg/kg BW‐day ThS‐dosed group. However, in the 5.0 mg/kg BW‐day group, a widened sinusoid along with diffuse microvascular degeneration of hepatocytes (yellow arrow) [26] was observed (Figure 7A). In the 10.0 mg/kg BW‐day ThS‐dosed group, degenerated hepatocytes and cytoplasmic vacuolar degeneration (red arrow) were visualized [27, 28]. In terms of kidney morphology, at 2.5 mg/kg BW‐day dose, no significant alterations were observed (Figure 7B). But, at 5.0 mg/kg BW‐day ThS dose, tubular vacuolization (as described by Celikel et al. [29]), acute tubular lesions with loss of brush border (yellow arrow, as described by Vidal et al. [30]), enlarged Bowman's capsules (orange arrow) with degenerated glomeruli (red arrow), and widened urinary space (green arrow) were visualized. In 10.0 mg/kg BW‐day ThS‐dosed mice, degenerated glomeruli (red arrow), degenerated tubular regions (purple arrow, as described by Al‐Sadoon et al. [31]), isometric vacuolization [32] with widened urinary space (green arrow) were observed.

FIGURE 7.

Histological analysis of mice liver and kidney treated with tetrahydroserpentine. (A, B) Images of hematoxylin and eosin stained mice tissue section showing histomorphological characteristics of control and ThS‐treated groups: (A) Liver: CV = central vein, PV = portal vein, BD = bile duct, HA = hepatic artery, S = sinusoid, H = hepatocytes, black arrow = normal morphology, yellow arrow = microvascular degeneration of hepatocytes, green arrow = widened sinusoids, red arrow = degenerated hepatocytes, orange arrow = indistinctive sinusoids; and (B) Kidney: BC = Bowman's capsule, G = glomerulus, PT = proximal convoluted tubule, DT = distal convoluted tubule, PC = peritubular capillary. Black arrow = normal morphology, yellow arrow = severe tubular vacuolation plus tubular brush border damage, red arrow = degenerated glomeruli, orange arrow = enlarged Bowman's capsule, green arrow = widened peritubular capillaries (urinary space), progressive loss of tubular brush (purple arrow).

2.13. Effect of ThS on the Blood Sugar Level and BW in Hyperglycemic Mice

Subacute toxicity assessment revealed that ThS > 2.5 mg/kg BW‐day dose, caused liver and kidney damage. Therefore, considering 2.5 mg/kg BW‐day as a safer dose, we investigated its effect on streptozotocin (STZ)‐induced hyperglycemic mice. The results showed that ThS did not reduce blood glucose in hyperglycemic mice (Figure 8B). Furthermore, ThS did not prevent the decrease in BW observed in STZ‐induced hyperglycemic mice (Figure 8C). These findings demonstrate that 2.5 mg/kg BW‐day of ThS failed to ameliorate both hyperglycemia and associated weight loss in STZ‐induced hyperglycemic mice.

FIGURE 8.

Effect of tetrahydroserpentine on body weight, blood sugar, and liver and kidney function in hyperglycemic mice. (A) The schematic diagram shows the plan of STZ and ThS administration and selected parameters for toxicity assessment. (B) The bar graph shows changes in whole blood glucose after 14 days of treatment. (C) The line graph shows the variation in body weight after STZ and/or ThS treatment. (D–H) Bar graphs show changes in the levels of markers of liver and kidney function: (D) SGOT, (E) SGPT, (F) SGOT to SGPT ratio, (G) ALP, and (H) urea. STZ: streptozotocin, ip: intraperitoneal, ThS: thyro serpentine, HyG: hyperglycemia, SGOT: serum glutamic oxaloacetic transaminase, SGPT: serum glutamic pyruvic transaminase, ALP: alkaline phosphatase. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 versus control or between the indicated groups; n = 5.

2.14. Effect of ThS on Liver and Kidney Function Parameters in Hyperglycemic Conditions

Liver and kidney function parameters were assessed through serum biochemical analysis. Hyperglycemic mice exhibited significantly elevated levels of serum glucose, SGOT, SGPT, ALP, and urea (Table 3). Furthermore, ThS was found to exacerbate SGOT (Figure 8D) and urea levels (Figure 8H) in hyperglycemic mice. Although it notably decreased SGPT levels (Figure 8E) in hyperglycemic mice, the SGOT:SGPT ratio was considerably higher than that of the hyperglycemic mice (Figure 8F), indicating increased liver damage. However, in hyperglycemic mice, the lack of a significant increase in the SGOT: SGPT ratio might be attributed to the elevation of both enzymes simultaneously. ThS appeared to significantly decrease ALP levels (p < 0.05), although they remained substantially higher compared to the control (p < 0.05) (Figure 8G). Therefore, ThS at 2.5 mg/kg BW‐day dose had no beneficial effects on STZ‐induced hyperglycemic mice, but rather worsened liver and kidney parameters.

TABLE 3.

Serum biochemical analysis comparing the impact of tetrahydroserpentine (2.5 mg/kg BW‐day) on hyperglycemic (STZ‐induced) mice.

Serum biochemical parameters	Control	ThS 2.5 mg/kg BW‐day	Hyperglycemic mice	Hyperglycemic + ThS
Fasting serum glucose (mg/dL)	130.3 ± 15.50	165.5 ± 30.1	477 ± 35.5****	413.5 ± 40.5****
Bilirubin total (mg/dL)	0.70	0.85 ± 0.07	0.85 ± 0.07	0.70
Bilirubin direct (mg/dL)	0.30	0.30	0.25 ± 0.07	0.25 ± 0.71
Bilirubin indirect (mg/dL)	0.40	0.55 ± 0.07	0.60	0.45 ± 0.07
SGOT (U/L)	143.5 ± 15.5	162.8 ± 40.3	270.5 ± 20.4****	324.0 ± 25.4****
SGPT (U/L)	55.0 ± 7.5	50 ± 9.55	94.5 ± 5.5*	50 ± 10.25
SGOT: SGPT	2.6 ± 0.45	3.25 ± 0.50	2.86 ± 0.85	6.48 ± 1.2***
ALP (U/L)	87.0 ± 5.5	81.0 ± 10.3	156 ± 15.25***	119.0 ± 12.85*
Total protein (g/dL)	5.6 ± 0.28	5.5 ± 0.30	5.2 ± 0.29	4.85 ± 0.70
Albumin (g/dL)	3.5 ± 0.85	3.0 ± 0.28	2.9 ± 0.28	2.45 ± 0.07
Globulin (g/dL)	2.1 ± 0.56	2.5	2.3	2.4
Albumin: Globulin	1.8 ± 0.88	1.2 ± 0.011	1.26 ± 0.13	1.02 ± 0.03
Urea (mg/dL)	28.8 ± 2.5	34.2 ± 0.42	38.52 ± 3.32*	44.944 ± 0.22**
Creatinine (mg/dL)	0.15 ± 0.04	0.14 ± 0.01	0.14 ± 0.02	0.18
Uric acid (mg/dL)	4.4 ± 1.77	3.7 ± 0.14	3.6	3.3 ± 0.57
BUN (mg/dL)	15.5 ± 3.53	16.0 ± 2.33	18.0	20 ± 1.41

Note: Statistical significance levels are denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, with comparisons made against the control group (n = 5 mice per group).

2.15. Effect of ThS on Liver and Kidney Morphology of Hyperglycemic Mice

Histomorphological analysis using H&E staining clearly revealed well‐structured hepatocytes, bile ducts, hepatic arteries, and sinusoids in the control group and mice treated only with ThS (2.5 mg/kg BW‐day) (Figure 9A,B). Conversely, degenerated hepatocytes were observed in the liver of both hyperglycemic and ThS‐treated hyperglycemic mice (red arrow, Figure 9C,D). This indicated that ThS did not improve liver damage in the hyperglycemic mice. H&E analysis of kidney sections showed normal kidney histology in the control group and mice administered with ThS (2.5 mg/kg BW‐day) (Figure 10A,B). On the contrary, hyperglycemic mice displayed tubular vacuolation and damaged brush borders in the tubular areas, degenerated glomeruli in enlarged Bowman's capsules, and complete abolition of glomeruli (Figure 10C). ThS‐treated hyperglycemic mice also displayed degenerated glomeruli, tubular vacuolation in both distal and proximal convoluted regions, degenerated brush borders, and widened peritubular capillaries (Figure 10D). This observation suggests that ThS failed to ameliorate kidney damage in hyperglycemic mice.

FIGURE 9.

Effect of tetrahydroserpentine on hyperglycemic mice liver. (A–D) Images of hematoxylin and eosin‐stained liver sections show histomorphological characteristics of control and ThS‐treated groups. CV = central vein, PV = portal vein, BD = bile duct, HA = hepatic artery, S = sinusoid, H = Hepatocytes. Black arrow = normal morphology, red arrow = degenerated hepatocytes. Image magnification 40×, scale bar = 20 µm.

FIGURE 10.

Effect of tetrahydroserpentine on hyperglycemic mice kidney. (A–D) Images of hematoxylin and eosin‐stained kidney sections show histomorphological features of control and ThS‐treated groups. BC Bowman's capsule, G = glomerulus, PT = proximal convoluted tubule, DT = distal convoluted tubule, PC = peritubular capillary. Black arrow = normal and well‐structured morphology, yellow arrow = significant tubular vacuolation, red arrow = degenerated glomeruli, orange arrow = enlarged Bowman's capsule, blue arrow = glomerular abolition, and green arrow = widened peritubular capillaries.

3. Discussion

High blood pressure (hypertension)is linked to a higher chance of death from 17 out of 67 causes, mostly related to heart attacks and strokes [33]. It is closely associated with hyperglycemia, one of the world's most prevalent chronic diseases. According to the WHO, almost 830 million people worldwide are living with diabetes. Diabetes can cause hypertension either through diabetic nephropathy, which impairs kidney function and leads to sodium and fluid retention [34], or by damaging arteries, causing them to narrow and stiffen [35]. Worldwide mortality due to diabetes is associated with the combined effects of both hyperglycemia and hypertension, leading to cardiovascular failure [36]. ThS, an indole alkaloid, is well known for its pharmaceutical benefits for high blood pressure and has been well studied for its antihypertensive effect [5, 37]. Previous studies reported that almitrine–ThS combination treatment raised arterial oxygen partial pressure and hemoglobin oxygen saturation and thus increased oxygen content in arterial blood [10]. This combination is also being used for improving mental condition in elderly people [38]. The reported LD₅₀ (acute) toxicity dose of ThS was 20 mg/kg (iv) and 400–450 mg/kg (oral) in mice, and 750 mg/kg (oral) and 20–26 mg/kg (iv) in rats [21]. While these studies primarily focused on acute toxicity, there is limited information on chronic toxicity. In a single study, it was reported that ThS showed no evidence of chronic toxicity up to 40 mg/kg over a 24‐week period in mice and rats (which strain not specified in either case) [21].

ThS was also reported to strongly inhibit CYP2D6 at a very low concentration of 3.30 nM [17]. CYP2D6 comes under the large family of CYP P450 enzymes responsible for metabolizing both exogenous compounds (such as drugs, toxins, and carcinogens) and endogenous compounds (including hormones and steroids). CYP2D6 metabolizes around 20% of commonly used drugs and is primarily found in the liver but also highly expressed in the central nervous system [18, 39]. Therefore, inhibition of any CYP450 enzymes (including CYP2D6) can result in increased plasma concentrations of the affected drugs, harmful intermediates, and endogenous metabolites, potentially leading to higher systemic exposure, impaired detoxification, and prolonged effect [19, 41]. This indicates that, despite having therapeutic benefits, ThS may have systemic toxicity in the body. Especially under hyperglycemic conditions, where liver and kidney functions may already be compromised, the use of ThS to control hypertension could be detrimental. Considering these facts, the present study aimed to investigate the antioxidant, toxicity, and the potential effects of ThS on hyperglycemic conditions.

At first, the vitro cytosafety assessment of ThS was performed in various mammalian cell types, including F111, HepG2, WRL‐68, and HEK293. ThS beyond 25 µM concentration showed cytotoxicity in all cell types, though at 25 µM ThS showed antioxidant activity against H₂O₂‐induced intracellular ROS generation. However, its antioxidant activity diminished over time, as 25 µM ThS failed to prevent H₂O₂ cell death at the later (12 h) time point (Figure 2D–F). H₂O₂ was reported to induce cell death by inducing oxidative stress [40, 41] and cell cycle arrest at G₀/G₁ [42] or G₂/M [43], or even multifaceted cell cycle arrest in G₀/G₁, G₂/M and S phase simultaneously [23]. Here, ThS was found to marginally reduce this H₂O₂‐induced G₂/M phase arrest but significantly intensified the H₂O₂‐induced S phase arrest. Therefore, the results suggested that ThS is not a strong antioxidant. Considering its cytotoxicity across both cancer and noncancerous cell types, its toxicity was further analyzed in depth using in silico and in vivo approaches.

From the network pharmacology analysis, it was found that ThS could regulate a total of 110 genes, and among them, AKT1, STAT3, GSK3B, EGFR, mTOR, HIF1A, MAPK14, PARP1, PIK3CA, and MCL1 were the hub genes. This suggests that ThS can influence biological pathways linked to these genes. Specifically, Akt1 supports neuronal survival [44], GSK3B regulates insulin signaling and glycogen metabolism while promoting inflammation via AMPK inhibition [46, 47], and EGFR drives cell growth and proliferation [46]. Hence, it can be inferred that ThS possesses the ability to regulate a broad spectrum of pathways, spanning from cell proliferation to immunity. To gain further insight into the potential outcomes associated with ThS, we conducted a functional analysis and the results indicated that ThS could regulate several pathways, including non‐odorant GPCRs, circulatory system processes, response to hormone, and positive regulation of phosphorylation. Non‐odorant GPCRs regulate diverse physiological functions, including hemostasis, metabolism, and neurotransmission [47], and their dysregulation is associated with various diseases such as liver disease, hepatic steatosis, chronic kidney disease, diabetic nephropathy, lupus nephritis, and liver and renal cell carcinomas. As GPCRs were identified as the most highly regulated pathway by ThS according to functional analysis, close clinical monitoring of ThS's effects is required. ThS was predicted to positively regulate phosphorylation, whose dysregulation is often implicated in a range of chronic diseases, including cancer [49], steatotic liver disease [50], and chronic kidney disease [51]. Also, it was found that ThS could impact behavior and serotonergic synapse that plays an overarching role in various physiological functions, including appetite, body temperature, learning and memory, cognitive control, motor activity, and motor function [52]. Furthermore, association analysis indicated that the primary target tissue for ThS is the liver.

Following this, we conducted in vivo toxicity assessments in Swiss albino male mice, a strain widely used for toxicological evaluation [53, 54, 55], to determine a safe dose for hyperglycemic mice. The recommended adult daily dose of ThS is 20 mg/24 h [37, 56], which corresponds to 4.02 mg/kg BW‐day in mice, calculated using the standard body surface area normalization method [57]. Based on this, in this study, mice were administered with three ThS doses: 2.5, 5.0, and 10.0 mg/kg BW‐day for 14 consecutive days, covering both lower and higher ranges relative to the mice‐equivalent human recommended dose. It was observed that 10.0 mg/kg BW‐day ThS caused a notable increase in mice's BW and fasting blood glucose, and a reduction in the liver and kidney weight. A decrease in the liver and kidney weight indicated significant alterations in liver and kidney function [58]. Variations in food and water intake were also observed following ThS administration. A slight increase in food intake occurred at 2.5 mg/kg BW‐day, whereas reduced intake was noted at 5.0 and 10.0 mg/kg BW‐day. In contrast, all treated groups showed consistently higher water consumption. These behavioral alterations may be associated with the predicted interaction of ThS with the serotonergic system, as indicated by our in silico analysis. ThS (ajmalicine) has been reported to interact with serotonergic receptors, including 5‐HT₁A [59], although its specific functional role at these receptors remains to be clarified. In addition to these behavioral changes, a notable increase in total leukocyte (WBC) count at 10.0 mg/kg BW‐day, together with elevated neutrophil and lymphocyte levels in the 5.0 and 10.0 mg/kg BW‐day groups, indicated a treatment‐related activation of immune or inflammatory responses.

The elevated levels of bilirubin, SGOT, and the SGOT/SGPT ratio in mice treated with 5.0 and 10.0 mg/kg BW‐day suggest ThS‐induced hepatic injury [60]. In contrast, ALP levels were significantly reduced in these groups, indicating altered liver function at higher doses. Although an increase in ALP activity is commonly associated with hepatic dysfunction [60], decreased ALP activity has also been reported in chronic liver disease [61]. The underlying mechanisms remain unclear, but may involve severe malnutrition [61] or secondary drug interactions [62]. Moreover, a reduction in ALP may further aggravate liver damage, as ALP contributes to endotoxin inactivation and hepato‐protection [61]. Further histopathological evaluation of the liver in the 5.0 mg/kg BW‐day group revealed diffuse microvesicular degeneration of hepatocytes and sinusoidal enlargement, indicative of hepatocellular injury and impaired systemic or portal venous flow that may drive chronic liver damage [26, 63]. These structural disruptions reflect early pathological alterations that could progress with continued exposure. Consistently, the more extensive hepatocyte degeneration and indistinct sinusoidal architecture observed in the 10.0 mg/kg BW‐day group indicate a greater severity of injury at higher doses, supporting a dose‐dependent hepatotoxic response to ThS.

In terms of kidney function, the increases in serum urea, BUN, and the BUN/creatinine ratio observed in the 5.0 and 10.0 mg/kg BW‐day groups suggested a decline in renal filtration efficiency and the onset of treatment‐related renal stress. Elevated serum urea is widely recognized as a biomarker of chronic kidney dysfunction [64], with reduced levels reflecting improved pathological status [65]. Similarly, increased BUN is associated with chronic kidney disease progression [66], while a higher BUN/creatinine ratio has been linked to acute tubular injury and impaired renal function [67]. Kidney histopathology further confirmed loss of the tubular brush border and enlarged Bowman's capsules, accompanied by glomerular degeneration in the 5.0 mg/kg BW‐day group, indicating early renal injury [30, 68, 69]. Enlargement of Bowman's capsules is linked to altered filtration dynamics in kidney stress conditions, including obesity‐related nephropathy [70, 71]. In the 10.0 mg/kg BW‐day group, more pronounced tubular degeneration, isometric vacuolization, glomerular damage, and widened peritubular capillaries suggested aggravated renal pathology consistent with acute injury [31, 32, 68, 69]. Collectively, these observations indicate a dose‐dependent hepatotoxic and nephrotoxic effect of ThS at higher doses, whereas 2.5 mg/kg BW‐day appears comparatively safer. Therefore, in this study effect of ThS on hyperglycemic mice was evaluated at a dose of 2.5 mg/kg BW‐day. However, ThS did not improve hyperglycemia‐related conditions, including elevated blood glucose or loss of BW. Metformin (250 mg/kg BW‐day), used as a reference hypoglycaemic agent, also showed no noticeable improvement in glycemic status in the hyperglycemic mice (data not shown), aligning with previous observations by Oliveira [72]. Analysis of liver and kidney function markers revealed elevated serum glucose, along with significantly increased SGOT, SGPT, ALP, and urea levels in hyperglycemic mice, confirming the onset of hepatic and renal impairment, consistent with previous reports [60, 64]. SGOT:SGPT ratio did not rise markedly due to the concurrent elevation of both enzymes; however, overall biochemical alterations clearly indicated liver dysfunction. Treatment with ThS failed to mitigate these changes. Instead, ThS further increased serum SGOT and urea levels in hyperglycemic mice, suggesting an exacerbation of liver and kidney injury. Despite a reduction in SGPT levels following ThS administration, the SGOT:SGPT ratio remained elevated relative to hyperglycemic controls, signifying no functional improvement. These biochemical findings were corroborated by histomorphological evaluation of liver and kidney tissues, which demonstrated persistent structural damage without evidence of protection or recovery in ThS‐treated hyperglycemic mice. Overall, ThS did not show any protective effect; instead, it appeared to further exacerbate hyperglycemia‐induced liver and kidney dysfunction, as evidenced by additional elevations in SGOT and serum urea levels and the lack of histological recovery. The lack of any beneficial effect of ThS and the appearance of dose‐dependent organ toxicity indicate that ThS offers no therapeutic benefit under hyperglycemic conditions and may, in fact, worsen hepatic and renal outcomes. Therefore, its use under such conditions should be approached with caution, and further mechanistic and safety evaluations are essential.

In summary, this study offers a comprehensive evaluation of ThS by assessing its antioxidant and toxicological profiles in both normal and hyperglycemic conditions using in silico in vitro and in vivo approaches. Recognizing its potential to interfere with the CYP2D6 liver enzyme can be a concern about hepatotoxicity under hyperglycemic stress. While ThS showed moderate in vitro antioxidant activity, the protective effects were limited during prolonged exposure. Although ThS interacted with a broad network of molecular targets, including those linked to metabolism, inflammation, and cell survival, this did not translate into meaningful therapeutic benefit in hyperglycemic mice. Significantly, ThS exhibited dose‐dependent toxicological effects; at higher concentrations, it adversely altered liver and kidney function markers and induced histological damage in these organs. In hyperglycemic mice, ThS failed to improve metabolic dysfunction or organ damage and, in fact, exacerbated elevated liver enzyme levels. These findings highlight ThS as a bioactive compound but one with a narrow safety margin and limited suitability for therapeutic use in chronic metabolic disorders such as diabetes. Further mechanistic investigations and chemical optimizations are therefore essential before ThS can be considered for broader pharmacological applications.

4. Experimental Section

4.1. Chemicals

DMEM (Sigma‐Aldrich‐D7777, D5030), fetal bovine serum (FBS) (Gibco‐10270106), streptomycin–penicillin (Thermo Fisher Scientific, 15140122), 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (SRL chemicals‐33611), 2′,7′‐dichlorofluorescin diacetate (Sigma‐Aldrich‐D6883), DPPH (SRL‐29128), propidium iodide (PI) (Sigma‐Aldrich‐P4170), dimethyl sulfoxide (SRL‐66120), H₂O₂ (Merck 107209, STZ (Sigma‐Aldrich S0130), ThS or ajmalicine (Sigma‐Aldrich R751618, CID 441975), carboxy methyl cellulose (CMC) (Sigma‐Aldrich C5678), mice feed (Golden Feeds Ltd., India), sucrose solution (Sigma‐Aldrich S5016), formalin (SRL chemicals 15776), hematoxylin (SRL chemicals 28394), and eosin (SRL chemcials 56605)

4.2. Cell Culture Maintenance and Treatment

Mammalian cells were cultured in a humidified incubator at 37°C under a CO₂ (5%) atmosphere according to the standard protocol [73]. Murine fibroblast F111 (RRID: CVCL_6C52) cells were kindly provided by Kasturi Datta, DBT Distinguished Biotechnology Professor at Jawaharlal Nehru University, New Delhi, India. Human hepatocellular carcinoma HepG2 (RRID: CVCL_0027), Human embryonic kidney HEK293 (RRID: CVCL_0045), and Human embryonic liver WRL‐68 (RRID: CVCL_0581) cell lines were obtained from the National Centre for Cell Science, Pune, India. Murine fibroblast F111 cells were cultured in low‐glucose DMEM, while HepG2 hepatocellular carcinoma cells and human hepatic cells were maintained in high‐glucose DMEM. All media were supplemented with 10% (v/v) FBS, 100 U/mL penicillin, and 10 µg/mL streptomycin. Cells were grown up to 80% confluency and then trypsinized with trypsin‐EDTA (trypsin 0.25% + EDTA 0.50 mM in phosphate buffer saline [PBS]) solution and subcultured. All experiments involved treatment at 50%–60% cell confluency. ThS stock solution (prepared in DMSO) was freshly diluted in serum‐free DMEM (SFM) for treatments, and all exposures were performed in SFM with a final DMSO concentration maintained at < 0.03%. In experiments assessing the effect of ThS against H₂O₂‐induced cellular stress, cells were pretreated with ThS for 2 h before H₂O₂ exposure. In all experiments, cells were washed with PBS, pH 7.2, before and after treatment and dye addition.

4.3. Maintenance of Animals

Animals were maintained in standard conditions in CLAR‐JNU (Central Laboratory Animal Resources‐Jawaharlal Nehru University). Swiss albino male mice were provided by CLAR‐JNU (Ethical clearance certificate no‐27/2019). Mice were maintained at 23°C–25°C, 50% humidity, and a 12‐h light/dark cycle. Standard feed (purchased from Golden Feeds Ltd.) was provided to the mice. Mice were acclimatized for 14 days before the experiment and were maintained under identical temperature and humidity conditions, housed in the same room and at the same rack height. The number of animals per group (n = 5; total N = 20 across four groups [74]) was determined using the resource‐equation method [75] (calculated DF = 16, which falls within the acceptable range of 10–20).

4.4. Cell Viability Assay

For cell viability assessment, 10 × 10³ cells per well were grown in a 96‐well plate for 24 h, followed by treatment with ThS and/or H₂O₂ in serum‐free DMEM media (SFM). After completion of treatment, cell viability was measured with MTT dye as per the protocol mentioned by Roy et al. [76].

4.5. Intracellular ROS Measurement

Intracellular ROS was measured with H₂DCFDA fluorescent dye by fluorimetry. H₂O₂ was used to induce intracellular ROS in the cells, a reliable and widely used method for triggering oxidative stress in vitro [77, 78]. Briefly, cells at ∼60% confluency were pretreated with ThS and then incubated with 100 µM H₂O₂ for 10 min. Thereafter, cells were washed twice with 1X PBS (room temperature) and incubated with 10 µM H₂DCFDA dye (in SFM) for half an hour. Following, intracellular ROS was measured by fluorometry at λ _ex = 488 nm, λ _em = 500–550 [76] using a Spectro‐fluorophotometer (RF‐5301PC, Shimadzu). Maximum fluorescence intensities (arbitrary units) were recorded for each sample for further statistical calculation. Fold changes in ROS levels, both increases and reductions, were calculated using the previously described formula [79].

4.6. Cell Cycle Analysis

Cell cycle analysis was done to investigate the alterations in cell cycle progression due to the treatments. At 50% confluency, cells were treated with ThS and/or H₂O₂ for 12 h. After treatment, cells were harvested and fixed in 70% (v/v) pre‐chilled ethanol to prepare a single‐cell suspension. After fixation, the ethanol solution was discarded and washed twice with precooled 1X PBS buffer. Cells were stained with PI staining buffer containing 50 µg/mL of PI and 40 µg/mL RNAse and incubated in a 37°C water bath for 30 min. Then, PI fluorescence intensity of a minimum of 1 × 10⁴ cells was acquired with a BD FACSVerse flow cytometer. ModFit LT 5.0 software (VSH, USA) was used to analyze the distributions of cells at different cell cycle stages [80].

4.7. In Silico Analysis

Network pharmacology was utilized to predict the toxicology of ThS. For this, gene targets of ThS were identified through SwissTargetPrediction (http://swisstargetprediction.ch/) and STITCH (http://stitch.embl.de/) database [81]. While selecting the target genes, Homo sapiens was chosen as the target organism, and the probability score was set at 0.30. Genes with a probability score of less than 0.30 were excluded from the analysis. A PPI network was constructed using the String 12.0 (https://string‐db.org/) platform, with a threshold score > 0.7 and H. sapiens as the model organism. After that, this was visualized in Cytoscape_v3.10.1, and the key targeted pathways were identified using the CytoHubba v0.1 plugin [82]. Furthermore, the ThS targeted genes were annotated and analyzed using the Metascape tool for functional enrichment analysis [83].

4.8. Subacute Oral Toxicity of ThS

For 15 days, subacute oral toxicity [84], three different doses of ThS 2.5, 5.0, and 10 mg/kg BW‐day were administered orally in Swiss albino male mice (8‐week‐old, weight 25 ± 2.5 g). The treatment groups included: control, ThS at 2.5 mg/kg BW‐day, ThS at 5.0 mg/kg BW‐day, and ThS at 10 mg/kg BW/day, with five animals in each group. ThS solution was prepared freshly by suspending it in 0.5% CMC. Control mice were fed with 0.5% CMC. Treatment was performed every 24 h, and mice were monitored closely for 2 h after every treatment. Daily food and water uptake per group was monitored.

4.9. Induction of Hyperglycemia and ThS Intervention

Hyperglycemic mice were established using STZ administered intraperitoneally (ip) as described in the protocol by Furman [85]. For this study, a total of 20 mice aged 6–8 weeks and weighing approximately 20–24 g were divided into control and treatment groups (five mice per group), with equal average BW in each group. The treatment groups were: ThS (2.5, 5, and 10 mg/kg/day), hyperglycemia (HyG), and HyG + ThS (2.5 mg/kg/day). For the generation of hyperglycemia, the mice were fasted overnight (with food but not water) before the administration of STZ. The next morning, STZ was administered at a dose of 50 mg/kg BW ip, and the mice were immediately provided with a 10% sucrose solution instead of water. This treatment was continued for five consecutive days. On the sixth day, the sucrose solution was replaced with regular water. Blood glucose levels were monitored at regular intervals (every 3 days) by Control D Glucometer (Haiden Technology Private Ltd., New Delhi, India). During blood glucose measurement, the blood sample (approximately 10 µL) was collected from the tail vein by a glucometer lancet. After 2 weeks, mice with blood glucose levels exceeding 200 mg/dL were considered hyperglycemic. Thereafter, ThS (2.5 mg/kg/day) was administered to mice for 15 days to check the effect of ThS on hyperglycemic mice.

4.10. OGTT in Mice

OGTT in mice was conducted according to the protocol described by Andrikopoulos et al. [86]. Briefly, mice were fasted (food only) for 6 h, and then 2 g/kg BW of glucose was administered orally. Thereafter, blood glucose level was measured at 0, 15, 30, 60, and 120 min by Control D Glucometer (Haiden Technology Private Ltd.).

4.11. Terminal Blood Collection and Analysis

Terminal blood collection from the mice after the end of the experiment was done according to the protocol as described by Sahagun et al. [87]. Briefly, immediately after the euthanizing of mice by CO₂, blood was collected by cardiac puncture. For hematological analysis, blood was collected in an EDTA‐containing vial, whereas for serum analysis, blood was collected in a normal vial and analyzed with an automated blood counter in a standard veterinary laboratory. For serum parameter analysis, blood was kept for 20 min at room temperature and then centrifuged at 2500 rpm for 5 min at 4°C to obtain serum. The serum samples were outsourced to AM Thyrovision Laboratories Pvt. Ltd., New Delhi, India, for the measurement of glucose, total protein, albumin, ALP, AST, SGPT, total bilirubin, direct bilirubin, creatinine, and uric acid.

4.12. Histomorphology Analysis of Mice Tissue

Mice liver and kidney histology were analyzed to investigate tissue morphological changes. Initially, the tissues were fixed in a 10% formalin solution to preserve their cellular architecture and prevent decomposition. Following fixation, the tissues underwent a series of processing steps, including dehydration (removal of water), clearing (replacement of dehydrating agents with a substance like xylene), and infiltration with paraffin wax. Then, tissues were embedded in paraffin wax. Thereafter, thin sections of the tissues, each 50‐µm thick, were meticulously cut using a microtome. These sections were then stained with H&E, a widely used technique where hematoxylin stains cell nuclei blue–purple and eosin stains the cytoplasm and extracellular matrix pink–red, providing a clear contrast between different cellular components. The stained sections were mounted on glass slides with a permanent mounting medium. After that, the tissue sections were analyzed with a microscope equipped with a camera, and images were captured at 10× and 40× magnifications [88].

4.13. Statistical Analysis

Statistical analysis was done using GraphPad Prism 8.0 software. One or two‐way ANOVA with Tukey's post hoc test was applied to compare between groups [89]. Statistical significance was denoted as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 versus control, or between the indicated groups.

Author Contributions

Ashim Chandra Roy: conceptualization, methodology, investigation, resources, data curation, writing – original draft, review and editing, validation, formal analysis, visualization, software. Komal Priya: methodology, investigation, data curation, validation, formal analysis, visualization, software. Abhinav Prasad: investigation, writing – review and editing, validation, formal analysis, visualization, software. Prabhat Kumar: investigation, validation, formal analysis, software. Ramandeep Sharma: investigation, validation, formal analysis, software. Anuradha Pandey: investigation, validation, formal analysis, software. Ilora Ghosh: conceptualization, resources, writing – review and editing, validation, supervision, project administration.

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflict of interest.

Supporting information

Supporting File 1: cbdv70849‐sup‐0001‐SuppMat.docx

Data Availability Statement

The data that support the findings of this study are available in the Supporting Information of this article.