The production of nano-formulated Bischofia javanica leaf involves High Energy Milling (HEM). The process commenced with preparing 2 kg of fresh Bischofia javanica leaves, which were meticulously washed with running water and dried in a shaded room for one week. Afterward, a grinding machine was utilized to grind the dried Bischofia javanica leaves into a coarse powder. The coarse powder was then placed in a grinding container, along with alumina grinding balls, in a ratio of 1:20 (powder mass to grinding ball mass). The grinding process was initiated at a speed of 350 rpm and followed a specific time variation pattern. This pattern encompassed grinding for 3 hours, a subsequent 1-hour pause, another grinding session lasting 6 hours, and another 1-hour pause. This sequence continued until a final grinding session of 9 hours was completed, resulting in the nano-formulation of Bischofia javanica leaves. The diameter of the particles was assessed using a Particle Size Analyzer to confirm their nano-sized range, certifying the suitability of the herbal material for various applications.

The study employed forty healthy adult male Mus musculus weighing between 20 and 40 g. To facilitate identification and data collection, each rodent received individual labeling and was distributed into five groups, each consisting of six mice (n=6). These groups were housed separately, and an initial ten-day acclimatization period in a laboratory environment was provided for all mice before the experiment. During this period, the animals were maintained at consistent room temperature and humidity levels, adhering to a 12-hour light and 12-hour dark cycle. They were supplied with standard pellet food and had access to water ad libitum. Among these groups, four received graded doses of nano-formulated Bischofia javanica leaves, while the fifth group, serving as the control, received only clean water and standard feed. Weekly weight measurements of the mice were recorded before dosing with nano-formulated Bischofia javanica leaves. On the 60^th day of the experiment, the animals were humanely euthanized under mild chloroform anesthesia.

For this investigation, four categories of nano-formulated Bischofia javanica leaves were designated as T1 (2 g/kg BW), T2 (4 g/kg BW), T3 (6 g/kg BW), and T4 (8 g/kg BW). These doses were selected based on our previous research, utilizing the LD50 method for nano-formulated Bischofia javanica leaves, which determined the LD50 dose to be 12.6 g/kg BW. Throughout the 60-day study period, the mice were orally administered daily doses of nano-formulated Bischofia javanica leaves and sterile drinking water. The living conditions, food, animal dosages, and daily observations strictly adhered to the recommendations outlined in the Organization for Economic Co-operation and Development (OECD) guideline 452. The allocation of mice to each group adhered to the principles of the 3R concept, emphasizing the reduction, refinement, and replacement of animal use. Furthermore, international principles and standards were strictly followed to prevent suffering or distress among the test animals. The guidelines for Animal Research: Reporting of In Vivo Experiments (ARRIVE) and the Helsinki Declaration (updated in 2013) were also adhered to, ensuring the ethical and scientific integrity of the study.

Daily monitoring of the animals was conducted to detect any potential adverse effects of nano-formulated Bischofia javanica leaves following dosing throughout the study period. This monitoring encompassed observations of rat feeding behavior, fur color, self-isolation signs, pain indications, and any instances of mortality.

Blood samples were obtained from the tail vein, incubated with anticoagulants, and analyzed utilizing automated hematology (Mindray BC-2800 Auto Hematology Analyzer). The following parameters were assessed through the Automatic Hematology Analyzer BF-6800: White Blood Cell (WBC) count, Red Blood Cell (RBC) count, hemoglobin (HGB) levels, hematocrit (HCT) levels, Mixed Cell Count (MXD), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, Red Cell Distribution Width-Standard Deviation (RDW-SD), lymphocyte count, MXD, Neutrophil count, Lymphocyte count, Neutrophil count, Red Cell Distribution Width-Coefficient Variation (RDW-CV), Platelet Distribution Width (PDW), mean platelet volume (MPV), and Platelet-Large Cell Ratio (P-LCR).

Mice were weighed 12 hours before euthanasia with chloroform, and blood specimens were collected through a cardiac incision. Five milliliters of blood were collected in gel separator tubes, allowed to clot, and then centrifuged for 15 minutes at 3000 rpm. The serum was separated and stored at -20 °C. Biochemical parameters were determined using the Automatic Biochemistry Analyzer NEUES480 (Brand: MedGroup). These parameters included Albumin, Globulin, Total Protein, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), Indirect Bilirubin, Direct Bilirubin, Total Bilirubin, Low-Density Lipoprotein (LDL), High-Density Lipoprotein (HDL), Very Low-Density Lipoprotein (VLDL), Total cholesterol, Triglycerides, Creatinine, Uric acid, Blood sugar, Gamma-glutamyl transferase (GGT), and Blood Urea Nitrogen (BUN).

The vital organs, including the liver, heart, kidneys, and lungs, were carefully removed, weighed, and preserved in 10% neutral buffered formalin. Subsequently, these organs underwent histological processing. A small section from each organ was precisely sliced, dehydrated in graded alcohol, and embedded in paraffin. Following preparation, the sections, ranging from 4 to 10 µm in thickness, were fixed using a neutral DPX medium, stained with hematoxylin and eosin, and then mounted. Images were magnified at 40×, 100×, and 400× using a light microscope.

The findings were subjected to a one-tailed analysis of variance (ANOVA) with a 95% confidence level. Subsequently, Tukey’s post hoc analysis was applied to the data utilizing SPSS version 21, and the results were presented in tables and graphs. Graphical analysis was additionally conducted using GraphPad Prism version 8.0.

Regular checks on drinking, eating, physical appearance, and activity were crucial in toxicology investigations. Macroscopic studies of the mice involved in this investigation revealed no significant changes in feeding, exploration, or drinking habits during treatment. Furthermore, we assessed incisor height, fur color, and overall appearance to maintain standards.

Certain xenobiotic substances can disrupt eating, drinking, and digestive patterns, leading to hormonal and enzymatic issues. Reduced appetite often leads to weight loss and delayed growth. This study examined the impact of graded nano-formulated Bischofia javanica leaf doses on mouse body weight over eight weeks. All groups showed increased body weight (Fig. 1), with no statistical difference between the control and comparison groups.

Figure 1. 

Weekly changes in body weight. Values are shown as mean ± SEM. K- (Control), T1 (2 g/kg BW), T2 (4 g/kg BW), T3 (6 g/kg BW), and T4 (8 g/kg BW).

Medicinal substances may adversely affect immune function, hormone and enzyme activity, and hematopoiesis (Lubran 1989). Bioactive substances can disrupt hematopoiesis, leading to various blood disorders. Xenobiotics increase white blood cell (WBC) production as part of the body’s defense mechanism (Wang et al. 2021; Han et al. 2016). Thus, this study evaluated hematological parameters to assess the toxicity of Bischofia javanica concerning hematological functions.

WBCs increased from 8.94 ± 0.56 in the negative control to 12.10 ± 0.31 in the T3 group (6 g/kg BW dose) (Fig. 2). The percentage of mixed cells (MXD), consisting of monocytes, eosinophils, and basophils, significantly increased from 14.43 ± 0.93 in the negative control to 19.29 ± 2.24 in group T1 (2 g/kg BW dose) and 16.62 ± 0.84 in group T4 (8 g/kg BW). Among all measured hematological parameters, only those related to neutrophils showed significant differences with the control group. Neutrophils increased in percentage from 8.83 ± 0.62 in the negative control group to 20.42 ± 1.20 in the T1 group, 15.57 ± 1.84 in the T3 group, and 14.22 ± 3.13 in the T4 group. Meanwhile, the percentage of neutrophils decreased in the T2 group (7.30 ± 0.29), showing no significant difference compared to the negative control group. Additionally, the number of neutrophils (×10³) exhibited significant differences between the negative control group and the T1, T3, and T4 groups, with a decrease observed in the T2 group (although not statistically significant from the negative control value).

Figure 2. 

The impact of nano-formulated Bischofia javanica leaf extract on hematological parameters. Values are shown as mean ± SEM. K- (Control), T1 (2 g/kg BW), T2 (4 g/kg BW), T3 (6 g/kg BW), and T4 (8 g/kg BW).

AST is typically found in the cytoplasm and mitochondria of the heart, liver, and skeletal muscle (Upur et al. 2009), while ALT resides mainly in the cytosol of liver cells. These liver enzymes enter the bloodstream during necrosis, liver injury, or changes in hepatocellular permeability, making their blood levels useful markers for hepatotoxicity, reflecting hepatocyte degeneration (Wong et al. 2017). Indirect Bilirubin, a product of HGB and red blood cell breakdown, is converted to direct Bilirubin in the liver and excreted via bile. In bioactive toxicity studies, bilirubin assays are crucial for evaluating red blood cell hemolysis and liver catabolic function. The kidneys handle urea, Bilirubin, and other waste substances excretion. The liver also produces body triglycerides and proteins, while approximately 80% of cholesterol in the body is endogenously synthesized. Liver cholesterol contributes significantly to transportation and other functions. Hence, liver cholesterol levels can describe the liver’s synthetic capacity after herbal administration.

Serum creatinine and blood urea nitrogen levels are vital indicators of renal function because they help eliminate creatinine, a byproduct of protein metabolism (Kluwe 1981). Bilirubin levels showed no immediate increase with T1, T2, or T3 dosing but significantly rose at T4. Albumin levels were similar to those in group K. Total Bilirubin and direct bilirubin levels increased in the T3 and T4 groups, while AST levels increased across all treatment groups. HDL levels decreased in all treatment groups, while LDL and total cholesterol increased. Notably, LDL levels significantly increased in groups T2, T3, and T4 compared to the control (Fig. 3). Most lipid parameters in the treatment groups were not significantly different from the control. Blood sugar levels did not significantly differ in any treatment group compared to the control, remaining within the normal range (Fig. 3).

Figure 3. 

The impact of nano-formulated Bischofia javanica leaf extract on the parameters of biochemical function. Values are shown as mean ± SEM. K- (Control), T1 (2 g/kg BW), T2 (4 g/kg BW), T3 (6 g/kg BW), and T4 (8 g/kg BW).

Certain bioactive substances can induce inflammation in various tissues and organs, potentially leading to body and organ weight changes. Organ and body weight comparisons between treated and control groups serve as a crucial measure of the toxic effects of these substances. In general toxicity studies, the Society of Toxicologic Pathology regards organ weight evaluation as a vital screening tool for assessing the toxicity of bioactive substances (Michael et al. 2007; Simanjuntak and Rumahorbo 2022; Rumahorbo et al. 2023b). This study assessed the heart, liver, kidney, spleen, and testes. Most organs decreased in the treatment group, but it is noteworthy that specific organs, including the liver, kidney, and heart, showed significant reductions in the negative control group (Fig. 4).

Figure 4. 

Organ weights relative to treatment with nano-formulated Bischofia javanica leaves. Values are shown as mean ± SEM. K- (Control), T1 (2 g/kg BW), T2 (4 g/kg BW), T3 (6 g/kg BW), and T4 (8 g/kg BW).

Microscopic analysis of vital tissues and organs is crucial to assess the safety of bioactive substances, revealing asymptomatic toxic effects often missed in biochemical studies (Mensah et al. 2020). This study exposed experimental animals’ hearts, lungs, livers, kidneys, and brains to nano-formulated Bischofia javanica leaf extract for histological evaluation. These organs were also examined in the control group for comparison.

In the control group, there was moderate to severe central venous congestion (Fig. 5). In contrast, the Bischofia javanica-treated group displayed subcapsular hemorrhage and congestion in central veins, sinusoids, and prominent veins beneath the capsule (Fig. 5). The T1 dose group exhibited areas of necrosis without signs of inflammation. The T3 and T4 groups also had scattered hepatocytes containing intracellular fat globules. Central venous congestion was evident in the livers of T1, T2, and T3-treated mice (Fig. 5). The hepatocyte cells in the T2 group displayed microvesicular conformation with noticeable areas of fatty change. Fig. 5 illustrates more pronounced liver conditions in the T2, T3, and T4 treatment groups, highlighting congestion in various liver regions, including under the capsule, sinusoids, large blood vessels, and central veins. Furthermore, the T4 group exhibited foamy hepatocytes.

Figure 5. 

Microscopic images of vital Mus musculus organs under chronic nano-formulated Bischofia javanica leaf treatment. K- (Control), T1 (2 g/kg BW), T2 (4 g/kg BW), T3 (6 g/kg BW), and T4 (8 g/kg BW).

In control and T1 mice, renal architecture appeared normal, showing typical glomeruli, renal tubules, and collecting ducts with minimal stromal congestion (Fig. 5). However, all treatment groups, except for T1, exhibited severe stromal congestion and congestion in glomeruli, renal tubules, and collecting ducts (Fig. 5). Notably, the T4 group, receiving the highest dose of nano-formulated Bischofia javanica leaves, displayed persistent inflammation (Fig. 5).

Dispersed, severely clogged cardiac arteries were observed at all nano-formulated Bischofia javanica dose levels, with no signs of inflammation, infarction, or fibrosis (Fig. 5). In the cardiac sections of mice in the T4 group, we observed highly eosinophilic muscle fibers, hemorrhage with accompanying amorphous exudates in isolated muscle fibers, and scattered, highly congested arteries (Fig. 5).

The control group exhibited airway epithelial sloughing in the lungs, while the T4 group displayed chronic inflammation with inflammatory cell clusters in the alveolar spaces (Fig. 5). In the T3 group, the lungs showed consistent inflammatory changes characterized by lymphocytes and macrophages, along with epithelial cell sloughing and debris covering the alveolar spaces (Fig. 5). Mice administered 8 g/kg BW of nano-formulated Bischofia javanica displayed moderate chronic inflammatory changes dominated by lymphocytes and macrophages, with some areas showing epithelial cell exfoliation in the lungs. Fig. 5 illustrates debris and inflammatory cells covering the alveolar space. In group T2, some areas in the alveoli exhibited chronic inflammation with clusters of inflammatory cells and airway exfoliation (Fig. 5). The T1 group showed minor inflammation in certain areas, with stable clusters of inflammatory cells filling the space (Fig. 5).

The brain histology of the adverse control group (K-) remained unchanged, showing no edema or congestion (Fig. 5). In the T1 group, mild edema and congestion were observed, along with focal congestion and edema lesions (Fig. 5). In contrast, the brain tissue in T2 exhibited more severe congestion lesions after 60 days of exposure to nano-formulated Bischofia javanica leaves compared to T1 (Fig. 5). Similar conditions were observed in the T3 group. Meanwhile, the T4 group displayed a high formation level of focal congestion lesions and severe edema. Nearly every neuron cell in this group’s cerebrum of the brain tissue exhibited similar changes (Fig. 5). This suggests that administering high doses of nano-formulated Bischofia javanica leaves for an extended duration strongly impacts rat brain histology.