Acute and 13 weeks subchronic toxicological evaluation of the flavonoid-rich extract of Sophora flavescens

Abstract The roots of Sophora flavescens have a long history of use in Chinese medicine for the treatment of various medical conditions. Flavonoids from the ethyl acetate extract of S. flavescens have shown anti-inflammatory, anticancer, and antidiabetic properties. The objective of this study was to evaluate the toxicological profile of a flavonoid-rich extract of S. flavescens (SFEA). We conducted acute and sub-chronic oral toxicity studies of SFEA in Kunming (KM) mice and Sprague-Dawley (SD) rats. Acute oral administration of 9.0 g/kg SFEA did not result in mortality, clinical signs of toxicity, or abnormal changes in the body weight or food consumption patterns. No significant changes in hematological, blood biochemical, or histopathological parameters were observed. A 13-week sub-chronic toxicity study was conducted in SD rats; the rats were orally administrated with various doses of SFEA (in mg/kg): 0 (control), 40, 80, 400, 800, and 1200. Mortality, clinical signs, or treatment-related changes in body weight, food consumption, hematological parameters, blood biochemical parameters, organ weights, or histopathological parameters were not observed. We found that SFEA is practically nontoxic to KM mice at a dose of 9.0 g/kg and that the no-observed-adverse-effect-level (NOAEL) of SFEA in SD rats is greater than 1200 mg/kg.


Introduction
Sophora is a genus within the Fabaceae family. More than 15 species categorized in this genus have received considerable attention (Aliyu et al. 2007). The roots of Sophora flavescens, also known as 'Kushen,' possess a strong, bitter taste and cold properties. It has long been used in Chinese medicine for the treatment of fever, jaundice, pruritus, dysentery, inflammatory disorders, ulcers, skin infections, and burns (State Administration of Traditional Chinese Medicine 1999, Chinese Pharmacopoeia Commission 2010. Phytochemical research has revealed that the main bioactive components of S. flavescens are flavonoids and alkaloids (Liu et al. 2010, He et al. 2015, Huang et al. 2016. Flavonoids extracted from S. flavescens have received considerable attention due to their various pharmacological activities including anti-inflammatory (Jin et al. 2010), anticancer (Sun et al. 2008), antibacterial (Kuroyanagi et al. 1999), neuroprotective (Park et al. 2009), anti-arthritic (Jin et al. 2010), antimicrobial (Zheng et al. 2008, Oh et al. 2011, and antiangiogenesis (Zhang et al. 2013). The flavonoids derived from S. flavescens showed antimyocardial fibrosis activity and protected the myocardium of rats (Fan et al. 2013). The flavonoids from S. flavescens EtOAc extract demonstrated promising antidiabetic properties in type-2 diabetic KK-Ay mice (He et al. 2015).
The roots of S. flavescens are reported to be harmful at excessive dosages. Matrine is one of the main alkaloids in S. flavescens. Intraperitoneal injection of matrine was found to induce irritability, restlessness, central nervous paralysis, and spasms in mice; the LD50 was found to be 157.13 mg/kg (Wang et al. 2010). Furthermore, long-term oral administration of matrine in mice resulted in the inhibition of sperm activity and sperm malformation (Jing-Li et al. 2007). Studies have reported the developmental toxicity and neurotoxicity of two matrine-type alkaloids isolated from S. flavescensmatrine and sophocarpinein the zebrafish model. Compared with the alkaloids, the flavonoids derived from S. flavescens have less pronounced adverse effects, and studies involving systematic toxicological and safety assessments are scarce. The intravenous LD 50 value of the total flavonoids of S. flavescens in mice was found to be 309 ± 233 mg/kg (Lu et al. 2014). Our previous study indicated that the LD50 value of the flavonoid-rich extract of S. flavescens (SFEA) administered orally in mice is higher than 7500 mg/kg (Yang et al. 2015). However, the sub-chronic toxicological properties of SFEA have not yet been scientifically evaluated.
Despite the broad spectrum of pharmacological and therapeutic properties of SFEA, only a few studies have been carried out evaluating the toxicological effects of SFEA. Hence, we report the results of oral acute toxicity studies of SFEA in KM mice and sub-chronic studies in SD rats. We aimed to evaluate the toxicological profile of SFEA and provide reference material promoting the further use of SFEA as a medicine or as a functional food ingredient.

Materials and methods
The study was conducted in accordance with the Basic & Clinical Pharmacology & Toxicology policy for experimental and clinical studies (Tveden-Nyborg et al. 2018).

Plant material and preparation of extracts
The roots of Sophora flavescens were collected from Lingyuan City, Liaoning province, PR China in September 2012, and identified by Dr. Dingrong Wan of School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan, PR China. A voucher specimen (no. SF20120520) was deposited in the School of Pharmaceutical Sciences, South-Central University for Nationalities. Air-dried roots of S. flavescens (30 kg) were ground and then extracted sequentially by maceration at room temperature with n-hexane (3 Â 50 L, 3 d each), followed by ethyl acetate (3 Â 50 L, 3 d each) and methanol (3 Â 50 L, 3 d each). The solvents were evaporated at reduced pressure to yield 183.8 g, 863.5 g, and 1426.9 g of n-hexane, ethyl acetate, and methanol fractions, respectively. The ethyl acetate fraction of S. flavescens was chemically characterized based on off-line semi-preparative HPLC and NMR, similar to our previous study (Yang et al. 2015).

Animals
Male and female Sprague-Dawley (SD) rats and Kunming (KM) mice, certified specific pathogen-free, were purchased from the Hubei Provincial Center for Disease Control and Prevention (certification no. SCXK (E) 2008-0005). The animals were housed in suspended plastic cages and maintained under controlled temperature (23 ± 2 C), 55 ± 15% relative humidity, and a constant 12 h light-dark cycle. Food and water were provided ad libitum. Prior to the administration of test materials, the animals were quarantined and acclimated for one week. All animals used in this study received care in compliance with the Guide for the Care and Use of Laboratory Animals of China/the National Institutes of Health. All the operations were carried out under the Good Laboratory Practice (GLP) Regulations of the State Food and Drug Administration of China. The experimental procedures and the animal use and care protocols were approved by the Institutional Animal Ethics Committee (IAEC) of South-Central University for Nationalities (approval no. 2014-SCUEC-AEC-0025).

Acute oral toxicity evaluation
Acute oral toxicity tests were conducted according to the technical guidelines of acute toxicity studies for natural and traditional Chinese medicine. The acute oral toxicity of SFEA was carried out to evaluate whether SFEA induces any mortality and adverse side effects when given intragastrically at maximal feasible dosage in KM mice. Forty mice (male to female ratio of 1:1), weighing 20-24 g at the outlet of treatment, were randomly divided into a control group and SFEA treatment group (male to female ¼ 1:1 per group). SFEA was administered to mice by oral gavage in 0.5% sodium carboxyl methyl cellulose (CMC) suspension at a dose of 9.0 g/kg body weight. The control group received intragastric administration of 0.5% CMC (0.1 mL/10 g) alone. Prior to dosing, the mice were fasted overnight for approximately 16 h. Observation of toxic signs was recorded periodically during the first 24 h after administration of test articles and then once a day for 14 days to record any delayed toxic effects. Survival, feed consumption, and body weights were also recorded daily. At the end of the study, blood samples were collected from the retrobulbar venous plexus using capillary tubes under ether anesthesia for hematological and biochemical analysis. After blood collection, mice were euthanized by cervical dislocation, and gross necropsy was conducted. The following hematology parameters were analyzed: white blood cell (WBC) count, hemoglobin (HGB) concentration, erythrocyte count (RBC), platelet (PLT) count, hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC). The biochemical parameters detected included serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), total serum protein (TP), albumin (ALB), total bilirubin (TBIL), alkaline phosphatase (ALP), urea (URE), creatinine (CRE), and blood glucose (GLU).

Subchronic (13 weeks) toxicity evaluation
The subchronic oral toxicity tests were conducted according to the technical guidelines for subchronic toxicity studies for natural and traditional Chinese medicine. One hundred and twenty rats, approximately six weeks old, were randomly divided into a vehicle group (control) and five test groups, each consisting of 10 males and 10 females. The rats were administered either 0.5% CMC (control group) or five different doses of SFEA (40, 80, 400, 800, and 1200 mg/kg body weight) once daily by oral gavage at 10 mL/kg for 13 weeks. SFEA was dissolved in 0.5% CMC suspension and administered to mice at five different doses (40, 80, 400, 800, and 1200 mg/kg body weight). SFEA was administered repeatedly by oral gavage at a dose volume of 10 mL/kg body weight for 13 weeks.
All rats were weighed twice per week in the first four weeks and once a week thereafter, and food consumption was measured weekly. The animals were observed daily for toxicological signs, and the behavioral changes were recorded. At the end of 13 weeks, all rats were fasted overnight and anesthetized. Blood samples were collected from the abdominal aorta for hematological and biochemical analysis.

Clinical observations, body weight, and food consumption
General conditions and mortality were checked twice daily (morning and afternoon), and body weights were measured and recorded weekly during the experimental period. The amounts of supplied and remaining food were weighed weekly to calculate the mean daily food consumption. The animals were observed daily for clinical signs and toxic symptoms, while clinical examinations, including changes in the skin, fur, eyes, behaviors, appearance, fecal characteristics, and respiration, were recorded.

Hematological and serum biochemical evaluation
All rats were fasted overnight at the completion of the treatment. The blood samples were collected from the abdominal aorta and used to determine hematological and biochemical parameters, as described in Section 2.4.3.

2.4.3.
Macroscopic examination and relative organ weights A complete necropsy was performed for all animals. Animals were euthanized with the collapse of the circulation by drawing blood from the abdominal aorta. The necropsies included an examination of the visceral organs, external surfaces, all orifices, and the cranial, thoracic, and abdominal cavities. The gross pathological examinations were conducted for all animals. The following vital organs were collected and weighed: heart, liver, spleen, lungs, kidneys, testes (males only), uterus (females only), brain, and pancreas. The paired organs were weighed together. The relative organ weights were calculated against the fasting body weights.

Histopathological evaluation
At the time of necropsy, the heart, liver, spleen, lungs, kidneys, bladder, testes, uterus, brain, and pancreas were collected and weighed. Testes were fixed in modified Davidson's fluid, while others were fixed in 10% neutral-buffered formalin. The collected samples were then embedded in paraffin. The sections (4-8 lm) were cut and stained with hematoxylin and eosin. Microscopic examinations were first performed on all tissues of the control and 1200 mg/kg SFEA treated groups. If treatment-related changes appeared at the highest dose (1200 mg/kg), we conducted extensive examination using the corresponding organs or tissues from mice treated with lower doses.

Statistical analysis
Data were expressed as mean ± standard deviation (SD) and analyzed by one-way ANOVA followed by Tukey's post hoc test using GraphPad prism 5.0 software (La Jolla, CA). A p values of less than 0.05 was considered statistically significant.

Acute oral toxicity of SFEA in Kunming mice
In the acute toxicity experiments, all animals survived the 14 days observation period and did not exhibit any clinical signs post-administration of 9.0 g/kg SFEA. Oral LD50 of SFEA in KM mice was found to be greater than 9.0 g/kg. We observed normal body weight gains (Supplementary Table 1) and food consumption (Supplementary Table 2) in males and females of both groups, indicating no toxic effects. The hematological parameters, including WBC, HGB, RBC, PLT, HCT, MCV, MCH, and MCHC in mice treated with SFEA, were not remarkably different to the parameters of the control group mice (Supplementary Table 3). Similarly, no significant differences in the biochemical parameters, including ALT, AST, TP, ALB, TBIL, ALP, URE, CRE, and GLU, were observed between the control and SFEA treatment groups (Supplementary Table 4). The macroscopic examination of the organs revealed no changes in the necropsies of the mice.

Clinical observations, body weights, and organ weights (absolute and relative)
No mortality or treatment-related clinical signs were observed at any of the SFEA doses throughout the experimental period. No differences in the functional behavior between male and female rats of the SFEA treatment groups and controls were observed during the observation period. In general, all groups of rats exhibited a similar level of movement during all intervals.
As shown in Figure 2(A,B), all treated rats in each of the dosage groups continued to gain weight. Compared with the male rats, the female rats demonstrated significantly lower mean body weight gain at different SFEA dosages each week. Mean body weights of male and female rats in SFEA 1200 mg/kg group at the tenth week and eighth week, respectively, were slightly less as compared with the mean body weights of the control group. However, the differences were not statistically significant. Meanwhile, no significant differences (p > 0.05) in the food consumption ( Figure 3) and absolute and relative organ weights were observed between the different groups of rats (Tables 1 and 2).

Hematology and serum biochemistry
Hematological analysis revealed that the MCHC in male rats was significantly reduced at 40 mg/kg SFEA (p < 0.05) dose, while other hematological parameters were unchanged even after 13 weeks of treatment (Table 3). Statistically significant (p < 0.05) reduction of ALB was observed in 80 mg/kg SFEA treated male rats (Table 4). These variations in hematological or biochemical parameters were considered nonspecific or incidental since the lack of any dose-response relationship suggests the absence of any association with test article exposure. Further, we observed no treatment-related abnormalities and significant histopathological changes.

Conclusions
In conclusion, the single orally administered dose of 9.0 g/kg SFEA did not produce acute oral toxicity in KM mice. These findings show that the single-dose oral LD50 for KM mice is greater than 9.0 g/kg. Hence, SFEA was classified as a relatively harmless or nontoxic substance. The results of the 13 week sub-chronic oral toxicity study suggested that daily oral administration of 40, 80, 400, 800, and 1200 mg/kg SFEA did not cause mortality or toxic clinical signs or other abnormalities in rats of either sex. The no-observed-adverseeffect-level (NOAEL) of SFEA is proposed to be greater than 1200 mg/kg in accordance with daily oral administrations to SD rats for 13 weeks.

Discussion
Considering the broad spectrum of pharmacological activities, potential health benefits, and scarce relevant toxicological Values are mean ± SD for 10 rats in each group. Values are mean ± SD for 10 rats in each group. studies of SFEA, there is a pressing need to conduct its toxicological evaluation and safety assessment. We conducted an acute oral toxicity study in KM mice and a 13-week subchronic study in SD rats to carry out a comprehensive safety evaluation of SFEA.
In the acute oral toxicity study, no mortality was observed in mice treated with a single dose of SFEA (9.0 g/kg). All mice exhibited normal behavior and normal physical condition with no significant abnormalities in terms of clinical signs or changes in body weight or food consumption. No Values are mean ± SD for 10 rats in each group. Ã Statistically significant compared to control (p < 0.05).  toxicologically relevant changes were observed in hematology, clinical biochemistry, or macroscopic findings. The results showed that SFEA is unlikely to be toxic at the dose of 9.0 g in KM mice, and the LD50 of administered orally SFEA in KM mice is greater than 9.0 g/kg.
In the 13-week sub-chronic oral toxicity study, we observed no SFEA-related deaths or abnormalities in clinical signs. All rats continued to gain weight throughout the study period, and no statistically significant differences in mean body weights were observed between normal control and different SFEA groups. Slight decreases in the body weights of male and female rats of the SFEA 1200 mg/kg group were observed after the tenth week and the eighth week, respectively. One possible explanation of the alterations in the body weights of rats is that S. flavescens has antidiabetic and antihyperlipidemic properties and regulates glucose and lipid metabolism (Kim et al. 2008, Yang et al. 2015. Furthermore, the reduction in body weights was not found to be associated with other clinical abnormalities. There were no significant differences between different SFEA groups and the control group in terms of food consumption and relative organ weights in either sex. Hematological and serum biochemical examinations were performed to evaluate those aspects of health that may not be visible during clinical observations. The hematopoietic system is sensitive and can be affected by toxic substances. The serum biochemical profile is an important index that indicates the function of the principal organs. Among the biochemical parameters, plasma GLU is used to indicate the effects of a test substance on glucose metabolism. The parameters that reflect hepatic and renal function -ALT, AST, ALP, TP, BUN, and Creare widely used to diagnose various hepatorenal diseases (Aliyu et al. 2007, Gnanamani et al. 2008, Chen et al. 2014, Kandhare et al. 2015. In this study, a significant decrease in the level of MCHC was observed in male rats treated with 40 mg/kg SFEA, but the level was still within the normal range (Petterino andArgentino-Storino 2006, Han et al. 2010). The reduction of ALB in the 80 mg/kg SFEA treated male mice was regarded as incidental due to the lack of any dose-response relationship. Due to the lack of a dose-response relationship, the low magnitude of the differences, and the absence of related abnormalities and significant histopathological changes, these alterations were assumed to be toxicologically irrelevant and unrelated to test article administration. Except for the changes in these parameters, there were no significant changes in hematological or clinical biochemical parameters. For instance, we observed no significant changes in ALT, AST, ALP, TP, or Cre in the SFEA treated groups as compared with the control group, demonstrating that SFEA shows no hepatorenal toxicity to the liver and kidney functions.
At the time of necropsy, internal organs, including the heart, liver, spleen, lungs, kidneys, bladder, testes, uterus, brain, and pancreas, were collected and examined. Some test substances may cause cellular damage to the internal organs or tissues without showing any significant abnormalities. To this end, a histopathological examination was conducted to identify any cellular damage. During the histopathological examination, histopathological lesions were sporadically detected in the liver, lung, kidney, and heart of the control rats and the rats administered with 1200 mg/kg of SFEA. They were slight rather than severe and isolated to individual rats. No consistent histopathological changes were observed in either sex. These lesions were considered spontaneous or incidental in nature and irrelevant to SFEA treatment.