Subchronic toxicity of herbal compound “Jiedu Huayu” granules in rats

Background “Jiedu Huayu” (JDHY) granules are traditional Chinese herbal compounds that have been used to treat severe liver injury for many years. The purpose of the current study is to evaluate the safety of JDHY granules. Methods Subchronic toxicity was tested in male and female rats that were orally administered three different doses (80, 100, and 130 g/kg/d) of JDHY for 13 weeks. Clinical signs, bodyweight, food consumption, hematological and biochemical parameters, organ coefficients, and histological changes were observed during the study. Results There were no significant changes in toxicity observed in either sex at any dose of JDHY granules treatment. Conclusions These results suggest that repeated oral administration of JDHY granules at dosage levels of ≤130 g/kg/d can be considered safe.


Background
Traditional Chinese herbal compounds have been widely used throughout history. They have been considered to be effective and have few side effects, particularly because of their natural origins [1]. The therapeutic effects of compounds depend on the components of each herb involved. In recent decades, the use of herbal compounds has rapidly increased. However, the safety of herbal compounds has been greatly debated [2].
"Jiedu Huayu" (JDHY) granules are traditional Chinese herbal compounds that are commonly used for the treatment of liver failure. These granules consist of six herbs: Artemisia capillaries, Radix paeoniae rubrathe, Rheum officinale, Oldenlandia diffusa, Radix curcumae, and Acorus gramineus. Previous clinical studies established that JDHY has hepato-protective effects in hepatitis Brelated acute chronic liver failure patients, improved liver function, and reduced complications [3]. Moreover, animal experiments demonstrated that JDHY decreased the expression of Caspase-3 mRNA in a model of acute liver failure. This suggests that JDHY may be an effective therapy for treating hepatocyte-related apoptosis [4]. However, there is currently insufficient investigation into the toxicity of JDHY granules. In this study, we evaluate the toxicity and safety pharmacology of an oral administration of JDHY granules to rats for 13 weeks to assess an optimal dose for clinical use.

Experimental animals
Animal experiments were approved by The First Affiliated Hospital of Guangxi University of Traditional Chinese Medicine. Male and female Wistar rats weighing 100 g-120 g were purchased from the Animal Experimental Center of Guangxi Medical University (Nanning, China, License No. SCXK Gui 2009-0002). All rats were fed a Specific Pathogen-Free (SPF) diet.
of Traditional Chinese Medicine. The herbal mixture underwent combined decoction. It was extracted twice in distilled water at 100°C for 2 h. Following filtration, the resulting mixture was concentrated to a cream with a relative density between 1.05-1.10 (70°C). Next, 60% ethanol was added for 24 h, and following filtration, the resulting mixture was then concentrated to a cream with a relative density of 1.30-1.35 (80°C). Dextrins were added to make the final weight 1000 g, and the equivalent dose was 3.2 g/g (total raw materials/ weight after concentration). Quality control was performed as previously reported [5]. Prior to usage, JDHY granules were configured into three concentrations (6.5 g/ml, 5.0 g/ml, and 4.0 g/ml) by distilled water.

Experimental design
Forty-eight rats were randomized into four groups: control group (gavaged with distilled water,a low dose group (gavaged with JDHY granules 80 g/kg/d), a middle dose group (gavaged with JDHY granules 100 g/kg/d),and a high dose group (gavaged with JDHY granules 130 g/kg/d). Each experimental group consisted of 6 males and 6 females. Following an environmental adaptation period of 1 week, all rats were fed the SPF diet, and their initial weight was recorded. Rats were administered an amount of 20 ml/kg, which was adjusted once a week to accommodate changes in weight. Each group was gavaged for 6 days and rested for 1 day each week for a total duration of 13 weeks.

Clinical observation, bodyweight, and food consumption
Clinical signs were observed twice daily for all rats. Clinical signs included changes in fur, mucous membrane, eyes, physical activity, behavior, fecal excretion, and mortality. Bodyweight and food consumption were recorded once a week [6].      All samples were collected from five rats in each group a Data are given as mean ± SD ANOVA analysis indicated no statistical significance P > 0.05 All samples were collected from five rats in each group a Data are given as mean ± SD ANOVA analysis indicated no statistical significance P > 0.05 MCH concentration (MCHC). All samples were measured using automatic blood cell analyzer (mindray, China).

Hematology and serum biochemistry
The following serum biochemistry parameters were also detected: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), total bilirubin (TBIL), total cholesterol (CHOL), triglycerides (TG), glucose (GLU), blood urea nitrogen (BUN), creatinine (Cr), creatine kinase (CK), sodium (Na), potassium (K), and chloride (Cl). All samples were measured using automatic biochemical analyzer (mindray, China). All samples were collected from five rats in each group a Data are given as mean ± SD ANOVA analysis indicated no statistical significance P > 0.05 All samples were collected from five rats in each group a Data are given as mean ± SD ANOVA analysis indicated no statistical significance P > 0.05
Relative organ weight was calculated as a percentage of body weight [7].

Histopathology
At the conclusion of the experiment, all animals were sacrificed using ether anesthesia. Tissues and organs were harvested and preserved in 10% formalin solution. All samples were sectioned and stained with hematoxylin and eosin [8]. We used a biological microscope and dp-20 All samples were collected from five rats in each group a Data are given as mean ± SD ANOVA analysis indicated no statistical significance P > 0.05 All samples were collected from five rats in each group a Data are given as mean ± SD ANOVA analysis indicated no statistical significance P > 0.05 digital imaging system (OLYMPUS, Japan) to conduct histopathological examination.

Statistical analysis
Spss 18.0 software was used for statistical analysis. Data are presented as means ± standard deviation. Data were analyzed by one way analysis of variance (ANOVA) followed by the Student-Newman-Keuls test. Statistical significance was set at P < 0.05 as compared to the control group.

Results
Clinical signs, food consumption, and body weight We did not observe any death or adverse clinical signs in the JDHY groups during the 13-week experiment. There were no statistically significant changes observed in food consumption (Tables 2 and 3, Fig. 1a) and body weight ( Table 4 and 5, Fig. 1b) between control and JDHF groups.

Hematology and serum chemistry
The results of the hematological detection and serum chemistry examination are shown in Tables 6, 7, 8, and 9. There were no statistically significant changes in hematological detection and serum chemistry examination between the control and JDHY groups in male or female rats.

Organ coefficient
As shown in Tables 10 and 11, JDHY groups did not generate any statistically significant changes in organ coefficients in either male or female rats when compared with the control group.

Histopathology
Following a 13-week treatment with JDHY granules, there were no treatment-related changes in histopathology detected between the control and JDHY groups. In fact, histopathological analysis of liver, lung, and kidney showed normal structure in all groups (Fig. 2).

Discussion
Changes in food consumption and body weight are considered to be an indicator of organ toxicity [9]. In our 13-week study, we observed no deaths or abnormal behavior performance in all groups. While increases in food consumption and body weight were observed for all groups, there were no statistically significant differences between those given different doses of JDHF and the control group. Therefore, different doses of JDHY granules below or equal to 130 g/kg/d had no side effects on growth in male or female rats. The circulatory system is essential for body function. Such functions include providing nutrients to different organs and carrying cytokine factors for immune reaction. Thus, the circulatory system is sensitive to toxicity of drugs [8]. In this study, we did not observe any statistically significant hematological differences between the JDHF groups and the control group. Levels of WBC, PLT, and HGB were in the normal range. Serum ALT, AST, BUN, and Cr can reflect liver and kidney function and are markers of  liver and kidney damage [10,11]. There were no statistically significant differences in these serum parameter levels between the JDHF groups and the control group. Thus, JDHY granules did not have a damaging effect on liver and kidney function. Organ coefficients can provide direct evidence for pathological changes and can indicate organs influenced by drugs. In our study, analysis of organ weights resulted in no statistically significant changes between the JDHF groups and the control group. Furthermore, we used histopathological analysis to determine any pathological changes in liver, kidney, or lung because these organs play a significant role in the accumulation of drugs. We observed no histopathological malignancies in liver, kidney, and lung at a high dose of JDHY group. These results indicate that JDHY granules have no chronic toxicity for critical organs.

Conclusion
Oral administration of JDHY granules to rats for 13 weeks did not have adverse side effects in both male and female rats of a dose up to 130 g/kg/d. Thus, no toxic effects were produced from treatment with JDHY granules.