The dry roots of Cimicifuga heracleifolia Komarov (500 g; Chungju Hospital of Korean Medicine, College of Korean Medicine, Semyung University, Chungju, Republic of Korea) were immersed in distilled water and boiled under reflux for 2 h 30 min, and the resulting extract was centrifuged at 5,000 × g for 10 min. The supernatant was concentrated to 300 ml using a rotary evaporator under reduced pressure (Eyela NE-1001; Tokya Rikakikai Co., Ltd, Tokyo, Japan). The concentrates were then freeze-dried using a lyophilizer (Labconco, Kansas, MO, USA) to obtain 92.8 g solid residue, resulting in a yield of 18.6% (w/w).

Healthy gingival tissues were obtained from healthy patients undergoing crown-lengthening procedures. This study was reviewed and approved by the Institutional Review Board of Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea (Seoul, Korea; KC11SISI0348), and informed consent was obtained from all patients.

The tissues were immediately placed in sterile phosphate-buffered saline (PBS; Welgene, Inc., Daegu, Korea) with 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 4°C. The gingival tissue was de-epithelialized, minced, digested with collagenase IV (Sigma-Aldrich) and incubated at 37°C in a humidified incubator with 5% CO₂ and 95% O₂. The non-adherent cells were washed with PBS after 24 h, supplied with essential minimal medium α (α-MEM; Gibco Life Technologies, Grand Island, NY, USA) containing 15% fetal bovine serum (Gibco), 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma-Aldrich), 200 mM L-glutamine (Sigma-Aldrich) and 10 mM ascorbic acid 2-phosphate (Sigma-Aldrich), and fed every 2–3 days. These cells showed the characteristics of stem cells, including colony-forming abilities, plastic adherence and multi-lineage differentiation (osteogenic, adipogenic, chondrogenic) potency. Approximately 3×10⁵ cells were incubated with specific PE-, APC-, BV421-, PerCP-cyTM5.5- or fluorescein isothiocyanate-conjugated mouse monoclonal antibodies for human CD44, CD73, CD90, CD105, CD14, CD45, CD34 and CD19 (BD Biosciences, San Jose, CA, USA). The cells expressed CD44, CD73, CD90 and CD105, but did not express CD14, CD45, CD34 and CD19 when examined by flow cytometry (FACSCanto II; BD Biosciences).

The stem cells were plated at a density of 2.0×10³ cells/well in 96-well plates. The cells were incubated in α-MEM (Gibco Life Technologies, Grand Island, NY, USA) that was composed of 15% fetal bovine serum (Gibco Life Technologies), 100 U/ml penicillin, 100 µg/ml streptomycin, 200 mM L-glutamine (Sigma-Aldrich) and 10 mM ascorbic acid 2-phosphate (Sigma-Aldrich) in the presence of the Cimicifugae Rhizoma extract at final concentrations that ranged from 0.001 to 1,000 µg/ml. The concentrations used were 0 (untreated control), 0.001, 0.01, 0.1, 1, 10, 100 and 1,000 µg/ml, respectively. The morphology of the cells was viewed under an inverted microscope (Leica DM IRM; Leica Microsystems, Wetzlar, Germany) on days 1, 3, 5 and 7. The images were saved as JPEG files.

The analysis of cell proliferation was performed on days 1, 3, 5 and 7. Viable cells were identified using a Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) assay. The spectrophotometric absorbance was measured with a Synergy MX microplate reader (BioTek Instruments, Inc.; Winooski, VT, USA), and the analysis was performed in triplicate.

The results are represented as the means ± standard deviation. A test of normality was performed, and a one-way analysis of variance (ANOVA) with post hoc test was performed to determine the differences between the groups using commercially available software (SPSS version 12 for Windows; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The morphology of the stem cells at day 1 is shown in Fig. 1. Under an optical microscope, the control group showed spindle-shaped, fibroblast-like morphology. The shapes of the cells treated with 0.001–10 µg/ml Cimicifugae Rhizoma were similar to the shapes of the control group. Significant alterations were noted in the 100 and 1,000 µg/ml groups when compared with the control group. The shapes of the cells in the 100 and 1,000 µg/ml groups were rounder, and fewer cells were present.

The morphology of the cells on day 3 is shown in Fig. 2. The shapes of the cells in treated with 0.001–10 µg/ml were similar to the shapes of the control group. Marked alterations in cytoskeletal organization were observed in the 100 and 1,000 µg/ml groups. The shapes of the cells in the 100 and 1000 µg/ml groups were rounder, and fewer cells were present, when compared with the control group.

The morphology of the cells on days 5 and 7 is shown in Figs. 3 and 4, respectively. The shapes of the cells in the 0.001–10 µg/ml groups were similar to the shapes of the untreated control group.

The results of the cell proliferation assay on days 1, 3, 5 and 7 are shown in Fig. 5, 6, 7 and 8, respectively. The cultures that were grown in the presence of Cimicifugae Rhizoma on day 1 showed an increase in the CCK-8 result at 0.001 µg/ml (Fig. 5). The relative value of the CCK-8 assay result for 0.001 µg/ml Cimicifugae Rhizoma was 110.6±15.2%, when the CCK-8 result of the untreated control group on day 1 was considered to be 100%; however, there were no significant differences (P>0.05). On day 3 (Fig. 6), the relative value of the CCK-8 result for 0.001 µg/ml Cimicifugae Rhizoma was 120.2±24.6%, when the CCK-8 result of the untreated control group on day 3 was considered to be 100%. On day 5 (Fig. 7), the cultures grown in the presence of Cimicifugae Rhizoma at concentrations of 0.001, 0.01, 0.1 and 1 µg/ml resulted in increased CCK-8 values (P<0.05). The relative values of the CCK-8 results for 0.001, 0.01, 0.1, 1 and 10 µg/ml Cimicifugae Rhizoma were 133.3±10.0, 132.7±4.2, 131.2±5.8, 131.2±7.3 and 115.5±4.0%, respectively, when the CCK-8 result of the untreated control group on day 5 was considered to be 100% (100.0±7.7%). On day 7 (Fig. 8), The relative value of the CCK-8 result for 10 µg/ml Cimicifugae Rhizoma was 59.2±1.5%, when the CCK-8 result of the untreated control group on day 7 was considered to be 100% (P<0.05).