**1. Introduction**

Colorectal cancer is the third-most commonly diagnosed cancer worldwide [1]. Although surgery plays a key role in the diagnosis and treatment of colorectal cancer, there are still increasing attempts to stop the progression of this cancer via the application of new synthetic and naturally-occuring compounds [2,3]. Bioactive compounds from plants have been screened for anticancer activities [4,5]. Approximately 50–60% of cancer patients in the United States utilize complementary and alternative medicines with traditional therapeutic regimens, such as radiation therapy and chemotherapy [6].

Apoptosis pathways are important targets in cancer-related therapies, and insufficient apoptosis results in uncontrolled cancer cell proliferation [7]. The use of natural phytochemicals for inhibiting cancer cell proliferation and inducing apoptosis contributes to promoting cancer cell death [8,9]. Natural phytochemicals are multiple-target molecules found in plants and microorganisms, and they exert strong anticancer activity [10,11]. Phytochemicals isolated from natural sources also exhibit various beneficial effects against inflammation, cancer, and neurodegenerative disorders [10]. This

broad spectrum of biological and pharmacological activities has made natural compounds suitable candidates for treating multifactorial diseases, such as colorectal cancer.

*Salviae miltiorrhizae* Radix (SMR) is one of the well-known traditional herbal medicines and has been used in Asian countries [8]. Recently, there has been increasing scientific attention towards SMR for its remarkable bioactivity against cardiovascular disease, renal damage, tumor angiogenesis, and tumor cell invasion [12–14]. In the last decade, accumulating evidence has shown that SMR exerts a significant anticancer e ffect against promyelocytic leukemia, breast cancer, ovarian carcinomas, and hepatocellular carcinoma (HCC) [15–17]. In a recent network pharmacology-based study on the anti-HCC e ffect of SMR, 62 chemical compounds form SMR yielded 101 putative targets that played a critical role in HCC via multiple targets and pathways, especially the EGFR and phosphatidyl-inositol 3-kinase (PI3K)/Akt signaling pathways [18]. However, the e ffect of SMR and its compounds on human colon cancer cells has not been fully elucidated. The aim of the present study was to simultaneously analyze the compounds of SMR and determine their cytotoxic e ffects on HCT-116 human colorectal cancer cells.

#### **2. Materials and Methods**

## *2.1. Plant Materials*

*Salviae miltiorrhizae* Radix (SMR) was obtained from Kwangmyungdag Medicinal Herbs (Ulsan, Korea) and identified by Dr. Goya Choi, Herbal Medicine Resources Research Center, Korea Institute of Oriental Medicine (HMRRC, KIOM; Naju, Korea). A voucher specimen (SMR-2-14-0073) was stored at the herbarium of the HMRRC, KIOM.

#### *2.2. Chemicals and Reagents*

Five reference standard compounds, salvianic acid A (98.0%), ca ffeic acid (99.0%), rosmarinic acid (97.0%), salvianolic acid B (98.0%), and tanshinone IIA (98.8%) were purchased from standard manufacturers: Acros Organics (Pittsburgh, PA, USA), Merck KGaA (Darmstadt, Germany), and ChemFaces Biochemical Co., Ltd. (Wuhan, China).

The solvents including methanol, acetonitrile, and water (HPLC-grade) and formic acid (≥98.0%, ACS reagent-grade) for quantitative analysis were obtained from Merck KGaA (Darmstadt, Germany) and J. T. Baker (Phillipsburg, NJ, USA), respectively.

#### *2.3. Preparation of 70% Ethanol SMR Extract*

Dried SMR (0.3 kg) was extracted with 70% ethanol (3.0 L, 3 times) for 1 h at room temperature by a Branson 8510 ultrasonicator (Denbury, CT, USA). The extract solution was filtered with 150 mm Ø filter paper (Whatman, Maidstone, Kent, UK) under vacuum, concentrated to remove the organic extract solvent (ethanol) using a Büchi rotary evaporator R-210 (Flawil, Switzerland), and then lyophilized using a Ilshin BioBase FD-5525L freeze-drier (Dongducheon, Korea) to obtain powdered extract. The yield of lyophilized 70% ethanol extract of SMR was 69.8 g (23.3%).

#### *2.4. HPLC Analysis of Five Components in SMR*

HPLC analysis was conducted using the Prominence LC-20A Series instruments (Shimadzu, Kyoto, Japan) consisting of a DGU-20A3 degasser, LC-20AT solvent delivery unit, SIL-20A auto sample injector, CTO-20A column oven, and SPD-M20A photodiode array detector. All chromatographic data were obtained and analyzed with the LabSolution software (Version 5.53; SP3, Kyoto, Japan). Five components were separated using a reverse-phase SunFireTM C18 analytical column (4.6 × 250 mm, 5 μm; Waters, Torrance, CA, USA) at 40 ◦C with gradient solvent condition. The mobile phases consisted of 0.1% (v/v) aqueous formic acid (A) and 0.1% (v/v) formic acid in acetonitrile (B) and were adjusted following the gradient condition: 0–30 min, 10–60% B; 30–40 min, 60–100% B; 40–45 min, 100% B; 45–50 min, and 100–10% B. The re-equilibrium time was adjusted for 10 min. The flow rate of the mobile

phase was 1.0 mL/min, and the injection volume of the standard and test solution was 10 μL each. For quantitative determination of five marker components (salvianic acid A, caffeic acid, rosmarinic acid, salvianolic acid B, and tanshinone IIA) in SMR, 200.0 mg of lyophilized SMR extract was liquefied with 20 mL of 70% methanol and sonicated for 30 min. It was also diluted 20-fold for quantification of salvianolic acid B. All samples were filtered using a membrane filter (0.2-μm, Pall Life Sciences, Ann Arbor, MI, USA) before analysis.
