Safety evaluation of Balanced Health Care Dan—A medicinal formulation containing traditional edible ingredients in lung tumor‐loaded mice

Abstract Chinese formulation‐based medicinal food has been widely used in clinical trials, but its safety is not well studied. In this research, the edible safety assessment of Balanced Health Care Dan—a formulation containing traditional edible ingredients that were initially formulated to reduce side effects for lung cancer patients—was studied in mice based on biochemical and gut microbial analyses. The experimental mice were subcutaneously loaded with lung tumor A549 cells and then administrated with Balanced Health Care Dan (200 mg/kg or 400 mg/kg b.w. in gavage feeding) for 4 weeks. The body weight, blood parameters, and pathogenic phenotype in tissues were examined. No toxicological symptom was found in experimental mice compared with the normal control. Comprehensive analyses were also conducted to evaluate intestinal microbiota that are associated with many diseases. Balanced Health Care Dan modified the gut microbiota structure in a positive way. In conclusion, the Chinese formulation‐based medicinal food has shown no toxicological effect in mice within 4 weeks of feeding experiment and has the potential to be used in clinical trials.

Traditional medicinal food (TMF) offers a promising option to reduce the side effects during cancer treatment (Luo et al., 2019;Zhang et al., 2022). "Balanced Health Care Dan" is a formula that is designed to improve patient's quality of life, and decrease chemotherapy-induced adverse effects. Although most of the TMFs are botanical and have been traditionally considered to be nontoxic, the ingredients of TMF are generally complex with certain substances that might cause additive toxicities (Lin et al., 2018;Zhang & Yuan, 2012). Therefore, edible safety evaluation and clinical studies of TMFs are equally important. Animal-based toxicity evaluation is necessary before the clinical application of these TMFs added to the diet of cancer patients (Shen et al., 2014;Wang, 2015). The current research focuses on the edible safety evaluation of "Balanced Health Care Dan," which can be considered a model for traditional formulabased medicinal food.

| Animals and cells
Totally, 72 BALB/C Nude mice (36 males and 36 females) of 5-week-old with an average body weight of 40-60 g were purchased from Vital River Laboratory Animal Technology Co., Ltd. The animal room was maintained at 23 ± 2°C, with relative humidity of 50 ± 5%. A 12 h light/dark cycle was provided by automated fluorescent illumination. All mice were provided with their diet and water ad libitum. The animal studies were approved by the Animal Care and Use Committee at China Agricultural University and all experiments were performed in accordance with relevant guidelines and regulations (Approval Number: AW02110202-4).
A549 lung cancer cells (ATCC) were cultured in a carbon dioxide cell incubator at 37°C. The medium was Dulbecco's modified Eagle medium basal medium, complemented with 10% fetal bovine serum and 100 U/ml penicillin and streptomycin. The cells were digested with 0.25% trypsin and passaged on alternate days.

| Traditional formula ingredients
The Balanced Health Care Dan was prepared using the following in-

| Mouse lung cancer tumor modeling
Each mouse was subcutaneously inoculated with A549 cells (5 × 10 6 cells/mouse) on the right back. The animals were used for the experiment when the average tumor volume of the group reached over 100 mm 3 . The successful model mice were randomly divided into groups as follows: Control model mice were gavaged with sterile water, while low-dose group and high-dose groups of mice were gavaged with 200 or 400 mg/kg of Balanced Health Care Dan solution for 4 weeks, respectively. The doses used were physiologically relevant to that administered to humans. The treatment was carried out in six consecutive days per week. The daily body weight of the mice was recorded.

| Blood biochemistry
At the end of the experiment, mice were fasted for 12 h before

| Pathology examination
The fresh tissues of the tumor, liver, kidney, spleen, and lung of mice were fixed with 4% paraformaldehyde for 24-48 h. The tissues were dehydrated, waxed, embedded, and sliced. Subsequent HE staining was performed and then examined microscopically by a professional staff.

| Fecal metagenomic analysis
The intestinal contents of mice were collected in sterilized tubes and frozen at −80°C. DNA was then extracted from the intestinal contents according to the instructions in the kit (FDA6512, Beijing Ford Press Technology Co., LTD). The 16S rDNA sequencing and data analysis were performed as reported Xu et al., 2020). Briefly, The V3-V4 region of the 16S rDNA was amplified by PCR with specific primers linked to the barcode. Thermal cycling consisted of initial denaturation at 98°C for 1 min, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 50°C for 30 s, and elongation at 72°C for 30 s. Finally, 72°C for 5 min.
Sequencing libraries were generated using TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina) following the manufacturer's recommendations, and index codes were added. The library quality was assessed on the Qubit@2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. At last, the library was sequenced on an Illumina NovaSeq platform. After data filtering, UPARSE software (uparsev7.0.1001) was used to cluster valid data into Operational Taxonomic Units. Mothur method and SILVA138 (http://www.arb-silva.de/)'s SSUrRNA database were used for species annotation analysis. Qiime software (Version 1.9.1) was used to analyze diversity. R software (Version 2.15.3) was used for PCA analysis. LEfSe software was used for LEfSe analysis, and the filter value of LDA Score was 4 by default (Segata et al., 2011).

| Statistical analysis
The experimental data were presented as mean value ± standard deviation, and the data were analyzed by GraphPad Prism 8. A one-way analysis of variance (ANOVA) was applied with Dunnet-1 post hoc analysis. Differences between values were considered statistically significant at *p < .05, and extremely significant at **p < .01.

| Tumor incidence
A total of 72 mice (36 female and 36 male mice) were used in this experiment, among which 54 mice (27 males and 27 females) were successfully loaded with tumors that met the requirement and were included in the follow-up experiment. The 54 mice were randomly divided into six groups (3 male groups and 3 female groups; 9 mice/ group) following a computerized randomization scheme based on body weight. Four weeks treatment with Balanced Health Care Dan did not significantly affect the body weight of these mice (Figure 1a).

| Analysis of clinical appearance
During the 4-week experiment, no treatment-related adverse effects in the clinical appearance of the animals were observed.
The body weight of male and female mice in the experimental groups was comparable with that of the control group on day 7, day 14, day 21, and at the end of the experiment. Balanced Health Care Dan slightly increased body weight nonsignificantly, demonstrating that the formula did not exhibit any acute toxicity effects on the animals' growth and development (Hamaguchi et al., 2019).

| Analysis of hematology
Hematology index is an important indicator in safety evaluation (Rosa et al., 2018). Serum biochemical profile derangement can re-

| Analysis of pathology in organs
A complete gross necropsy and microscopic anatomic pathological analysis of organs were conducted on all animals after the 4-week feeding study. The liver, kidney, lung, and spleen showed no pathological lesions (data not shown) in the low-dose or high-dose groups.
From the pathological point of view, Balanced Health Care Dan does not have any toxic or adverse effects.

| Analysis of microbiota
Intestinal flora is known as the "second fingerprint" of the body (Duffy et al., 2015;Ursell et al., 2012), which has an enormous impact on the nutrition and health status of the host. Diet directly affects the balance of intestinal microbiota. For edible safety evaluation, analyzing the changes in intestinal microbiota can directly reflect the effects of tested materials on body health (Barko et al., 2017). Therefore, the evaluation of intestinal microbiota is an important part of edible safety evaluation. Thus, in this study, the intestinal contents were used to evaluate the effects of Balanced Health Care Dan.
Based on metagenomic sequencing, in both male and female mice, the relative abundance of intestinal microbiota changed after TMF treatment (Figure 4a,b). Principal coordinates analysis showed significant changes in the intestinal microbiota of male mice in the TMF-treated group compared with the control group, while the effect of TMF on the intestinal microbiota of female mice was relatively small (Figure 4c,d). The Shannon index was used to measure species diversity (Nielsen, 2021). From Figure 4e was negatively correlated with proinflammatory factors (Chung et al., 2020), and the increasing abundance of Muribaculaceae in the study indicated that it could protect intestinal health. The abundance of Lachnospiraceae was increased in the TMF treatment groups. As a potentially beneficial bacterium, Lachnospiraceae participates in the metabolism of a variety of carbohydrates, among which acetic acid, the fermentation product, is the main source of energy for the host (Vacca et al., 2020). In contrast to the control groups, the proportion of Firmicutes to Bacteroidetes in male mice groups did not change but significantly increased in female mice group, suggesting that after high-dose treatment, female mice were more likely to absorb heat to maintain body weight (John & Mullin, 2016;Zhao et al., 2021).
Therefore, to a certain extent, Balanced Health Care Dan improved the intestinal microbiota of mice and then might increase body immunity. This effect was more obvious in male mice. Therefore, from the point of view of intestinal health, Balanced Health Care Dan is not potentially harmful to the body. It needs to be noted that there are huge differences in gut microbiota between human and animal models, which are caused by species-specific differences, evidenced by host-microbial interactions, environment, diet, and genetic responses (Nguyen et al., 2015). Further clinical trial is still necessary for accurate risk assessment.

| CON CLUS ION
In a 4-week animal feeding test, we evaluated edible safety of a traditional formula-based medicinal food called Balanced Health Care Dan by examining behavior performance, body weight, relevant blood parameters, pathological phenotype, and intestinal microbiota in lung tumor-loaded mice. We found no treatment-related adverse effects in this short-term toxicity evaluation experiment. This research provides a new strategy for the edible safety evaluation of TMFs which have the potential to be used for cancer patients.

ACK N OWLED G M ENT
None.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest in this study.