## 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, caffe 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, caffe 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. 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 \(\upmu\)M SMR showed approximately 50% suppression. Figure 1: Three-dimensional high-performance liquid chromatogram of 70% ethanol extract of _Salviae mitliorrhizae_ Radix. 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, oligonucleotidesomal 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 \(\upmu\)M (Supplementary Figure S1). Figure 2: Cytotoxic effect of 70% ethanol extract of _Salviae miliorrhiae_ Radix (**SMR**), salvianic acid A (**1**), caffeic acid (**2**), rosmarinic acid (**3**), salvianic acid B (**4**), and tanshinone IIA (**5**) on HCT-116 cells. 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 3: Effects of 70% ethanol extract of _Salviae milliorrhizae_ 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. 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].