Acute and Subchronic Oral Toxicity Evaluation of Herbal Formulation: Piper crocatum Ruiz and Pav., Typhonium flagelliforme (Lodd.) Blume, and Phyllanthus niruri L. in Sprague–Dawley Rats

Background The product combination of Piper crocatum Ruiz. and Pav., Phyllanthus niruri Linn., and Typhonium flagelliforme (Lodd.) BL ethanolic extract (SKM) exerts immunomodulatory activity. However, the toxicity profile of the combination has never been investigated. Objective This study aimed to establish the acute toxicity profile of the SKM product on Sprague–Dawley (SD) rats and its subchronic toxicity profile on female SD rats. Method The acute and subchronic toxicity tests were conducted in accordance with OECD 423 and OECD 408, respectively. Result The SKM product was safe up to 5000 mg/kg b.w. in male and female SD rats. In repeated doses of SKM for 90 days, the administration of 22.5, 45, and 90 mg/kg b.w. per day of the SKM product to female SD rats did not affect clinical signs, body weight, food and water consumption, hematological parameters, clinical chemical parameters, urinalysis, relative organ weights, and gross pathological and histopathological features compared with the control group. Conclusion Analyses of these results suggest that the long-term oral administration of the SKM product for 90 days does not cause subchronic toxicity.

An acute toxicity study showed that the lethal dose 50 (LD50) of P. niruri ethanolic extract in mice is 18,000 mg/kg b.w. [5]. In addition, the lethal dose of T. fagelliforme, which causes 50% of death, is 48.081 g/kg b.w. [6]. Halim performed an acute toxicity study using Balb/c mice and found that the LD50 value of P. crocatum extract is 1,588,781 mg/kg b.w. [7].
Several studies examined the toxicity profle of each plant. Hartini et al. showed that neolignan Pc-2 isolated from P. crocatum leaves demonstrates hydropic degeneration and histopathological features of necrosis [8]. Te Brine Shrimp Lethality Test results demonstrated that T. fagelliforme tuber hexane extract is slightly toxic, with an LD50 value of 762,086 ppm [6].
A toxicity study reported that P. niruri extract has no toxic efect on aminotransferase (AST) and alanine aminotransferase (ALT) levels in diabetes mellitus rats at 50 and 100 mg/kg b.w. [9]. A subchronic toxicity test showed that the ethanolic extract combination of Piper crocatum leaves, Typhonium fagelliforme Lodd. Blume. tuber, and Phyllanthus niruri Linn. herbs is nontoxic on liver function [10].
However, the toxicity profle of combined extracts remains unknown. Terefore, the potential acute and subchronic toxicity profles of combined extracts in Sprague-Dawley (SD) rats should be investigated. Tis study analyzed the acute and subchronic safety profles of the combination product of P. crocatum, P. niruri, and T. fagelliforme ethanolic extract (SKM product).

Experimental Animals.
Male and female SD rats aged 6-8 weeks old with an initial body weight of 100-150 g were used for the acute toxicity study, and only female rats were used for the subchronic study. Female rats were selected based on the OECD guideline test No. 423 and No. 408 precisely due to the better sensitivity of female rats than males. All the animals for the acute toxicity study were obtained from the Laboratory of Pharmacology and Toxicology, Faculty of Pharmacy, Universitas Gadjah Mada, Indonesia. Te subchronic study used female rats supplied by the Animal Experimental Unit, Animal Research, and Development Center, Universitas Gadjah Mada. SD rats were randomly divided, marked, and acclimated to laboratory conditions. Te rats were acclimatized for 5 days to avoid stress before administration. Te temperature was set to approximately 25 C-30 C. Humidity was about 40%-70%, and the light condition was 12 h bright and 12 h dark. Temperature and humidity were controlled using an air conditioner, a room thermometer, and a room hygrometer.

Acute Toxicity Study.
Te study was conducted in accordance with OECD Guidelines test number 423 [11]. SKM product was suspended in 0.5% sodium CMC solvent and administered to the rats by single gavage doses after 24 h fasting. Te animals were divided into three groups of six rats each: control, 2000 mg/kg b.w. dose, and 5000 mg/kg b.w. dose. Te rats from diferent groups were placed in diferent cages. Cages were plastic boxes with chaf as the base.
Te rats in the control group received sodium CMC solvent only to evaluate the efect of the vehicle. Te initial dose in this study was 2000 mg/kg b.w., considering the acute toxicities of every single extract. All deviations in general behaviors were observed for 4 h after dosing, and then the monitoring was conducted daily for 14 consecutive days. Body weights were measured before and after administration daily. Necropsy was performed on all animals at the end of the 14-day observation to fnd apparent changes in major organs and tissues.

Subchronic Toxicity.
Te study was performed following OECD Guidelines test number 408 [12]. Forty rats were randomly separated into four groups of 10 each: control (received CMC-Na 0.5% as a vehicle), 22.5 mg/kg b.w., 45 mg/kg b.w., and 90 mg/kg b.w. Additional 10 rats for the recovery period were divided into the satellite control group and the satellite 90 mg/kg b.w. group. Te test material was suspended with CMC-Na 0.5% and orally administrated for 90 consecutive days. Te test material was mixed with a vehicle every 5 days. All animals were observed for clinical signs and food and water consumption once daily. Individual body weights were recorded every 3 days. Rats in control and dose groups were sacrifced at day 90. Satellite groups were continuously observed without treatment in the next 28 days for the recovery period and then sacrifced.

Urinalysis.
Urine was collected for 24 h using metabolic cages. Te animals fasted overnight, but the water was provided ad libitum during the collection period. Volume, pH, and color were observed.

Organ Weight and Histopathological Evaluation.
Te relative organ weights of the heart, spleen, liver, lungs, kidneys, stomach, and intestine were calculated by dividing organ weight by body weight. Te organs were washed in an isotonic solution, fxed in 10% formalin, and embedded in parafn to ease the slicing. Te samples were stained with hematoxylin-eosin dye and then examined microscopically.

Statistical Analysis.
Quantitative data were presented as mean value ± standard deviation. All results of the treatment groups were compared with those of the control group. Data were analyzed using SPSS 16.0 for Windows (SPSS Inc., Chicago, IL, USA). Statistical methods were used to analyze the body weight, relative organ weight data, food and water consumption, hematological parameters (RBC count, WBC count, Hb, HCT, MCV, MCH, MCHC, and PLT), and biochemical parameters (AST, ALT, TP, TC, GLU, UREA, and CRE). Te one-way ANOVA was used to show differences among the groups, whereas a paired sample T-test was used to show signifcant diferences between days 0 and 90. An independent sample T-test was used to show signifcant diferences between days 90 and 118. Statistical signifcance was considered at P < 0.05.

Total of Death and Toxic Syndrome.
Te animals used in the main study were observed 14 consecutive days after dosing, and none showed any toxic signs or died. No male or female rats died during the 14 days of observation. Te general appearance, grooming, posture, gait, behavior, and all other observational parameters were normal during the study.

Body Weight.
Body weight data were presented as average changes in body weight. It is one of the parameters to evaluate the general health condition of animal experiments. It was obtained by reducing the body weight of test animals on the 14th day with body weight on the frst day and then dividing by 14. Te average of the body weight changes is shown in Figures 1 and 2. No signifcant difference in body weight was found between the treated and control groups.

Organ Weight.
Organ weight data were expressed as a relative weight (Tables 1 and 2). No signifcant diference in organ weight was found between the treated and control groups (P > 0.05).
Tese results of the animal experiments indicate that the SKM product does not cause any changes in the relative weights of organs. Te average relative organ weights of male and female animals are shown in Tables 1 and 2. 3.1.4. Histopathological Evaluation. Histological examination showed no morphological alteration for 14 days (Figure 3).

Clinical Signs and Mortality.
No toxicity symptom or death related to SKM administration was observed during the treatment and recovery periods at any dose.

Body
Weight. Fluctuated mean body weights were noted for all groups from week 2 ( Figure 4). Reduction in mean body weights occurred at week 7 and rose again at week 8. No signifcant diference in the mean body weights during the experimental period was observed. Furthermore, the observed diferences in body weight were not of toxicological relevance.

Food and Water Consumption.
Overall, food and water consumption varied for all groups (Figures 5 and 6). However, the variation observed was similar and showed no attributable changes after the administration of the product.

Hematology.
Te hematological changes are shown in Table 3. No signifcant changes were observed in comparison with the control group. By the end of the recovery period, no treatment-related diferences were detected in these hematological parameters.

Clinical Chemistry.
Clinical chemistry or biochemical parameters of female SD rats exposed to SKM product (22.5; 45; 90 mg/kg b.w.) by oral route and control rats during the 90-day followed by a 28-day recovery period are shown in  3.2.6. Urinalysis. Urinalysis did not indicate any treatmentrelated changes in the volume, pH, and colors. Several observed changes were considered incidental (shown in Table 5).

Organ
Weights. Relative organ weight changes are shown in Table 6. Statistical analysis showed no signifcant diferences in relative liver, spleen, stomach, and heart weights compared with the control group. Signifcant differences in relative lung weight were found between the 45 mg/kg b.w. and control groups.

Gross Pathology and Histopathology.
In the gross pathological and histopathological examination, the appearance of the heart, lung, spleen, stomach, kidney, liver, and intestine was normal in all groups.

Discussion
For centuries, natural products, such as medicinal plants, have been the basis for the treatment of various ailments [15]. Tis research tries to reveal the efect of several natural products, specifcally from Indonesia. It is an SKM product that contains the combination of Piper crocatum Ruiz. and Pav., Phyllanthus niruri Linn., and Typhonium fagelliforme. Typhonium fagelliforme is a medicinal plant native to Indonesia and can be used as an immunomodulator, antioxidant, and hepatoprotective agent. In addition, Piper crocatum Ruiz. and Pav. and Phyllanthus niruri Linn are the natural products that are originally from Asia. Tese plants have an activity to modulate immune responses including innate and adaptive immune systems, which will give therapeutic benefts for immune-related diseases, such as infection, rheumatoid arthritis, and cancer [16]. Phyllanthus niruri water extract increased the proliferation of B cells and T lymphocytes, production of specifc cytokines such as TNF-α, IFN-c, and IL-4 release. Phyllanthus niruri was also able to stimulate macrophage phagocytosis activity and lysosomal enzyme activity and modulate nitric oxide retention by macrophages [17]. Hartini et al. reported that red betel leaf extract and neolignan compounds isolated from these leaves have an immunostimulating efect [18]. Based on the research by Nurrochmad et al., the ethanol extract of Typhonium fagelliforme tuber was able to increase the phagocytosis ability of macrophages and the proportion of CD8+ T cells in cyclophosphamide-induced rats [2]. Te efect given by these plants is due to their main respective bioactive component. Specifcally, pharmacological properties of Piper crocatum Ruiz. and Pav. are widely known that consist eugenol and hydroxychaviol as bioactive components, including eugenol and hydroxychavicol [19], while the main compound in Phyllanthus niruri Linn. is phyllanthin [2].
In screening natural products for pharmacological activity, the assessment and evaluation of toxic characteristics of a natural product extract, fraction, or compound are usually an initial step. Regardless of the pharmacological benefcial efects of SKM products, detailed knowledge about the acute and subchronic toxicology of this product is lacking. Hence, the current study evaluated the acute and subchronic toxicity of SKM products in an animal model.
In the acute toxicity study, SKM was administered to test animals in high doses (2000 and 5000 mg/kg b.w.) [20]. Tere were no signifcant changes in the central nervous system, somatic motor system, autonomic nervous system, digestive system, skin and hair, mucous membrane, eyes, and organs of the animals during the study.
Mortality is a critical parameter in the toxicity study [21,22]. In this study, the single administration of SKM product up to 5000 mg/kg b.w. did not cause death or any toxic syndromes in the rats. Te acute toxicity test results indicated that the estimated tolerated dose of SKM was greater than 5 g/kg in SD rats. Tus, the LD50 of SKM products was categorized into the unclassifed category in accordance with OECD.
Te toxicity of SKM was evaluated by performing 90-day subchronic oral toxicity studies in female SD rats. Clinical signs could be used to describe the toxic efects caused by the administration of this product. Tis study observed no signifcant changes in general behavior, fur color, eyes, mucous membrane, movement, posture, secretion, and excretion.   Mean body weights were measured to determine the efect of SKM on animal growth. Body weights are closely related to nutrition obtained from food and water consumption. Stress factors could also afect body weight. Te decline in body weight gain is an uncomplicated and sensitive indicator after exposure to a substance [23,24]. Statistical analysis using oneway ANOVA showed no signifcant diferences among the groups (P > 0.05), indicating that the administration of SKM did not afect the mean body weights of all animals.
Changes in organ weight can be used as sensitive indices to evaluate substance toxicity [25]. A signifcant diference in relative kidney weights occurred in the control group. Te diferences in the relative organ weights were considered an incidental change and did not correlate with the administration of the product because of the lack of diferences in the microscopic observation.
No obvious infuences on food and water consumption were noted. Changes in food and water consumption occurred in all groups. Food intake could be afected by the individual needs for energy and environmental factors, such as temperature or light cycle [26]. If the environmental temperature is lower than the animal's average temperature, food consumption will increase to maintain a constant body temperature. Te factors leading to a decrease in food consumption are high temperature, stress, housing condition, and humidity. Te external factor afecting water consumption is the housing condition. High temperature and humidity will decrease water consumption.
Hematological analyses are the essential indices to evaluate the toxicity of substances in humans and animals [27][28][29][30]. Evaluation of hematological parameters shows an efect in the blood due to the administration of a test material. No signifcant diferences were found between the dose and control groups, indicating that the product did not afect hematological parameters.
Biochemical parameters are also the fundamental diagnostic criteria in clinical practice. It may indicate the adverse efect caused by substances [31]. Biochemical  : Food consumption (g/day) of SD female rats exposed to SKM product (22.5; 45; 90 mg/kg b.w.) by oral route and control rats during the 90-day followed by a 28-day recovery period.  analyses are used to defne, detect, and characterize the toxic efects caused by toxic compounds. Te analyses are also crucial for the evaluation of the specifc target organ of toxic compounds and provide valuable information for understanding the disease [32]. UREA and CREA values are the parameters used to evaluate kidney damage. Te liver function can be evaluated by the levels of markers, including ALT, AST, and TP [29]. Te liver and kidney, which are involved in the elimination of xenobiotics, are sensitive organs that can be altered by substances, including plants and drugs [32]. Te signifcant diference between the control group and TP is considered as an individual variation because no changes were found in the histopathological observation. Te levels of UREA, CRE, AST, ALT, TP, TC, and GLU were not afected by SKM compared with the control group. Te urine examination also did not show any change in the group. Tose data indicated that SKM did not alter the functions of the kidney and the liver. Te results of biochemical analyses were confrmed by the histopathological study.
Te histopathological study is intended to observe any abnormalities in the gross pathology and organ histopathology [33,34]. Te histopathological evaluation of the vital organs showed that the structures were normal and that no microscopic changes occurred in the lung, heart, kidney, liver, stomach, intestine, or lymph.   : Water consumption (mL/day) of SD female rats exposed to SKM product (22.5; 45; 90 mg/kg b.w.) by oral route and control rats during the 90-day followed by a 28 day recovery period.

Conclusion
Tis study analyzed the toxicological profle of SKM. Te results suggest that the single administration of SKM is nontoxic to the SD rats. Tus, it is categorized into the unclassifed category in accordance with OECD. Te daily administration of SKM for 90 days did not afect the clinical signs, mean body weights, food and water consumption, hematological parameters (WBC count, RBC count, platelet count, MCH, MCHC, MCV, Hb, and HCT), clinical biochemistry (AST, ALT, TP, TC, and GLU), urinalysis, relative organ weights, gross necropsy fndings, and organ histopathology of the rats.

Data Availability
All data generated or analyzed during this study are included within the article.

Additional Points
Highlights. (i) Te SKM product exerts immunomodulatory activity. (ii) Te SKM product was safe up to 5000 mg/kg b.w. in male and female SD rats. (iii) Long-term oral administration of the SKM product for 90 days does not cause subchronic toxicity.

Disclosure
Part of this work's results and the manuscript has been presented as a Bachelor of Pharmacy thesis in the Faculty of Pharmacy, Universitas Gadjah Mada. Table 5: Urinalysis parameters of female SD rats fed exposed to SKM product (22.5; 45; 90 mg/kg b.w.) by oral route and control rats during the 90-day followed by a 28-day recovery period. Values are mean ± SE.