*3.4. Preparation of the Phlorethol Fraction (CcPh) for Bioassays*

**CcPh** was dissolved in sterile PBS to prepare stock concentrations of 10 mg/mL. Cells were treated with serially diluted phlorethols (culture medium used as diluent) to give the intended final concentrations (5–1000 μg/mL). The vehicle control was the cells treated with an equivalent volume of PBS for all presented experiments.

#### *3.5. MTS Assay*

HT-29 and HCT 116 or MCF-7 and SK-MEL-28 cells (1.0 <sup>×</sup> <sup>10</sup>4) were seeded in 200 <sup>μ</sup>L of complete McCoy's 5A/10% FBS or MEM/10% FBS media, respectively, and incubated for 24 h at 37 ◦C in 5% CO2 incubator. The attached cells were treated by phlorethols **CcPh** at concentrations ranging from 0 to 1000 μg/mL for an additional 24 h. Subsequently, the cells were incubated with 15 μL MTS reagent for 3 h, and the absorbance of each well was measured at 490/630 nm using PowerWave XS Microplate Reader (BioTek, Winooski, VT, USA). All samples were tested in triplicate.

#### *3.6. Soft Agar Assay*

To estimate the effect of phlorethols **CcPh** on colony formation (phenotype expression), human colorectal carcinoma cells HT-29 and HCT 116 (2.4 <sup>×</sup> <sup>10</sup>4) were treated with PBS (control) or phlorethols **CcPh** (10, 20, and 40 μg/mL) in 1 mL of 0.3% BME agar containing 10% FBS, 2 mM l-glutamine, and 25 μg/mL gentamicin. The cultures were maintained in a 37 ◦C, 5% CO2 incubator for 14 days, and the cell colonies were scored using a Motic microscope AE 20 (XiangAn, Xiamen, China) and ImageJ software bundled with 64-bit Java 1.8.0\_112 (NIH, Bethesda, MD, USA).

#### *3.7. Cell Irradiation Assay*

Human colorectal carcinoma cells HT-29 and HCT 116 were exposed to X-ray radiation using an XPERT 80 X-ray system (KUB Technologies, Inc., Milford, CT, USA). The absorbed dose of radiation was measured by a DRK-1 X-ray radiation clinical dosimeter (Axelbant LLK, Moscow, Russia).

To determine the sensitivity of HT-29 or HCT 116 cells to radiation, the cells (5.0 <sup>×</sup> 105) were exposed to X-ray at a dose rate from 2 to 10 Gy and recovered at 37 ◦C in a 5% CO2 incubator for 3 h. The cells were harvested with 0.25% trypsin/0.05 M EDTA solution and subjected to the "Soft agar assay" as described above.

To determine the radiosensitizing activity of phlorethols, HT-29 or HCT 116 cells (5.0 <sup>×</sup> 105) were exposed to 2 Gy of X-ray and incubated for 3 h. Then, cells were treated with either PBS (control) or phlorethols **CcPh** (5, 10, and 20 μg/mL) for an additional 24 h. The cells were harvested and used for the "Soft agar assay" as described above.

#### *3.8. Combination Index (CI) Calculation*

The calculations of drug concentration/X-ray irradiation dose-effect were performed by CompuSyn software version 1.0 (ComboSyn, Inc., Paramus, NJ, USA) using the median effect method described by Chou and Talalay [22].

#### *3.9. Data Analysis*

All assays were performed in at least three independent experiments. Results are expressed as the mean ± standard deviation (SD). The Student's t-test was used to evaluate the data with the following significance levels: \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

#### **4. Conclusions**

This study evaluated the in vitro anticancer and radiosensitizing effects of phlorethols (**CcPh**) from *C. costata* on human colorectal carcinoma HCT 116 and HT-29 cell lines. **CcPh** at non-toxic concentrations was found to significantly inhibit the colony formation in HT-29 and HCT 116 alone and in combination with X-ray irradiation. The combinatory effect of radiation and **CcPh** was synergistic (combination index CI < 0.7). The molecular mechanism of this action requires further research. In future studies, we plan to determine the effects of polyphenols on the detoxification of enzymes, the activation of endogenous protective systems, and the repair of DNA strand breaks induced by X-ray exposure.

**Supplementary Materials:** The following are available online, Figure S1: 13C-NMR spectra of the **CcPh** fraction, Figure S2: HMBC NMR spectra of the **CcPh** fraction, Figure S3: HSQC NMR spectra of the **CcPh** fraction, Table S1: The data of elemental compositions, monoisotopic masses and ions of the phlorethol **CcPh** fraction, Figure S4: The effect of phlorethols from *C. costata* (**CcPh**) on viability of normal mouse epidermal cells JB6 Cl41.

**Author Contributions:** O.S.M. and T.I.I.: the conceptualization, design, and methodology of the work, data curation, writing-original draft preparation. T.I.I.: the preparation of the phlorotannins. O.S.M. performed the biological assays. S.P.E.: the editing of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** The reported study was funded by RSF grant # 16-14-10131.

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