Evaluation of Oral Subchronic Toxicity of Soshiho-Tang Water Extract: The Traditional Herbal Formula in Rats

Soshiho-tang (Xiao-chai-hu-tang in Chinese and Sho-saiko-to in Japanese) has been widely used for its various pharmacological effects, which include anti-inflammatory, antioxidant, antihepatic fibrosis, and antitumor properties. To evaluate the safety of Soshiho-tang water extract (SST), we tested its subchronic toxicity in male and female Crl:CD (SD) rats. Rats were orally treated with four different doses (0, 500, 1000, and 2000 mg/kg/day) of SST administered for 13 weeks. Mortality, clinical signs, body weight changes, food and water consumption changes, ophthalmology, urinalysis, hematological and biochemical parameters, gross findings, organ weights, and histological markers were monitored during the study. The SST treatment did not result in any toxicologically significant changes in mortality, clinical signs, body weights, food and water consumption, ophthalmoscopy, urinalysis, hematological and serum biochemical parameters, gross findings, organ weights, or histopathology. Histological analysis did not show any liver or kidney alteration. We concluded that the 13-week repeated oral administration of SST did not cause any adverse effects in rats at dosage levels of ≤2000 mg/kg/day. Under these experimental conditions, the no-observed-adverse-effect level was concluded to be 2000 mg/kg/day for both sexes.


Introduction
Medicinal herbs are used for the prevention and treatment of disease and have a long history. Because traditional herbal medicines have few side effects and are very effective, the consumption of these medicines has increased substantially in recent decades [1]. With increasing herbal medicine consumption, concerns have been raised over the lack of quality control and scientific evidence of the efficacy and safety of herbal medicine [2][3][4].
Soshiho-tang (Xiao-chai-hu-tang in Chinese and Shosaiko-to in Japanese) is frequently used for the treatment of liver disorders. It is composed of seven herbs: Bupleuri Radix, Scutellariae Radix, Ginseng Radix, Pinelliae Tuber, Glycyrrhizae Radix et Rhizoma, Zingiberis Rhizoma Crudus, and Zizyphi Fructus (Table 1). According to previous studies, Soshiho-tang exhibits various pharmacological properties including anti-inflammatory [5], antioxidant [6], immunomodulating [7], hepatoprotective [8], antihepatic fibrosis [9], and antitumor effects [10]. Although Soshihotang has various pharmacological properties, few scientific studies have investigated its safety and toxicity. Recently, the complexity of herbal prescriptions in general, and their inherent biological variations, was demonstrated; it is now necessary to evaluate their safety, efficacy, and quality [1]. However, the toxicity and quality control of herbal formulas to the public is becoming a problem because of the lack of scientific evidence about herbal formulas. Because few studies have explored the safety and toxicity of herbal formulas, public concern has been raised regarding their potential adverse effects [11].
The present study examined the safety of the oral use of SST for 13 weeks. The study was conducted in accordance with the guidelines established by the Organization for Economic 2 Evidence-Based Complementary and Alternative Medicine Cooperation and Development (OECD, TG 408) [12] for the testing of chemicals and recent Good Laboratory Practice (GLP) Regulations.

Reagents and Materials.
Liquiritin, baicalin, and glycyrrhizin were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The purity of each component was determined to be above 98% by high-performance liquid chromatography (HPLC) analysis. HPLC-grade reagents, methanol, acetonitrile, and water were obtained from J. T. Baker (Phillipsburg, NJ, USA). Glacial acetic acid was of analytical reagent grade, procured from Junsei (Tokyo, Japan). The materials used to form Soshiho-tang were purchased from Omniherb (Yeongcheon, Republic of Korea) and HMAX (Chungbuk, Republic of Korea). A voucher specimen (2008-KE26-1∼KE26-7) was deposited at the Basic Herbal Medicine Research Group, Korea Institute of Oriental Medicine.

Preparation of Soshiho-Tang Water Extract (SST) and
Sample Solutions. A decoction of Soshiho-tang was prepared in our laboratory from a mixture of chopped crude herbs (Table 1), extracted in distilled water at 100 ∘ C for 2 h. The solution was evaporated to dryness, and freeze-dried (yield: 22.9%). Lyophilized Soshiho-tang extract (200 mg) was dissolved in distilled water (20 mL). The solution was then diluted tenfold with distilled water. The solution was filtered using a SmartPor GHP syringe filter (0.2 m, Woongki Science, Seoul, Republic of Korea).

Preparation of Standard Solutions.
A methanol standard stock solution containing liquiritin, baicalin, and glycyrrhizin (all 1.0 mg/mL) was prepared and diluted to the appropriate concentration range for the establishment of calibration curves. The calibration curves were calculated using the peak areas of standard solution in the range 3.91-250.00 g/mL for the three compounds.

HPLC Analysis of SST.
A Shimadzu LC-20A HPLC system (Shimadzu Co., Kyoto, Japan) consisting of a solvent delivery unit, an online degasser, a column oven, an autosampler, and a PDA detector was used. The data processor employed LCsolution software (version 1.24). The analytical column used was a Gemini C18 (250 × 4.6 mm; particle size 5 m, Phenomenex, Torrance, CA, USA). The mobile phases consisted of solvent A (1.0%, v/v, aqueous acetic acid) and solvent B (acetonitrile with 1.0%, v/v, acetic acid). The gradient flow was as follows:  Healthy male and female rats were randomly assigned to four experimental groups: SST 0, 500, 1000, and 2000 mg/kg/ day groups and a vehicle control group. Each experimental group consisted of 10 rats of each sex.
In the previous 4-week repeated-dose study of SST, no treatment-related toxic changes were observed at the highest dose of 2000 mg/kg/day. Therefore, the highest dose level of 2000 mg/kg/day, with a common ratio of 2, was chosen for this 13-week repeated-dose study. Oral administration was selected in the present study, because the oral route is the clinically intended route for the administration of Soshihotang. The SST was suspended in distilled water and was freshly prepared daily before treatment for 13 weeks. The daily application volume (10 mL/kg of body weight) of SST was calculated in advance, based on the most recently recorded body weights of individual animals.

Clinical Observations and Mortality.
All animals were observed twice daily and clinical signs and mortality (if any) were recorded. Body weight and food and water intake were monitored weekly. After a one-week acclimation period, rats were observed twice daily (before and after the daily treatment) for the presence of common symptoms and mortality. Food and water consumption was recorded once before treatment onset and approximately once a week thereafter by measuring the difference between the amounts provided, and amounts remaining on the following day. This was regarded as daily consumption. External eye examinations were performed during the pretest period and all animals in each

Urinalysis, Hematology, and Serum Biochemistry.
Fresh urine was collected from all animals, and urinalysis was conducted to assess urine volume, specific gravity, pH, and the levels of protein, ketone bodies, glucose, bilirubin, nitrite, urobilinogen, color, cast, epithelial cells, erythrocytes, leucocytes, and occult blood using a Multistix 10SG instrument (Bayer, USA) and a urine chemical analyzer (CliniTek 500, Bayer, USA). Fresh blood samples were analyzed to determine the red blood cell count, hemoglobin concentration, hematocrit level, mean corpuscular cell volume, mean corpuscular cell hemoglobin, mean corpuscular cell hemoglobin concentration, platelet count, white blood cell count (WBC), differential WBC count, reticulocyte count, prothrombin time, and activated partial thromboplastin time. All parameters were measured using an ADVIA 120 Hematology System (Bayer, USA). In addition, prothrombin time and activated partial thromboplastin time were determined from blood samples treated with 3.2% sodium citrate, using a coagulometer (Coagrez-100s, Japan).

Linearity, Range, Limit of Detection (LOD), and Limit of Quantification (LOQ).
Calibration curves of liquiritin, baicalin, and glycyrrhizin were obtained using standard solutions. The coefficients ( 2 ) of the calibration curves for the three constituents were ≥0.9993 (Table 2). LOD and LOQ were determined based on signal-to-noise (S/N) ratios of 3 and 10, respectively. The LOD and LOQ values were 0.09 and 0.29 g/mL for liquiritin, 0.41 and 1.37 g/mL for baicalin, and 0.21 and 0.70 g/mL for glycyrrhizin.

HPLC Analysis of SST.
Our analysis method was applied to the simultaneous determination of three compounds in SST: liquiritin, baicalin, and glycyrrhizin. The retention times of the three compounds were 12.99, 19.64, and 32.71 min for liquiritin, baicalin, and glycyrrhizin, respectively. The stability test was analyzed using the sample solution at 0, 1, 2, 5, 7, and 10 days. The relative standard deviation values of retention time and peak area were within 1.12% and 3.09%, respectively ( Table 2). These results suggest that the solution was stable for at least 10 days. Figure 1 shows chromatograms of reference components and SST, with detection of eluents at 254 and 275 nm. The concentrations of the three components identified in SST were 2.33-61.58 mg/g. These results are summarized in Table 3.

Mortality and Clinical
Signs. There was no mortality attributed to any effect of SST during the 13 weeks of drug administration. No SST treatment-related clinical signs were detected, with the exception of loss of fur, bite wounds, scratch wounds, eye discharge, and scabs.

Body Weight and Food Consumption.
There were no significant differences in body weight (Figures 2(a) and 2(b)) or food consumption between control and treatment groups.  Also, there was no clear food consumption in SST-treated groups compared with control group (data not shown).

Ophthalmological Examination and Urinalysis.
There were no abnormal ophthalmologic findings among the SSTtreated groups compared with each sex of the control group in the present study (data not shown). There were no significant changes in urinalysis results in any of the SST-treated groups compared with each sex of the control group (Table 4).

Hematology.
The results of the hematological examination are shown in Table 5. No treatment-related changes in hematological parameters were observed in the present study. A prothrombin time delay was observed in the 2000 mg/kg/day male groups.

Serum Biochemistry.
The results of the examination of serum biochemical values in the SST-treated and control groups are shown in Table 6. In the male 1000 mg/kg/day group, the levels of TBIL were significantly decreased. No other toxic-related serum biochemical value changes were observed in any group.

Relative Organ Weight.
The results of relative organ weights are shown in Table 7. There were no significant differences related to SST treatment in the relative organ weight of male or female rats. In females, the relative kidney weight was significantly increased in the 1000 and 2000 mg/kg/day groups, and the relative liver and heart weights were significantly increased in the 2000 mg/kg/day group.

Histopathology.
No treatment-related histopathology findings were observed after 13 weeks of SST treatment, and there were no histologic correlates for the organ weight changes (data not shown). Histopathological changes included minimal cardiomyopathy of the heart and foamy macrophages of the lungs in the male 2000 mg/kg/day group, and genital organs and estrus cycles were increased in the female group compared with the control group. These changes were not considered to be treatment related, because these microscopic changes are commonly observed in untreated laboratory animals and the incidence and severity were comparable between the treated and control animals. Histopathological features of the kidney (Figure 3(A)) and liver (Figure 3(B)) in the control group and the highest dosage SST-treated groups showed normal structure.

Discussion
Herbal medicines have become increasingly popular in modern societies around the globe, and many research projects have conducted the studies on herbal medicine's pharmacological properties to establish its scientific evidence. Evidence of therapeutic materials is assessed throughout various sources including toxicity, pharmacological properties, clinical trials, and systematic reviews [13]. Traditional herbal medicine is the most important part of 6 Evidence-Based Complementary and Alternative Medicine  complementary and alternative medicine and has been practiced for thousands of years. Although there are many traditional herbal medicines available, and some have been verified by clinical trials, their efficacy and safety are still questioned by consumers [14].
Some traditional herbal preparations in Korea and Japan are officially recognized as approved ethical drugs, and, in some cases, are covered by the National Health Insurance Program. Therefore, to confirm the safety of SST, the present study assessed its repeated-dose oral toxicity for 13 weeks in Evidence-Based Complementary and Alternative Medicine 7  female and male Crl:CD (SD) rats. The mortality and changes in body weight, food consumption, clinical signs, urinalysis, ophthalmological examination, hematology, serum biochemistry, gross observation, organ weight, and histopathology were monitored in accordance with the KFDA and OECD guidelines. Overall, SST was not associated with adverse effects in doses up to 2000 mg/kg/day administered for up to 13 weeks in both sexes. Herbal medicine, a mixture of several herbs are considered to decrease its toxicity and enhance or prolong the pharmacological effects of any component. Toxicity assessment of SST has been studied in Japan. Previous study demonstrated that administration of SST did not cause any adverse effects at 1000 mg/kg/day [15]. However, dose of SST in previous study was comparatively lower than that used in our study, and toxicity study has been conducted under non-GLP conditions. Therefore, the present study has been conducted to clarify the safety of SST at GLP Regulations.
Clinical signs of salivation were observed in female ( = 10) and male ( = 2) 2000 mg/kg/day SST-treated rats. However, these were not considered to be related to SST treatment because incidence was low and no dose-response relationship was observed.
There were no changes in body weight, food consumption, urinalysis, or ophthalmology. No abnormal changes were detected regarding hematological and serum biochemical parameters up to the highest dosage of SST, 2000 mg/kg/day; the changes in both sexes of treated rats were comparable to the changes in both sexes of control group rats.
Statistically significant increases in relative liver, kidney, and heart weights were observed in female rats treated with SST. However, these changes were not correlated with the SST dosage and were within the normal range. Moreover, no gross pathological findings or histopathological changes were observed. Therefore, these changes in body weight were not considered to be SST-induced abnormalities.
Histopathological findings showed cardiomyopathy of the heart and foamy macrophages in the lungs in the male 2000 mg/kg/day rats. In the female 2000 mg/kg/day rats there was an increase in the estrus cycle compared with the control group. However, there were no histopathological findings for the ovaries or uterus. Although histopathological lesions were sporadically observed in the organs in male and female SSTtreated rats, the degree and incidence were similar to those of the control groups. Moreover, these findings are commonly observed in normal rats [16][17][18]. Therefore, it is reasonable to conclude that they were of little toxicological significance.
In conclusion, repeated oral dose administration of SST to SD rats for 13 weeks did not cause any adverse effects in rats of either sex, up to and including doses of 2000 mg/kg/day. Thus, the no-observed-adverse-effect level was concluded to be 2000 mg/kg/day for male and female rats.