

I Nyoman Arsana1*, Ni Ketut Ayu Juliasih1
, Anak Agung Ayu Sauca Sunia Widyantari1
, I Gede Widhiantara2
and Putu Angga Wiradana2
1Department of Biology, Faculty of Information Technology and Science, University of Hindu Indonesia, Jl. Sangalangit, Tembau, Penatih, Denpasar, Bali Province, Indonesia
2Department of Biology, Faculty of Health and Science, Universitas Dhyana Pura, Jalan Raya Padangluwih, Dalung, North Kuta, Badung Regency, Bali Province, Indonesia
Corresponding Author E-mail: arsanacita@gmail.com
DOI : https://dx.doi.org/10.13005/bpj/3138
Abstract
Usadha is a traditional Balinese medicine system that has long been used in ancient medicine systems. However, so far there have been no reports on its safety. This study aims to examine the effect of polyherbal Usadha on hematology and blood biochemistry of Wistar rats. The study used a Completely Randomized Design with six treatments, namely polyherbal Usadha supplementation with doses of 0 mg/kg bw/day (E0), 100 mg/kg bw/day (E1), 200 mg/kg bw/day (E2), 300 mg/kg bw/day (E3), 400 mg/kg bw/day (E4), and 500 mg/kg bw/day (E5). The treatments were given for four weeks. The variables measured were complete hematology and blood biochemistry. Data were analyzed by One-Way ANOVA and continued with the Least Significant Difference test at a 95% confidence interval. Polyherbal Usadha appears to be safe for consumption without significant risk of side effects on blood parameters and liver function. Additional studies including histological analysis and mechanism of action will provide deeper insight into the therapeutic effects of polyherbal Usadha. In conclusion, polyherbal usada is relatively safe for liver and kidney function in Wistar rats. The results of this study support the safety of using Poliherbal Usadha as a candidate herbal medicine for long-term consumption.
Keywords
Blood biochemistry; Citrus amblycarpa (Hassk.) Ochse; Haematology; Piper nigrum L; Polyherbal Usadha
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Copy the following to cite this article: Arsana I. N, Juliasih N. K. A, Widyantari A. A. A. S. S, Widhiantara I. G, Wiradana P. A. Subacute Oral Toxicity Study of a New Polyherbal Formulation of Usadha on liver and Kidney Function of Wistar Rats. Biomed Pharmacol J 2025;18(1). |
Copy the following to cite this URL: Arsana I. N, Juliasih N. K. A, Widyantari A. A. A. S. S, Widhiantara I. G, Wiradana P. A. Subacute Oral Toxicity Study of a New Polyherbal Formulation of Usadha on liver and Kidney Function of Wistar Rats. Biomed Pharmacol J 2025;18(1). Available from: https://bit.ly/42l2LRN |
Introduction
Traditional medicine is gaining popularity because it supplies phytopharmaceuticals for the development of pharmacological medicines with desired qualities.1 Herbal plants have been utilized for thousands of years to treat a variety of ailments across the world. According to the World Health Organization (WHO), 80% of the world’s population continues to rely largely on traditional medicines for basic treatment2. Herbal medications are more popular than allopathic therapies because they are less expensive, have less side effects, are as effective as conventional pharmaceuticals, are more readily available, and are ecologically benign.2
Indonesia has the world’s sixth most diverse flora, with 20,000 species, 40% of which are indigenous to the country.3 The existence of native Indonesian flora is fascinating to research, particularly in the health industry. Furthermore, research on medicinal plants has increased dramatically in recent years, suggesting a growing interest in natural-based traditional medicine.4 Indonesia is well-known for its sustainable use of traditional medicine5. Traditional Indonesian herbal medicine has been used for hundreds of years in Indonesian society and is still widely used to maintain health and cure ailments due to its perceived safety over chemical pharmaceuticals.6,7 These herbs have historically played a significant role in Indonesian culture and medicine.8–10 Herbal plants, such as ginger, turmeric, and betel leaves, have been employed in traditional “Jamu” (herbal medicine) for thousands of years, demonstrating their long-term relevance in sustaining health and well-being. In addition to Jamu, the terminology for herbal medicinal beverages in Indonesia differ widely by location or ethnicity.11,12
Bali Province has a long history of treating various diseases with herbal plant components in various forms such as powder, decoction, paste, and oil contained in the Balinese traditional medicine system known as “Usadha”. The formulation of these medicines is based on two principles, namely utilizing one plant or using many herbs. Combining many herbs, often known as “Polyherbal”, is hypothesized to increase therapeutic efficacy because various bioactive chemicals work on various therapeutic targets to accelerate the healing process.13 Polyherbal has been used for thousands of years in many countries to treat various types of disorders such as diabetes, as an antioxidant, hepatoprotective, anti-inflammatory, and anxiety disorders.
Polyherbal derived from a mixture of Murraya koenigii L. Spreng leaves, Allium sativum L., Garcinia quaesita Pierre fruits, and Piper nigrum L. seeds is a common herbal remedy in Sri Lanka for treating diabetes mellitus and dyslipidemia.14 It has been found to be non-toxic and safe for long-term use. The polyherbal, which includes plant extracts including Boerhavia diffusa, Solidago virgaurea, Vitex negundo, and thymoquinone compounds, helps reduce hepatorenal damage caused by CCl4 through antioxidant and anti-inflammatory processes.15 AYUSH-64 is an Ayurvedic herbal polyphenol formulation consisting of many herbal components such as Alstonia Scholaris, Picrorhiza kurroa, Swertia chirata, and Caesalpinia crista, has been used for various disorders including malaria, chronic fever, and joint pain.16 Herbal mixture consisting of Cassia absus, Gymnema sylvestre, Nigella sativa, and Piper nigrum has high potential in treating diabetic complications, including liver and kidney damage, dyslipidemia, and oxidative stress.17 Arthralgex Ayurvedic Polyherbal consisting of 18 plant species did not cause serious side effects on biochemical, hematological, or organ parameters of rats during and after the treatment period.18 One of the polyherbal of usada formulas is made from a mixture of lime leaves (Citrus amblycarpa), 11 white peppercorns (Piper nigrum L.), and vinegar. These ingredients are ground until smooth, then mixed and used by drinking. This formula is used to treat paresthesia. Pepper (Piper nigrum L), has been known to have many active compounds, two of which have a high abundance of Piperidine, 1-[5-(1,3-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]-,(Z,Z)-; and Caryophyllene.19
The toxicity of herbal medicines has raised widespread concerns, and various side effects (including allergic responses, hepatotoxicity, nephrotoxicity, and cardiac toxicity) have been described in recent years.20 Plants with medicinal properties should have minimal toxicity due to their long-term use in humans.21 However, many therapeutic plants used in traditional medicine have been associated with harmful consequences. Conducting toxicity testing in appropriate animal models is essential to ensure the safety of treatments and is at the heart of toxicology. According to OECD standards, acute toxicity studies are an advanced approach to administer single and multiple doses of chemicals and report the primary safety profile of a particular compound.22
There are several articles that discuss in an exploratory manner the types of medicinal plants collected in Usadha in the province of Bali 23–25; but no empirical facts have been found regarding the safety or benefits of polyherbal Usadha. On the other hand, there is a high demand for polyherbals in health services. Therefore, this study was designed to evaluate the acute toxicity properties of polyherbal Usadha formulations on kidney and liver function in Wistar rats. The results of this study will be an excellent effort to encourage manufacturers and researchers to examine the optimal composition of raw materials when designing herbal medicines.26–28
Materials and Methods
Choice of plant combinations
The dried leaves and seeds were then ground and sieved and filtered with a 600 mesh sieve size. The powdered plant samples were stored in sterile airtight zip lock bags at 4 ℃ until used.28
Preparation of crude extract
Each of the simplicia was then extracted by maceration method using 96% ethanol solvent for 48 hours, and re-macerated twice, filtered using filter paper. The filtrate was then concentrated in a vacuum rotary evaporator at a temperature of 45℃ until a thick extract was obtained and then dried by the freeze-dry method. Usada poliherbal is made from a mixture of three ingredients, namely; white pepper seed extract, lime leaf extract, and vinegar in equal proportions.
Animals
A total of 24 male Wistar rats (Rattus norvegicus), aged three months, weighing 200 g to 250 g were used in this study. The rats were grouped into six groups of four each and then acclimatized for one week to adjust to the temperature and humidity of the research room. The rats were kept in cages containing four rats each, given food and drink as much as they wanted. The rats were given polyherbal usada at a dose according to the treatment group for four weeks through the stomach.
Sub-acute toxicity test
The experiment was conducted following OECD guidelines number 407 in the study.29 The study used a Completely Randomized Design with six treatments. The treatment was the administration of polyherbal usadha with six dose levels, namely; 0 mg/kgbb/day (E0), 100 mg/kgbb/day (E1), 200 mg/kgbb/day (E2), 300 mg/kgbb/day (E3), 400 mg/kgbb/day (E4), and 500 mg/kgbb/day (E5). Each treatment was repeated four times so that there were 24 research units. The treatment was given for four weeks. Twenty-four hours after the last treatment, the mice were euthanized using ketamine, then blood was taken from the orbital sinus cantus using a capillary pipette and stored in a tube.
Blood biochemical analysis
Biochemical studies were conducted to evaluate the safety profile of the polyherbal extract of usadha. Biochemical investigations included aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), Blood urea nitrogen (BUN), SC, and Total protein (TP). Blood biochemical parameters were determined using a medical analyzer with fuitest whose working procedure was in accordance with the company’s instructions.1
Hematological analysis
Blood samples from the treatment and control groups were taken and placed into 3 mL vaculab tubes containing EDTA. The parameters used to indicate hematological conditions were RBC (Red Blood Cell), HGB (Hemoglobin), HCT (Hematocrit), MCV (mean cell volume), MCH (Mean cell hemoglobin), MCHC (mean cell hemoglobin concentration), RDW (Red cell distribution width); WBC (white blood cells), Eo (eosinophils), Baso (basophils) Lymp (Lymphocyte), Neut (Neutrophils), Mono (Monocytes), PTL (Platelets), PDW (Platelet distribution width), MPV (Mean platelet volume), PCT (Plateletcrit). These parameters were calculated using the Sysmex pocH-100i™ Haematology Analyzer and the CBC line kit.1
]Statistical analysis
The measurement data were then tabulated in Microsoft Excel and evaluated statistically using One Way ANOVA with Duncan’s Test. Results are presented as mean ± SD. The statistical significance value between groups was at p≤0.05.29
Results
Blood Biochemical Levels
The results showed that the administration of Polyherbal Usadha had a significant effect (p≤0.05) on ALT and ALP, but not significantly (p≥0.05) on AST, BUN, SC, and TP. The average ALT, AST, BUN, TP, and SC tended to decrease after administering Polyherbal Usadha, while ALP increased after a 300 mg/kg bw dose. The mean ALT appeared to be significantly lower than the control after administration of 100 mg/kg bw to 500 mg/kg bw doses. Meanwhile, ALP appeared to be significantly higher than the control at a dose of 400 mg/kg bw (Table 1).
Table 1: Blood Biochemical Levels of Wistar Rats After Administration of Polyherbal Usadha (Mean ±SE mean)
Dose(mg/ kg bw) | Parameter | |||||
AST(U/L) | ALT(U/L) | ALP(U/L) | BUN (mg/dl) | SC (mg/dl) | TP (mg/dl) | |
0 | 160.75±27.55 | 85.75±9.73 | 300.50±62.98 | 36.95±9.05 | 0.95±0.02 | 5.25±0.02 |
100 | 150.00±23.98 | 66.25±5.91 * | 215.00±29.90 | 34.30±5.41 | 0.63±0.11 | 5.13±0.21 |
200 | 97.75±7.28 | 61.25±5.68 * | 266.25±46.38 | 32.05±5.93 | 0.88±0.06 | 4.85±0.18 |
300 | 98.40±7.98 | 61.72±3.84 * | 378.88±31.40 | 27.29±3.04 | 0.82±0.07 | 5.03±0.16 |
400 | 107.82±12.06 | 64.08±2.11 * | 460.90±36.40 * | 32.53±6.11 | 0.92±0.01 | 4.90±0.10 |
500 | 108.33±9.63 | 54.95±0.81 * | 341.75±22.31 | 21.79±3.45 | 0.85±0.06 | 5.01±0.13 |
*Significant with control at 0.05 level. ALT (Alanine aminotransferase), AST (Aspartate aminotransferase), ALP (Alkaline phosphatase), SC (Serum creatinine), BUN (Blood Urea Nitrogen), TP (Total protein (TP) |
The results showed that ALP levels showed a pattern that varied depending on the dose. The administration of polyherbal usadha at doses of 100 and 200 mg/kg bw resulted in lower ALP than the control. Still, it then increased significantly compared to the control at the dose of 400 mg/kg bw (Table 1). This condition indicates that administering polyherbal usadha at a dose of 400 mg/kg bw causes a certain metabolic response. The results showed that SC, BUN, and TP were not significantly different from the control (Table 1).
Blood Hematology Levels
Research shows that several hematological parameters after administration of Polyherbal Usadha show a pattern of changes that vary and depend on the dose. The administration of Polyherbal Usadha had a significant effect (p≤0.05) on MCV, PDW, Basophil, and MPV, but not significantly (p≥0.05) on WBC, Lymphocytes, Monocytes, Eosinophils, RBC, HGB HCT MCH, MCHC, RDW, Neutrophils, PLT. The administration of polyherbal usada at doses of 400 and 500 mg/kg bw resulted in a significantly higher MCV value (p≤0.05) compared to the control, while the doses 100; 200, and 300mg/kg bw resulted in a significant lower PDW (p≤0.05) compared to the control. Dose 300; 400, and 500mg/kg bw resulted in significantly lower basophils (p≤0.05). compared to controls, and doses of 100, and 200mg/kg bw also resulted in significantly lower MPV (p<0.05) (Table 2).
Table 2: Blood Hematology Levels of Wistar Rats After Polyherbal Usadha Supplementation (Mean ±SE mean)
Parameter | Dose mg/kg bw | Normal values | |||||
Control | 100 | 200 | 300 | 400 | 500 | ||
WBC (103/uL) | 11.80 ± 1.31 | 12.60 ± 1.6 | 12.13 ± 0.74 | 10.99 ± 0.82 | 10.56 ± 1.00 | 10.80 ± 0.85 | 4.80 – 17.50 |
Neut (103/uL) | 2.60 ± 0.25 | 2.94 ± 0.47 | 3.28 ± 0.76 | 4.08 ± 0.69 | 3.80 ± 1.05 | 3.35 ± 0.71 | 1.50 – 7.00 |
Lymph (103/uL) > | 7.51 ± 1.01 | 8.04 ± 1.31 | 7.37 ± 0.58 | 5.61 ± 0.23 | 5.42 ± 0.80 | 6.13 ± 0.20 | 1.00 – 3.70 |
Mono (103/uL) | 0.81 ± 0.10 | 0.93 ± 0.17 | 0.85 ± 0.06 | 1.11 ± 0.04 | 1.08 ± 0.14 | 0.87 ± 0.04 | 0.00 – 0.70 |
Eo (103/uL) | 0.34 ± 0.13 | 0.42 ± 0.08 | 0.42 ± 0.13 | 0.22 ± 0.04 | 0.16 ± 0.07 | 0.34 ± 0.08 | 0.00 – 0.40 |
Baso (103/uL) | 0.55 ± 0.12 | 0.27 ± 0.16 | 0.22 ± 0.21 | 0.00 ± 0.00 * | 0.00 ± 0.00 * | 0.11 ± 0.06* | 0.00 – 0.10 |
RBC (106/uL) > | 7.65 ± 0.19 | 7.72 ± 0.17 | 7.72 ± 0.09 | 7.35 ± 0.24 | 6.93 ± 0.27 | 7.56 ± 0.21 | 3.90 – 6.10 |
HGB (g/dl) | 13.45 ± 0.23 | 13.65 ± 0.50 | 13.45 ± 0.27 | 13.09 ± 0.42 | 12.50 ± 0.54 | 13.70 ± 0.31 | 11.1 – 18.0 |
HCT (%) | 40.25 ± 0.79 | 41.38 ± 1.00 | 41.03 ± 0.32 | 38.85 ± 0.89 | 38.12 ± 1.45 | 41.74 ± 0.74 | 31.0 – 52.0 |
MCV (fL) < | 52.68 ± 0.60 | 53.60 ± 0.40 | 53.13 ± 0.31 | 52.87 ± 0.60 | 54.99 ± 0.36 * | 55.24 ± 0.68 * | 86.0 – 110.0 |
MCH (pg) < | 17.63 ± 0.20 | 17.65 ± 0.31 | 17.42 ± 0.30 | 17.79 ± 0.18 | 18.02 ± 0.17 | 18.13 ± 0.21 | 26.0 – 38.0 |
MCHC (g/dl) | 33.43 ± 0.20 | 32.95 ± 0.41 | 32.78 ± 0.50 | 33.63 ± 0.34 | 32.77 ± 0.39 | 32.86 ± 0.25 | 31.0 – 37.0 |
RDW_SD (fL) | 33.93 ± 2.40 | 28.95 ± 0.10 | 29.83 ± 0.49 | 32.56 ± 1.42 | 34.31 ± 3.13 | 32.41 ± 1.46 | – |
RDW_CV (%) | 20.85 ± 0.94 | 18.25 ± 0.25 | 18.90 ± 0.09 | 20.09 ± 0.42 | 19.86 ± 0.88 | 19.28 ± 0.28 | 11.0 – 16.0 |
PLT (106/uL) > | 701.25 ± 170.42 | 946.25 ± 60.98 | 956.00 ± 168.27 | 1066.08 ± 68.13 | 1201.58 ± 234.00 | 1077.33 ± 118,77 | 150 – 450 |
MPV (fL) < | 7.80 ± 0.13 | 7.35 ± 0.12 * | 7.38 ± 0.13 * | 7.46 ± 0.08 | 7.55 ± 0.18 | 7.90 ± 0.13 | 9.0 – 13.0 |
PCT (%) | 0.54 ± 0.13 | 0.70 ± 0.03 | 0.70 ± 0.11 | 0.80 ± 0.04 | 0.90 ± 0.17 | 0.85 ± 0.10 | 0.17 – 0.35 |
PDW (fL) | 9.20 ± 0.11 | 8.35 ± 0.25 * | 8.35 ± 0.15 * | 8.61 ± 0.11 * | 8.91 ± 0.26 | 9.23 ± 0.15 | 9.0 – 17.0 |
P_LCR (%) | 11.23 ± 1.49 | 7.23 ± 0.40 | 7.48 ± 0.77 | 7.56 ± 0.74 | 8.50 ± 1.35 | 10.26 ± 0.81 | – |
* Significant with control at 0.05 level; > exceeding normal value, < less than normal value. Note: RBC (Red Blood Cell), HGB (Hemoglobin), HCT (Hematocrit), MCV (mean cell volume), MCH (Mean cell hemoglobin), MCHC (mean cell hemoglobin concentration), RDW (Red cell distribution width); WBC (white blood cells), Eo (eosinophils), Baso (basophils) Lymp (Lymphocyte), Neut (Neutrophils), Mono (Monocytes); PTL (Platelets), PDW (Platelet distribution width), MPV (Mean platelet volume), PCT (Plateletcrit). |
Discussion
The administration of polyherbal usadha resulted in an average lower ALT, and AST than the control, and even significantly lower ALT than the control. This condition is due to the influence of the active ingredients contained in polyherbal usadha, namely pepper and lime. The active compounds of pepper (Piper nigrum L) are mostly in the form of alkaloid compounds, including Piperidine, 1-[5-(1,3-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]-, (Z,Z)-; and Caryophyllene.19 Meanwhile, lime leaves (Citrus amblycarpa) have also been known to have many active compounds, most of which are essential oils, including Citronellol; Caryophyllene; Hexadecanoic acid, ethyl ester; 1-heptatriacotanol; Phytol; Ethyl 9,12,15-octadecatrienoate; Methyl glycolate,3TMS derivative; 3,7-dimethyl oct-6-en-1-yl stearate; Methyl iso-allocholate; Rhodopine; and Tricyclo [20.8.0.0(7,16) triacontane,1(22),7(16)-diepoxy-.30 Caryophyllene has significant pharmacological benefits, including antioxidant, anticancer, cardioprotective, anti-inflammatory, hepatoprotective, nephroprotective, gastroprotective, antimicrobial, and immunomodulatory properties.31 These active ingredients have a good effect on liver and kidney function so that polyherbal usadha does not cause hepatocyte damage.
Hepatotoxicity is a side effect that often occurs in treatment. This happens because the liver functions as a center for the disposition of drug metabolism and foreign substances in the body. Drugs given orally to pass through the intestinal cell membrane must be fat-soluble, then taken to the liver to be converted into water-soluble (more polar), then excreted in urine (if the molecule is small), or through bile (if the molecule is large). Liver lesions can occur due to hepatocellular reactions through the production of enzyme-drug complexes. This complex will then cause cell dysfunction, membrane dysfunction, and, cytotoxic response of T cells.32 A similar study using a mixture of Asparagus racemosus, Cajanus cajan, Cassia fistula, and Carissa spinarum, was able to protect the liver from damage induced by carbon tetrachloride (CCl4).33 Other studies also found that Piper nigrum stems provided protection against hepatotoxicity and dyslipidemia in tenofovir/lamivudine/efavirenz-induced wistar rats.34
ALP enzyme runoff in the blood can be used as a marker of cell damage.40,41 Lime and pepper mint can protect the kidneys so that they do not cause problems with kidney function. Caryophyllene with high concentrations in polyherbal 19,30; is known to have significant pharmacological benefits, including antioxidant, anticancer, cardioprotective, anti-inflammatory, hepatoprotective, nephroprotective, gastroprotective, antimicrobial, and immunomodulatory properties.31
Thus, it can be said that the administration of polyherbal usadha does not cause changes in the liver and kidney function of Wistar rats, so it is relatively safe when used as a medicinal ingredient up to a certain dose.
A slightly lower MCV value after low-dose polyherbal supplementation could indicate that at low doses, this supplement has the potential to lower red blood cell size, but this effect does not lead to anemia or iron deficiency, given that MCH and HGB remain stable.43
Red blood cell production is a complex and multi-layered process that is primarily regulated by erythropoietin (Epo). The main physiological regulator of EPO production is renal hypoxia or hypoxemia which both reduce the oxygen-carrying capacity of the blood. EPO gene expression is controlled by a transcription factor known as hypoxia-inducible factor (HIF) which plays an important role in cellular adaptation to decreased oxygen levels. Once released from the kidneys, the EPO circulates to the hematopoietic marrow which binds to and activates its receptors (EPOR). EPOR in turn phosphorylates and activates the signal transduction of Janus kinase-2 (JAK-2), and Signal Transducer and Activator of Transcription-5 (STAT-5) in erythroid progenitors, as well as phosphatidylinositol-3 (PI-3) kinase. Signals transduced through JAK-2/STAT-5 and PI-3 kinase cause changes in gene expression that promote effective erythropoiesis by increasing proliferation and reducing red blood cell progenitor apoptosis.44
An increase in neutrophils indicates a mild immune response, while a decrease in lymphocytes at high doses signals an adjustment of the immune system to polyherbal components without any indication of serious immune damage or suppression.45
Platelet counts at all doses of supplementation, and especially at high doses reaching 1201.58 10⁶/uL, well above the normal range (150–450 10⁶/uL). This response may be caused by polyherbal components that promote platelet formation. At low doses, it suggests that polyherbal may affect the average size of platelets, but this is not accompanied by changes that compromise platelet function. This condition is likely the result of infection, chronic inflammation, or drug reactions, which are the most common causes of thrombocytosis.46,47
Overall, polyherbal usadha supplementation exerted a significant influence on several hematological parameters in Wistar rats, specifically on the increase in platelets and RBCs without any visible hematological side effects. This supplementation appears to be able to support the hematopoiesis process, which is potentially beneficial in certain conditions that require an increase in platelet count or erythrocytes. However, given the fairly high increase in platelets, more research is needed to monitor the potential risk of hypercoagulability or increased blood clots. This study provides a solid basis for further studies regarding the benefits and risks of using polyherbal usadha in clinical applications.
Conclusion
Polyherbal usadha supplementation in Wistar rats affects several hematological and biochemical parameters. The main effects appear to be immune system stimulation and platelet formation. In addition, the positive impact of ALT shows potential benefits in supporting liver function. However, the increase in ALP at high doses should be looked at further, as it could indicate the potential for liver stress at higher doses. Polyherbal usadha appears consumable without the risk of significant side effects on blood parameters and liver function. Additional studies that include histological analysis and mechanisms of action will provide deeper insights into the therapeutic effects of Polyherbal usadha.
Acknowledgement
The author would like to thank the Hindu University of Indonesia (UNHI) for supporting the implementation of this research. The author would also like to thank the Study Program of Biology, Faculty of Health Sciences, Universitas Dhyana Pura (UNDHIRA-BALI) for helping to implement this research and writing this manuscript.
Funding Source
This research was funded by the Institute for Research and Community Service (LPPM) of the Hindu University of Indonesia (UNHI) through the 2024 Research Funding Grant Program with Contract Number: 001.PA/KPEN-LPPM/UNHI/VIII/2024
Conflict of Interest
The author(s) do not have any conflict of interest.
Data Availability Statement
This statement does not apply to this article.
Ethics Statement
The use of experimental animals in this study has received ethical approval from the animal ethics committee of the Faculty of Veterinary Medicine, Udayana University (UNUD) with Number: B/110/UN14.2.9/PT.01.04/2024.
Informed Consent Statement
This study did not involve human participants, and therefore, informed consent was not required.
Clinical Trial Registration
This research does not involve any clinical trials.
Permission to reproduce material from other sources
Not Applicable
Author Contributions
- I Nyoman Arsana: Conceptualization, Methodology, Data collection, Data analysis, Project administration, and Funding Acquisition;
- Ni Ketut Ayu Juliasih and Anak Agung Ayu Sauca Sunia Widyantari: Data collection, Data analysis, and Project administration,
- I Gede Widhiantara and Putu Angga Wiradana: Writing – Original Draft, Editing, Supervision, and Visualization.
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