Research Article |
Corresponding author: Syafruddin Ilyas ( syafruddin6@usu.ac.id ) Academic editor: Magdalena Kondeva-Burdina
© 2023 Cheryl Grace Pratiwi Rumahorbo, Syafruddin Ilyas, Salomo Hutahaean, Cut Fatimah Zuhra, Putri Cahaya Situmorang.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Rumahorbo CGP, Ilyas S, Hutahaean S, Fatimah Zuhra C, Situmorang PC (2023) Chronic toxicity assessment of nano-formulated Bischofia javanica leaves: Implications for pharmacological use. Pharmacia 70(4): 1101-1109. https://doi.org/10.3897/pharmacia.70.e110640
|
This study explores the chronic toxicity of nano-formulated Bischofia javanica leaves, a plant with pharmacological significance in Indonesia, following OECD guidelines 452. Graded doses (2, 4, 6, and 8 g/kg BW) of nano-formulated Bischofia javanica were administered to the treatment groups for 60 days, while the control group (K-) received only water. On the 61st day, the mice were euthanized, and samples for biochemical, hematological, and histopathological analysis were collected. Statistical analysis involved one-way ANOVA and Tukey’s post hoc tests. Graded doses of nano-formulated Bischofia javanica leaves caused significant alterations in liver function. Doses between 2 and 4 g/kg BW improved liver histology and physiological markers. Higher doses (6 to 8 g/kg BW) led to liver dysfunction and histological degeneration, characterized by substantial fatty alterations, intracellular fat accumulation in hepatocytes, and compromised central blood vessels and sinusoids. The lungs showed signs of alveolar inflammation, epithelial exfoliation, debris, and the accumulation of inflammatory cells in alveolar spaces. Lower doses showed uniformly distributed cardiac blood vessels, while higher doses resulted in heart hemorrhages and amorphous exudates. Except for high doses, which significantly elevated specific liver damage indicators, no other levels of Bischofia javanica caused treatment-related mortality or significant alterations in hematological and biochemical parameters. Blood sugar levels remained stable across the dose range. In conclusion, moderate doses of nano-formulated Bischofia javanica leaf extracts can enhance physiological functions positively. However, caution is necessary when contemplating high doses, as they can cause dysfunction or damage vital organ systems.
Bischofia javanica, toxicity, biochemical, hematological, microanatomy, proper dosage
Traditional medicinal therapies are widely practiced worldwide, especially in middle- and low-income countries. They offer several advantages over modern medicine, primarily due to their lower likelihood of causing harmful effects when used appropriately. When utilizing herbal therapies, it is crucial to consider the correct dosage, timing, administration method, ingredient selection, comprehensive information review, and appropriate indications. Traditional medicines often contain multiple chemical compounds, enabling complementary or synergistic effects to achieve treatment goals. Additionally, traditional medicine offers flexibility in administration, with options ranging from brewing and infusion to incorporation into food. This highlights the versatility and potential benefits of traditional medicine as a complementary or alternative approach to healthcare.
Pharmaceutical research into herbal medicines has gained recent prominence due to several factors, as outlined above. An essential initial step in developing these medicinal plants is the investigation of their toxic properties. Toxicity tests conducted on test animals provide crucial supporting evidence for the safety of these preparations. The choice of tests depends on the substance’s intended use and the potential risks associated with human exposure. One medicinal plant has garnered significant attention from researchers regarding its efficacy is Bischofia javanica. Several advanced studies have explored its pharmacological effects. For instance, locomotor activity has been assessed using different tests, including the Open Field (OP) test (
While previous studies on Bischofia javanica toxicity have primarily focused on the cytotoxic test LC50 method (
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 60th 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.
Medicinal substances may adversely affect immune function, hormone and enzyme activity, and hematopoiesis (
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.
AST is typically found in the cytoplasm and mitochondria of the heart, liver, and skeletal muscle (
Serum creatinine and blood urea nitrogen levels are vital indicators of renal function because they help eliminate creatinine, a byproduct of protein metabolism (
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 (
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.
In control and T1 mice, renal architecture appeared normal, showing typical glomeruli, renal tubules, and collecting ducts with minimal stromal congestion (Fig.
Dispersed, severely clogged cardiac arteries were observed at all nano-formulated Bischofia javanica dose levels, with no signs of inflammation, infarction, or fibrosis (Fig.
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.
The brain histology of the adverse control group (K-) remained unchanged, showing no edema or congestion (Fig.
Bischofia javanica, a commonly used herbal plant in Indonesia for its potential therapeutic benefits, has been the focus of this study to evaluate its safety, particularly in the nano form. The limited scientific data on its potential harm prompted this investigation into chronic toxicology. The results obtained from the test groups did not reveal significant deterioration in hematological function or any treatment-related mortality. All groups that received graded doses of nano Bischofia javanica leaves exhibited no signs of macrocytic or microcytic anemia, as evidenced by red blood cell and WBC counts. Although some groups showed a slight increase in red blood cell counts, these differences were statistically insignificant compared to the control group and were considered non-toxicologically relevant. In practical terms, medium and high doses of Bischofia javanica led to increased WBC counts, which were also statistically similar to the control group. However, upon euthanasia and examination, mice receiving a high dose of nano Bischofia javanica leaves (T4) exhibited slight ulceration in the small intestine during macroscopic digestive tract inspection.
Our results suggest high doses of nano-formulated Bischofia javanica leave significantly elevated AST levels in mice, indicating potential long-term toxicity or hepatocyte cell damage. This finding aligns with previous phytochemical screenings that have suggested moderate toxicity for these plants, emphasizing the importance of appropriate dosages (
Mice treated with a moderate dose of nano-formulated Bischofia javanica (T3; 6 g/kg BW) exhibited elevated blood sugar levels compared to the control group, potentially signaling impaired glucose metabolism, pancreatic injury, or a predisposition to diabetes mellitus. Previous studies have extensively explored hyperglycemia and diabetes mellitus (
This study revealed that mice treated with nano-formulated Bischofia javanica leaves experienced gastrointestinal (GIT) issues, particularly in the small intestine. Their lung morphology exhibited similar changes, with alveolar inflammation dominated by lymphocytes and macrophages and alveolar spaces filled with debris and chronic inflammatory cells. These effects are attributed to high doses of herbal Bischofia javanica leaves exceeding 8 g/kg BW. Drugs, administered through various routes, undergo absorption, distribution, and binding to exert their effects. With its high metabolic rate, the brain demands a continuous supply of calories and oxygen, receiving approximately 20% of the cardiac output, equivalent to around 750 ml of blood per minute. Our findings demonstrate that administering nano-formulated Bischofia javanica leaves induces histological changes in the cerebrum of white mice (Mus musculus), evident in congestion and perivascular edema lesions. While such pathological conditions are expected, even in control mice, differences lie in lesion distribution and treatment object health status. Experimental animals not bred specifically as pathogen-free (SPF) may exhibit unexpected changes (
Mice given low doses of nano-formulated Bischofia javanica leaves (T1) displayed mild kidney toxicity, while those receiving 4 g/kg BW showed moderate kidney toxicity. This was characterized by capillary blockages in the glomerular system, kidney tubules, and collecting duct blood vessels. Additionally, chronic inflammation was observed in mice given 6 g/kg BW, and the kidney weight in the 8 g/kg BW group was notably lower than in the control group. This decreased kidney weight in T4 might be attributed to chronic inflammation and potential atrophy (
Consequently, administering this nano-formulated leaf only for 60 days is advisable. Mice regularly receiving nano-formulated Bischofia javanica leaves showed minor cardiac damage, characterized by dispersed yet heavily clogged blood vessels. Rodents exposed to this herb also exhibited highly eosinophilic muscle fibers, hemorrhages containing unstructured excrement, isolated muscle tissue, and congested veins. These effects may be attributed to the high concentration and prolonged exposure to Bischofia javanica leaf nano herbs. Toxicity is influenced by toxin concentration, exposure duration and frequency, composition, and environmental conditions (
Our study indicates that prolonged exposure to high doses (> 4 g/kg BW) of nano-formulated Bischofia javanica leaves for ≥ 60 days may potentially damage critical organ systems. Conversely, at doses below 4 g/kg, these herbs could be considered for therapeutic purposes, showing positive effects on liver, blood, kidney, and heart functions, with minimal impact on the brain and lungs. We advise caution when using nano-formulated Bischofia javanica leaves, and we recommend further safety assessments, particularly in teratogenic toxicity models, especially for pregnant individuals. To ensure the quality of the plants, we advocate for improved processing and safe agricultural practices to prevent contamination with medicinal plant pesticides.