Safety Evaluation of a New Traditional Chinese Medical Formula, Ciji-Hua'ai-Baosheng II Formula, in Adult Rodent Models

Background Ciji-Hua'ai-Baosheng II Formula (CHB-II-F) is a new traditional Chinese medical formula that has been shown to reduce toxicity and side effects of chemotherapy and increase the probability of cancer patient survival. Whether CHB-II-F is safe as an adjunctive therapy for cancer patients receiving chemotherapy has yet to be determined. Purpose To evaluate the acute and subchronic toxic effects of CHB-II-F in rodent models. Methods In acute toxicity test, 24 Kunming mice were divided into 2 groups: untreated control and CHB-II-F 1.05 g/mL (31.44 g/kg) treated group. Treatment was administered to the treated group 3 times a day for 14 days. The overall health, adverse reactions, and mortality rate were documented. In subchronic toxicity test, 96 Sprague-Dawley rats were divided into 4 groups: untreated control, high dose CHB-II-F (H) (26.20 g/kg), medium dose CHB-II-F (M) (13. 10 g/kg), and low dose CHB-II-F (L) (6.55 g/kg) [equal to 24.375 g (dried medicinal herb)/kg] treated groups. Treated groups were given the treatments once a day for 4 weeks. The overall health and mortality rate were recorded every day. Body weight and food consumption were measured once a week. Hematologic and biochemical parameters, organ weights, and histopathologic markers were analyzed after 4 weeks. An additional 2 weeks were given as the treatment recovery period before end-point euthanization, and biochemical analyses were performed. Results The maximum tolerated dose (MTD) of CHB-II-F on mice was found to be 94.31 g/kg [equal to 351 g (dried medicinal herb)/kg], which is 108 times the human adult dose. In the acute toxicity test, administration of CHB-II-F 31.44 g/kg showed no adverse effect and did not cause mortality. In the subchronic toxicity test, after 4 weeks of treatment, compared to the controls, total cholesterol (TCHO) level, cardiac and splenic indexes, body weights of female rats, and mean corpuscular hemoglobin concentration (MCHC) in the CHB-II-F (H) group were significantly increased; triglyceride (TG) in the CHB-II-F (M) group and liver and splenic indexes in the CHB-II-F (L) group were increased. After the two-week recovery period, biofluid analyses, food consumption, and histopathologic examinations showed no abnormalities. Conclusion Administration of CHB-II-F had no obvious adverse effect on the overall health of rodent models. A daily maximum dose of less than 94.31 g/kg or 6.55 g/kg CHB-II-F for 4 continuous weeks was considered safe.


Introduction
Traditional Chinese medicine (TCM), rooted in ancient Chinese medical practices, has evolved over the past thousands of years and gained popularity worldwide. In particular, Chinese herbal medicine is a branch of TCM that prescribes formulations containing naturally occurring substances to treat diseases. The application of TCM is also versatile [1,2]. Compared to chemical synthesized or pure extracted drugs, herbs utilized in TCM are often considered to have fewer side effects when used in accordance with the principle of TCM [3], but potential adverse reactions may exist under certain contexts. For instance, improper formulation and processing of the herbal formula, as well as unwanted interactions between TCM and other medicines, can all lead to unfavorable responses [4][5][6]. Therefore, even though Chinese herbal medicine has been approved and used extensively in clinics, it is still necessary to evaluate its toxicology in order to ensure the highest quality and safety for usage in patients [7].
In China, the incidence and mortality rates of malignant tumor are increasing drastically. Since 2015, malignant tumor has become the leading cause of death and a major burden of health care costs [8]. At present time, chemoradiation and surgical removal of the tumor are the major therapeutic methods to treat malignant tumor in clinics. However, in addition to killing cancer cells, chemotherapy drugs are also damaging to healthy cells especially the ones that are actively dividing. According to the principle of TCM, chemotherapy drugs can induce toxicity and side effects that further decrease the body's health qi and blood in a cancer patient, which disharmonize the body equilibrium [9,10]. The phenomenon has been described in The Yellow Emperor's Inner Classic (Huang Di Nei Jing): "if healthy qi can be kept interior, pathogens cannot invade; and in order for pathogens to invade, qi must (first) be deficient." To the body's healthy qi, chemotherapy side effect is considered one kind of pathogens. TCM is therefore used to strengthen health qi and eliminate pathogens, increase drug's efficiency and decrease drug's toxicity, reduce toxicity of chemotherapy, and ameliorate unwanted symptoms.
According to TCM, cancer is a malignant disease of the internal organs, four limbs, and head, which is caused by multiple factors such as deficiency of healthy qi, invasion of pathogens and toxin, depression, and disorders in drinking and eating. These factors can induce functional disorder of the internal organs, abnormal circulation of blood and body fluids, and stagnation of qi and blood. Moreover, pathogenic dampness can generate phlegm and its accumulation induces toxic heat in the viscera. All of these can contribute to tumor formation in the long run [11]. Department of Traditional Chinese Medicine of Xiang'an Hospital of Xiamen University has created a new hypothesis for cancer formation based on experience in cancer diagnosis and expertise in TCM and pointed out that tumor formation begins with the imbalance of internal environment, which causes accumulation of multiple pathological factors such as phlegm, dampness, and blood stasis. Although patients undergo surgical operation and chemoradiation therapy, the imbalance of internal environment is not corrected. Therefore, the pathological factors that are still present in the body can still induce relapse of tumor or cancer metastasis [12]. Based on this hypothesis, Xiang'an Hospital of Xiamen University proposed the Ciji-Hua'ai-Baosheng Formula (CHBF), which focuses on reinforcing the body's immunity and removing pathological factors. It has been shown to attenuate the side effects of chemotherapy and restore the balance of internal environment. Clinical observations supported the beneficial effect of CHBF on cancer patients receiving chemotherapy, which reported in a Chinese patent that CHBF was used for the treatment of dozens of lung cancer and primary liver cancer patients, and after years of observation, the results indicated that it could relieve discomfort symptoms and prolong survival time [13]. Laboratory studies have also revealed that CHBF can prolong the lifespan of mice with ascitic H 22 hepatocellular carcinoma, inhibit tumor growth, prevent decrease of white blood cells and platelets, and improve the immune function of H 22 tumor bearing mice receiving chemotherapy [14,15].
Ciji-Hua'ai-Baosheng II Formula (CHB-II-F), as a new Chinese medical formula for reducing the recurrence rate of cancer patients [13], is a second generation formula refined from the original Ciji Hua'ai Baosheng Decoction (CHBD) [15] without changing the principles of treatment in order to better facilitate its subsequent applications and further development. CHB-II-F retains the most important eight medicinals in CHBD and is composed of Radix Codonopsis, Semen Ziziphi Spinosae, Fructus Hordei Germinatus, Pericarpium Citri Reticulatae, Poria, Concha Ostreae, Bulbus Fritillariae Ussuriensis, and Radix Salviae Miltiorrhizae. Radix Codonopsis can fortify the spleen and supplement the deficiency. Fructus Hordei Germinatus, Pericarpium Citri Reticulatae, and Poria can promote digestion, invigorate the stomach, move qi, strengthen the spleen, and dissolve dampness. Concha Ostreae, Bulbus Fritillariae Ussuriensis, and Radix Salviae Miltiorrhizae can soften the hardness, dissipate masses, invigorate blood, and dispel stasis. The formula is designed to remove pathogens and restore healthy qi, which reestablishes balance of the internal environment and decreases the recurrence rate of tumor [13].
Although CHB-II-F has been prescribed extensively in TCM clinics, its toxicity and safety have not been investigated. Therefore, the current study focuses on examining the acute and subchronic effects of CHB-II-F using mice and rats, respectively.  [17]. 96 rats of both sexes were randomly divided into 4 groups: control and three different concentrations of CHB-II-F (26.20 g/kg, 13.10 g/kg, and 6.55 g/kg). Each group contained 24 rats. CHB-II-F groups received intragastric injection of CHB-II-F in a volume of 1 mL/100 g body weight at 2.62 g/mL, 1.31 g/mL, and 0.65 g/mL [CHB-II-F (H), CHB-II-F (M), and CHB-II-F (L), respectively] once per day for 4 weeks. The doses of the three CHB-II-F groups were equivalent to 30, 15, and 7.5 times of the clinical recommended human daily dose, respectively. Distilled water (1 mL/100 g) was used for the control group. Body weight was measured once a week and the injection volume was adjusted according to weight change. After 4 weeks, 12 rats were selected randomly and anesthetized by peritoneal injection of 10% chloral hydrate at 0.3 mL/100 g body weight. Blood was collected from the abdominal aorta. Primary organs including cerebrum, heart, liver, spleen, lung, and kidney were quickly isolated, cleaned with physiological saline, and then weighed. The relative organ weight (ROW) indices (g/g) of rats were determined by organ weight (g) / body weight (g) ×100. The other 12 rats in each group were left without further treatment for another 2 weeks to observe possible reversible toxicity reactions. The behavior and biochemical characteristics of the rats were monitored daily.

Routine Analysis of Stool.
Stool samples from rats were collected during the 4-week treatment and 2-week recovery periods and used in stool saline smear. Presence of helminths, bacteria, cysts, and crystals was examined under the BL203LED Biological Microscope (Chongqing Optec Instrument Co. Ltd., Chongqing, China).

Histopathologic Analysis.
After tissues and organs were isolated and weighed, they were immediately fixed in 10% neutralized formaldehyde solution for at least 24 h and embedded in paraffin. Paraffin sections were cut in 5 m thickness and went through gradient dehydration. The sections were then stained with Hematoxylin and Eosin (H&E). Histologic changes were observed by Intellective Biological Microscope (Olympus Optical Co. Ltd., Tokyo, Japan).

Statistical Analysis.
Parametric data were expressed as mean ± standard deviation (SD) ( ±s). GraphPad Prism 5.0 software (GraphPad Software Inc., La Jolla, USA) was used for one-way analyses of variance (One-Way ANOVA [analysis of variance]). The least significant Evidence-Based Complementary and Alternative Medicine 5 difference (LSD) method was chosen as the post hoc analysis. Difference of P<0.05 was considered statistically significant.

Acute Toxicity Test Results.
After successive intragastric injection of CHB-II-F for 14 days, all animals were alive in control and CHB-II-F treated groups. There were no abnormalities in appearance including hair color and gloss, behaviors and activities, food and water intakes, and excretions of these mice. As shown in Tables 2, 3, and 4, at the end of the experiments, compared to the control group, body weights, food consumption, and weights of organs in the CHB-II-F treated group were not significantly different from controls (P>0.05). Overall, the gross anatomy of primary organs had no abnormalities through macroscopic observation. The clinically recommended daily dosage of CHB-II-F was 3.25 g (dried medicinal herb)/kg. The maximum tolerated dose (MTD) of CHB-II-F on mice was determined by 30 mL/kg × 3 times × 1.05 g/mL [3.9 g (dried medicinal herb)/mL] = 94.31 g/kg [351 g (dried medicinal herb)/kg], which is equivalent to 108 times the adult human dose. Even at such dosage, mouse median lethal dosage (LD 50) could not be measured due to the low toxic effect of CHB-II-F. After successive administration for 14 days, mice weights, food consumption, and indices of primary organs had no statistical difference compared to those of the control group (P>0.05) (see Tables  2, 3, and 4).

Observation on General State of Health.
After 4 weeks of treatment, all animals were alive. There were no abnormalities in the appearance, behavior, and activities of rats in all three CHB-II-F treated groups. No abnormal secretions were found from the eyes, ears, or genitals. Compared to the control group, color and texture of primary organs in the three CHB-II-F treated groups by macroscopic observation had no evidence of abnormalities. Weekly food intake in all three CHB-II-F treated groups also had no statistically significant difference from the controls (P>0.05) (see Table 5).

Body Weight Changes.
After 4 weeks of treatment, body weights of female rats in CHB-II-F (H) treated group were significantly higher than that in the control group (P<0.01), while body weights of other CHB-II-F treated groups had no significant differences (P>0.05) (see Table 6). After the two-week recovery period, body weight changes between the CHB-II-F treated groups and control group remained statistically insignificant (P>0.05) (see Table 7).

Relative Organ Weight (ROW) Indices of Rats.
After 4 weeks of treatment, compared to the control group, the heart and spleen indices of male rats in CHB-II-F (H) treated group were increased (P<0.05; P<0.01). In CHB-II-F (L) treated group, the spleen index of female rats was also increased (P<0.05), but the liver index of male rats in the same group was decreased (P<0.05) (see Table 8). After the two-week recovery period, the ROW indices of rats in all three CHB-II-F treated groups had no statistical differences compared to the control group (P>0.05) (see Table 9).

Hematological Cytologic Analysis.
After 4 weeks of treatment, compared to the control group, MCHC of female rats in CHB-II-F (H) treated group was decreased (P<0.05). All other hematological markers in rats treated with three different concentrations of CHB-II-F had no statistical significance (P>0.05) (see Table 10). After the two-week recovery period, all hematological markers in CHB-II-F treated groups compared to the controls remained statistically insignificant (P>0.05) (see Table 11).

Blood Biochemical Analysis.
After 4 weeks of treatment, CHO of male rats in CHB-II-F (H) treated group compared to the control group was decreased (P<0.05); TG of male rats in CHB-II-F (M) treated group was increased (P<0.05). Other blood biochemical markers showed no statistical difference compared to controls (P>0.05) (see Table 12). After the twoweek recovery period, all blood biochemical markers in all three CHB-II-F treated groups were not statistically different from controls (P>0.05) (see Table 13) 3.3.6. Urinalysis. After both the 4-week treatment period and 2-week recovery period, urine parameters in all three CHB-II-F treated groups had no abnormalities and no statistical differences compared to the control group (P>0.05) (see Tables 14 and 15).

Routine Analysis of Stool.
After both the 4-week treatment period and 2-week recovery period, the stools for routine in different groups were detected, respectively. There were no statistical differences (P>0.05) in the parameters analyzed of stool smears between the control group and the three CHB-II-F treated groups (see Tables 16 and 17).

Histopathologic Analysis of Primary Organs.
To determine if CHB-II-F treatments affected the primary organ tissues of rats, histological analyses were performed. As shown in Figures 1, 2, 3, and 4, after receiving the treatment of CHB-II-F, tissue structure and morphology of the rat immune system including lymph gland, thymus, and 6 Evidence-Based Complementary and Alternative Medicine Note: Data were presented as the mean ±SD from 6 mice. No statistically significant differences were found ( > 0.05). Note: Data were presented as the mean ±SD from 6 mice. No statistically significant differences were found (P > 0.05). Note: Data were presented as the mean ±SD from 6 mice. No statistically significant differences were found (P > 0.05). Note: Data were presented as the mean ±SD from 12 rats after the former 4 weeks and 6 rats after the later 2 weeks. No statistically significant differences were found (P > 0.05). Note: Data were presented as the mean ±SD from 12 rats. Statistical analysis: * P < 0.05, * * P < 0.01 compared with control group (untreated controls).
Evidence-Based Complementary and Alternative Medicine 7 Note: Data were presented as the mean ±SD from 6 rats. No statistically significant differences were found (P > 0.05). Note: Data were presented as the mean ±SD from 12 rats. Statistical analysis: * P < 0.05, * * P < 0.01 compared with control group (untreated controls).
spleen, the digestive system including stomach, large intestine, small intestine, and liver, the urinary system including kidney and bladder, the nervous system including brain, epencephala, brainstem, cervical cord, thoracic cord, and waist marrow, and the reproductive system organs including testis, epididymis, ovary, and uterus all were still normal, as well as that of heart, lung, and adrenal glands. There were no evidence of pathological changes and no abnormalities in all three CHB-II-F treated groups compared to controls.  Note: Data were presented as the mean ±SD from 6 rats. No statistically significant differences were found (P > 0.05).

Discussion
In certain countries around the world, traditional medicine is an integral part of the healthcare system. Medications formulated based on principles of traditional medicine are routinely prescribed to patients by healthcare practitioners. Most components in traditional medications such as those in TCM are naturally occurring and made from animals and/or plants. With the increased recognition of TCM especially Chinese materia medica (herbs) in the world, more and more patients with different health issues have accepted the treatment of Chinese materia medica and/or medical formulas [18]. Although clinical efficacy of TCM has been widely supported, its safety has not been validated by molecular analysis. Unlike allopathic medicine, toxicity and safety of TCM herbs have been based on a process of trial from early records [19]. It is therefore necessary to employ modern tools to identify potential toxicity associated with TCM herbal formulae for their successful application around the world [20]. The present study revealed that several chemical compounds such as 3,4-dihydroxybenzaldehyde, caffeic acid, baicalin, rosmarinic acid, salvianolic acid B, salvianolic acid C, and lithospermic acid were presented in CHB-II-F as determined by UHPLC. These compounds were all watersoluble components and most of them have antioxidant activities, which help remove free radicals [21][22][23][24]. 3,4-Dihydroxybenzaldehyde was reported to have vasculoprotective effects both in vitro and in vivo [25]. Spinosin may attenuate inflammation and regulate memory disorders of Alzheimer syndrome in mice [26]. Hesperidin and nobiletin were the main active components in Pericarpium Citri Reticulatae. Hesperidin is the major component of the flavonoids [27]. Nobiletin has been shown to have anticancer effects in vitro [28] and hesperidin has a similar effect [29]. Nobiletin, often found in citrus fruits, is an innoxious ingredient and the major component in dietary poly (methoxy) flavones. Nobiletin has been shown to induce various biological effects such as reducing inflammation and chemotherapy injury and protecting neuronal cells in mice/rats [30][31][32].
Based on the acute toxicity study of CHB-II-F, there was no death or adverse reactions associated with mice after intragastric injection. The maximum tolerated dose of CHB-II-F was 94.31 g/kg [equal to 351 g (dried medicinal herb)/kg], which is equivalent to 108 times of the daily recommended Based on the subchronic toxicity study, there were no significant changes in the overall health, molecular markers, and survival rate of male and female rats. Changes in body and relative organ weights are used as parameters to evaluate the toxicity of drugs [34], and losing more than 10% of body weight was considered a sign of adverse reactions [35]. In the present study, after 4 weeks of CHB-II-F treatment, the average body weight of female rats in CHB-II-F (26.20 g/kg) group was significantly higher than the controls. After the two-week recovery period, body weights were not significantly different among the three CHB-II-F groups and the controls. Although a large dose of CHB-II-F orally could increase body weight, it is not considered a toxic reaction and may possibly be a good response to CHB-II-F medication. Changes in organ index mark a good indication of drug toxicity [36]. In the present study, most of the organ indices in the three CHB-II-F treated groups had no remarkable difference compared to the controls. Exceptions include an increase in the heart and spleen indices of male rats in the CHB-II-F (H) treated group and the spleen index of female rats in CHB-II-F (L) group. The liver index of male rats in CHB-II-F (L) group was decreased. Changes in organ weight may not directly reflect their functional state, but a decrease in size may signify tissue damage, which hamper drugs metabolism and its therapeutic effect [37]. When the heart is seriously damaged, there will be prominent changes in some routine biomarkers such as LDH, AST, CK, and CK-MB. These biomarkers could Note: Data were presented as the mean ±SD from 6 rats. No statistically significant differences were found (P > 0.05).
be employed for evaluating early cardiac toxicity [38]. Spleen is an important immune organ, and the change of spleen function is an important indicator of the body's immune system [15,39]. The decrease of organ indices indicated that the organs were atrophic or degenerated; and increase of organ indices possibly showed that the organs were engorged, edematous, proliferative, and hypertrophic [40]. There are many factors affecting the changes of organ weights and indices of experimental animals, such as age, batch/lot, gender, and feeding season, as well as whether absolute diet before the removal of animal organs was complete, whether the removal operations were standardized, and whether the weighing was timely to avoid the evaporation of the surface water of the organs [41]. Existing reports pointed out that conceptual data of murine organ indices and biochemical indicators were close to each other on the whole but there was a slight difference, which may be related to the source and breeding environment of animal in different laboratories [42,43]. In our present study, knowledge of the concrete medicinals and bioactive components in CHB-II-F cannot be used to explain these phenomena. Combined with the present results of blood analysis and pathological examination, the above changes of organ indices should not be directly induced by CHB-II-F, and the underlying causes need further study. Biochemical markers in the blood provide valuable information on the effect of drug toxicity to the physiological status within the body [44]. In the subchronic toxicity test for CHB-II-F, only few biochemical markers were altered in the treatment groups compared to control. For example, in the CHB-II-F (H) group, MCHC was decreased in female rats and TCHO was decreased in male rats. In the CHB-II-F (M) group, TG was increased only in the male rats. The blood routine parameters of laboratory animals were less affected Note: Data were presented as the mean ±SD from 12 rats. Statistical analysis: * * P < 0.01 compared with control group (untreated controls).
by environment and other conditions [45]. Previous research has reported that male rats had a significant higher level of MCHC compared to female rats of the same age [46], and our present study also found the same results. However, with additional statistical analysis, it was found that the MCHC change observed had no clinical significance. Blood components such as WBC, RBC, and HGB were similar compared to control. Blood biochemical values can vary based on the genotype and sex of laboratory animals [47]. Blood TG is the main biochemical indicator for internal lipid metabolism, and elevated TG is a risk factor for arteriosclerosis, coronary heart disease, and fatty liver [48]. TCHO is the sum of all cholesterols within lipoproteins in blood, which is a reflection of the lipid synthesis and reserve in the liver [49]. Although TG and TCHO were both increased in some CHB-II-F groups, it was unlikely that these changes were due to toxicity effect. According to research, TG and TCHO can fluctuate due to stress, and these changes were within the normal range of data [50]. Blood ALT and AST are the cardinal indicators of liver injury [51], and ALP is often used as an indicator of liver and gallbladder diseases, especially the obstruction of common bile duct [52]. In the present study, ALT and AST had no significant changes, and ALP was increased in female rats in the CHB-II-F (L) group, which was not considered clinically significant. In addition, all parameters measured between CHB-II-F treated groups and the controls were not significantly different after the 2-week recovery period. In summary, the observed changes during the 4week treatment period may be due to other factors, and the specific reasons needed to be further researched and verified.
Urine samples are used to examine the health and function of the urinary system and used as supporting evidence for the diagnosis of kidney diseases [53]. Stool samples are also used to analyze pathological changes of the digestive tract based on color, microbiota, and the presence of blood. In the subchronic toxicity test, there were no abnormalities found in the urine or fecal matter of the three CHB-II-F groups.
The parameters analyzed showed no significant difference compared to the control group.
The pathological changes of animal organs were evaluated macroscopically and microscopically based on guidelines from medicinal safety regulations [54]. In both the acute and subchronic toxicity tests, no pathological changes were observed in the gross anatomy. Although the organ indices of liver, spleen, and heart were changed in some CHB-II-F treated groups, analysis of tissue structures revealed no abnormalities. No pathological changes were found in the other organs. Taken together, these data strongly suggested that CHB-II-F would not induce toxicity in the body and can be safely administered to patients at regulated dosage.
Due to the complexity of Chinese medical formulas and the time constraint on the experiments, there are several limitations to the present study. First, potential bioactive components of CHB-II-F in addition to the ones identified from the results will need to be carefully characterized. Second, although drugs with a treatment course of 4 weeks [51,55] are adequate in replicating long-term toxicity effect in Phase I clinical trials according to Research Methods in Pharmacology of Chinese Materia Medica [17], drug treatment of more than 6 months will be needed for subchronic toxicity test in Phases II and III clinical trials.

Conclusion
The maximum tolerated dose (MTD) of CHB-II-F was found to be 94.31 g/kg body weight [equal to 351 g (dried medicinal Note: Data were presented as the mean ±SD from 12 rats. No statistically significant differences were found (P > 0.05). And "-" represents that no positive results need to be reported. Note: Data were presented as the mean ±SD from 6 rats. No statistically significant differences were found (P > 0.05). And "-" represents that no positive results need to be reported.  Note: Data were presented as the mean ±SD from 12 rats. No statistically significant differences were found (P > 0.05). And "-" represents no positive results need to be reported.  herb)/kg] in mice in acute toxicity test. No significant visceral pathological change was observed in rats after administration of CHB-II-F at various concentrations for 4 weeks in the subchronic toxicity test, and no adverse reactions were observed in the two-week recovery period after CHB-II-F discontinuance. A daily dose of CHB-II-F less than 94.31 g/kg body weight or 6.55 g/kg body weight administered for 4 continuous weeks was considered safe.