*2.8. Western Blotting*

The apoptosis signaling pathways of HCT-116 cells induced by samples were performed using Western blot analysis [21,22]. Briefly, the cells were seeded in a 6-well plate at 4 × 10<sup>5</sup> cells/well and then incubated. After 24 h, the cells were treated with the indicated concentrations of each sample, and the cells were then incubated for 24 h. Following incubation, the cells were harvested with a scraper and lysed with radio-immunoprecipitation assay buffer (Elpis Biotech, Daejeon, Korea). The protein concentrations were calculated with the Pierce BCA Protein Assay Kit (Thermo Scientific, Carlsbad, CA, USA). The protein samples were separated by electrophoresis in a SDS-PAGE. Then, the proteins were transferred to PVDF membranes (Merck Millipore, Darmstadt, Germany). The membranes were conducted blockading by 5% skim milk. Then, the membranes were probed with primary antibodies for Bax, B-cell lymphoma 2 (Bcl-2), cleaved caspase-7, cleaved caspase-8, cleaved caspase-9, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and poly(ADP-ribose) polymerase (PARP) followed by incubating with secondary antibodies for anti-rabbit IgG (Cell Signaling Technology, Inc., Danvers, MA, USA).

## *2.9. Statistical Analysis*

All experiments were performed in triplicate, and the quantitative data were shown as mean ± SD. Statistical analysis using Student's t-test was conducted and considered statistically significant based on *p*-values less than 0.05.

#### **3. Results and Discussion**

In the present study, we analyzed five bioactive marker components found in SMR, consisting of four phenolic acids (salvianic acid A, caffeic acid, rosmarinic acid, and salvianolic acid B) and one terpenoid (tanshinone IIA). These compounds were separated with resolution >5.0 within 45 min and retention times of 5.87, 11.11, 17.72, 20.42, and 42.51 min, respectively (Figure 1). The content of rosmarinic acid, caffeic acid, salvianic acid A, salvianolic acid B, and tanshinone IIA in the samples was 3.72, 0.123, 1.27, 64.36, and 4.96 mg/g, respectively.

**Figure 1.** Three-dimensional high-performance liquid chromatogram of 70% ethanol extract of *Salviae miltiorrhizae* Radix.

We initially performed a cytotoxic evaluation using HCT-116 human colorectal carcinoma cells. As shown in Figure 2, only tanshinone IIA significantly decreased cell viability in a concentration-dependent manner, whereas 61.6 μM SMR showed approximately 50% suppression.

**Figure 2.** Cytotoxic effect of 70% ethanol extract of *Salviae miltiorrhizae* Radix (**SMR**), salvianic acid A (**1**), caffeic acid (**2**), rosmarinic acid (**3**), salvianolic acid B (**4**), and tanshinone IIA (**5**) on HCT-116 cells.

Many clinical anticancer drugs are known to exert their effects by inducing apoptosis [23]. Apoptosis is a gene-regulated response and, from the morphological point of view, is distinguished by the specific structural changes in cells, such as plasma membrane bleb formation, cell and nuclear shrinkage, oligonucleosomal DNA fragmentation, and chromatin condensation [24]. Morphological analyses showed that both SMR and tanshinone IIA decreased the number of cells and induced signs of cellular apoptosis, such as cellular shrinkage (Figure 3). Moreover, as shown in Figure 4, Hoechst staining also showed that both SMR and tanshinone IIA increased nuclear condensation, suggesting that SMR and tanshinone IIA successfully induced apoptosis, not necrosis, in human colorectal cancer cells. However, tanshinone IIA was not cytotoxic to LLC-PK1 pig kidney epithelial cell, which is normal cell lines, up to 100 μM (Supplementary Figure S1).

**Figure 3.** Effects of 70% ethanol extract of *Salviae miltiorrhizae* Radix (SMR) and tanshinone IIA on apoptosis in HCT-116 cells. (**A**) Morphology changes in HCT-116 cells. (**B**) Fluorescence microscopic images of apoptotic HCT-116 cells stained with Hoechst 33342.

Two major molecular pathways that trigger programmed cell death are the caspase-mediated intrinsic pathway, which is induced by cellular stresses, and the extrinsic pathway, which is related to the death receptor [25]. Both pathways activate the apoptotic caspases, resulting in morphological and biochemical cellular alterations related to apoptosis [26]. In addition, the extrinsic pathway controls cell turnover by decreasing mutant cells. In the extrinsic pathway, cancer cell death is triggered by the interaction with death ligands (such as tumor necrosis factor) and its death receptors. The cancer cell death-initiating complex stimulates the activation of caspase-3 and -8, which are effector and starter caspases, respectively [27,28]. The intrinsic pathway, which is typically activated in response to DNA or cellular damage, stimulates the expression of proteins in mitochondria, such as cytochrome c, which then activates caspase-3 and -9. [27,29]. It was also reported that after cleavage by caspase-9, caspase-3 inhibits reactive oxygen species production and is thus required for efficient induction of apoptosis, whereas caspase-7 is required for apoptotic cell elimination [30]. In our present study, the expressions of cleaved caspase-7 and -8 were significantly increased by tanshinone IIA, but there was no change in that of cleaved caspase-9 (Figure 4).

**Figure 4.** Effects of 70% ethanol extract of *Salviae miltiorrhizae* Radix (SMR) and tanshinone IIA on apoptosis in HCT-116 cells. (**A**) Protein expression of PARP, Bax, Bcl-2, cleaved caspase-7, cleaved caspase-8, cleaved caspase-9, and GAPDH. (**B**) Graph of relative protein expression. Data are the means of experiments performed in triplicate. Data are presented as the mean ± SD. and were analyzed using the Student's *t*-test. \* *p* < 0.05 versus non-treated cells.

Furthermore, anti- and pro-apoptotic Bcl-2 members play critical roles in the mitochondria-mediated pathway. That is, the ratio of anti- and pro-apoptotic proteins (e.g., Bax/Bcl-2) is considered as a determinant of survival or apoptosis of cancer cells [31]. Earlier studies have reported that the anti-apoptotic Bcl-2 members, which consist of Bcl-xl, Bcl-2, Bcl-w, and Mcl-1, exert an important role in the resistance of cancer cells to chemotherapy. Therefore, a reduction in Bcl-2 and an increase in Bax stimulate the apoptosis process and eliminate cancer cells [32]. Our western blotting analysis results showed increased Bax expression and decreased Bcl-2 expression in cells co-treated with tanshinone IIA, which was stronger than SMR (Figure 4); however, no difference was observed in poly (ADP-ribose) polymerase (PARP) expression, which is a parameter for stress and DNA damage in cells.

In summary, we simultaneously analyzed five compounds (salvianic acid A, rosmarinic acid, salvianolic acid B, caffeic acid, and tanshinone IIA) from SMR, and determined their cytotoxic effects on HCT-116 human colon cancer cells. Among the five compounds in SMR, only tanshinone IIA significantly decreased cell viability in a concentration-dependent manner. Both SMR and tanshinone IIA increased nuclear condensation, suggesting that SMR and tanshinone IIA successfully induced apoptosis. We also found that tanshinone IIA induced apoptotic cell death and significantly increased cleaved caspases-7, -8, and Bax expression, as well as decreased Bcl-2 expression in the course of apoptosis. Taken together, our data show that tanshinone IIA is an active ingredient of SMR and may be a useful chemotherapeutic strategy for patients with colorectal cancer.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-3417/10/4/1304/s1.

**Author Contributions:** Conceptualization, Y.-K.C. and K.S.K.; performing experiments and analyzing data, B.K., S.L., and C.-S.S.; C.-S.S.; validation, B.K. and K.S.K.; writing—original draft preparation, Y.-K.C. and K.S.K.; writing—review and editing, K.S.K.; funding acquisition. All authors have read and agreed to the published version of the manuscript.

**Funding:** The present study was also supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) (2019R1F1A1059173).

**Conflicts of Interest:** The authors declare no conflict of interest.
