Bombycis Faeces were purchased from Kwong-Mung-dang Company (Ulsan, Korea) and authenticated by Dr. Goya Choi of the Herbal Medicine Resources Research Center, Korea Institute of Oriental Medicine (Naju, Korea). A voucher specimen (medicinal ID: 2-18-0114) was deposited at the same institution. The Bombycis Faeces extract was prepared by refluxing in distilled water at 100 ± 2°C for 3 hours. The extract was subsequently filtered, concentrated using a rotary vacuum evaporator, and lyophilized (yield, 6.78%). Finally, the resulting powder was stored at 4°C until use (extract ID: 3-18-0044).

Male and female Sprague–Dawley (SD) rats (7-week-old) were purchased from Orient Bio (Seongnam, Korea) and acclimatized for 6 days. During the acclimatization and experimental periods, the animals had ad libitum access to tap water and standard rodent pellets (Teklad Certified Irradiated Global 18% Protein Rodent Diet; Envigo, USA). Rats were housed under controlled environmental conditions (temperature, 22 ± 2°C; relative humidity, 55 ± 10%; 12-h light/dark cycle). All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Korea Conformity Laboratories (Incheon, Korea) in accordance with the Animal Protection Act of the Republic of Korea (approval no.: IA20-02462).

Forty rats (20 males and 20 females) were randomly assigned to four groups (n = 10 per group; 5 males and 5 females): 0 (vehicle control), 500, 1000, and 2000 mg/kg oral doses of Bombycis Faeces extract.

After a 4-h fasting period, the animals were orally gavaged with bombycis fecal extract or vehicle at the designated doses (10 mL/kg body weight). Notably, the administration volume was calculated based on the individual body weights on the day of dosing. Oral administration was selected because it reflected the intended clinical route of administration.

All animals were observed for clinical signs immediately after dosing, and at 30min and 1, 2, 3, 4, 5, and 6 h after administration. Daily observations were conducted for 14 days to monitor clinical signs and mortality. Observations included posture, gait, coat condition, skin, eyes and pupils, mucous membranes, respiratory patterns, response to handling, and any convulsions, stereotypies, or abnormal behaviors.

Individual body weights were recorded immediately before administration and on days 1, 3, 7, and 14 after treatment.

On day 14, all surviving animals were anesthetized using isoflurane and euthanized by exsanguination of the abdominal aorta and posterior vena cava. A comprehensive macroscopic examination was performed on the external surface, cranial cavity, thoracic cavity, abdominal cavity, and their contents.

Blood samples were collected on day 14 under anesthesia for hematological analyses. Hematological parameters, including total white blood cell (WBC) count, differential WBC count, red blood cell (RBC) count, hemoglobin (Hb) level, hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and platelet (PLT) count, were measured using an ADVIA 2120i hematology analyzer (Siemens Ireland, Dublin, Ireland).

Data on body weight and hematological parameters were analyzed using one-way analysis of variance (ANOVA) followed by post hoc comparisons, where appropriate. All statistical analyses were performed using SPSS software (SPSS Inc., Chicago, IL, USA). Statistical significance was set at p < 0.05. All data are expressed as mean ± standard deviation (SD).

No mortality was observed in any of the treatment groups during the 14-day observation period (Table 1). All animals (5/5 in each group of both sexes) survived until the scheduled termination date. Additionally, no abnormal clinical signs were detected in male or female rats at any dose (500, 1000, or 2000 mg/kg). All rats exhibited normal behavioral patterns, including normal posture, gait, coat condition, skin integrity, eye appearance, mucous membranes, and respiratory patterns. Moreover, no instances of lacrimation, convulsions, piloerection, diarrhea, stereotypical behavior, or other abnormal movements were observed in any of the groups.

Body weight changes are shown in Fig. 1 and Table 2. In male rats, initial body weights ranged from 234.17 ± 11.78 to 240.32 ± 6.88 g across all groups, with no significant baseline differences observed. At day 14, body weight range increased to 360.15 ± 13.50 to 374.82 ± 17.89 g, representing weight gains of 120.29 ± 16.63 to 138.86 ± 17.80 g. Notably, the percentage weight gains in the 500, 1000, and 2000 mg/kg groups were 96.09, 96.21, and 99.20%, respectively, compared with that in the control group, indicating no treatment-related effects on body weight gain.

Fig. 1.  Changes in body weight of male (A) and female (B) Sprague-Dawley rats after single oral administration of Bombycis Faeces aqueous extract.

Animals were administered a single dose of the extract at 0 (control, G1), 500 (G2), 1000 (G3), or 2000 mg/kg (G4) and monitored for 14 days. Body weight was measured on days 0, 1, 3, 7, and 14. Data are expressed as mean ± standard deviation (SD), n=5 per sex per group. No statistically significant differences in body weight or weight gain were observed among treatment groups in either sex throughout the study period (p > 0.05).

In female rats, initial body weights ranged from 186.33 ± 5.95 to 188.29 ± 5.56 g. However, the final weights on day 14 ranged from 230.80 ± 15.10 to 244.57 ± 10.36 g, representing weight gains of 42.51 ± 12.20 to 58.23 ± 5.08 g. Importantly, the percentage weight gains in the 500, 1000, and 2000 mg/kg groups were 96.62, 102.38, and 101.22%, respectively, compared with that in the control group. Collectively, these results indicate that there were no significant differences in body weight or weight gain, regardless of treatment or sex.

Table 3 summarizes the gross necropsy findings. Notably, no treatment-related macroscopic abnormalities were detected in any of the examined organs. All animals in the dose groups showed normal gross findings for all organs examined, including the external surface and cranial, thoracic, and abdominal cavities. Collectively, these results indicate the absence of overt organ toxicity following a single oral administration of the extracts.

Tables 4 and 5 present the hematological parameters of male and female rats, respectively. In male rats, no significant changes in any of the hematological parameters were observed across all treatment groups compared with the control group. All measured parameters, including total and differential WBC counts, RBC parameters, and platelet counts, remained within the normal range.

In female rats, statistically significant increases were observed in absolute monocyte counts at all dose levels (500, 1000, and 2000 mg/kg) compared with those in the control group (p < 0.01). Monocyte percentages were significantly elevated in all treatment groups (p < 0.01). Specifically, the absolute monocyte count increased from 0.13 ± 0.04 K/μL in the control group to 0.23 ± 0.04, 0.19 ± 0.05, and 0.20 ± 0.02 K/μL in the 500, 1000 and 2000 mg/kg groups, respectively. Additionally, the monocyte percentage increased from 1.3 ± 0.3% in the control group to 2.7 ± 0.8, 2.3 ± 0.2, and 2.2 ± 0.4% in the 500, 1000 and 2000 mg/kg groups, respectively.

All other hematological parameters in female rats, including total WBC count, neutrophil count, RBC parameters (hemoglobin, hematocrit, MCV, MCH, and MCHC), and platelet count, showed no significant differences compared with those in the control group. Although statistically significant, the observed monocyte counts and percentages were within the normal physiological range in Sprague–Dawley rats.