CO was purchased from Dongsuh Foods Co. (Seoul, Republic of Korea). PF was purchased from Dongui Herb Co. (Seoul, Republic of Korea). BS was provided by Sambou bamboo salt Inc. (Jeonbuk, Republic of Korea). Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 17β estradiol (E2) were purchased from Sigma chemical Co. (St. Louis, MO, USA). RPMI 1640 and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). PF was boiled at 100°C for 2 h and then lyophilized. The yield of PF was about 18% (54 g). E2 was dissolved in DMSO.

Each sample was prepared according to the various proportions and placed in refrigerated conditions until testing. About 20 ml of each sample was provided in a white 50 ml paper cup with a random 4-digit number written on it. All samples were evaluated for overall quality (color, smell, taste, bitterness, acidity, and preference) by 15 trained sensory evaluation personnel from the Department of Food Science and Technology, Hoseo University. Sensory evaluation results were used in the 7-point hedonic scale (low, 1 indicates a strong hate, while high, 7 indicates a strong preference).

Blind consumer test was conducted with the finally determined experimental groups and control groups. Similarly, about 20 ml of each sample was provided in a white 50 ml paper cup with a random 4-digit number written on it. The preference of the samples was evaluated by 34 people. Blind consumer test results used a ranking method.

MCF-7 cells were bought from the Korean Cell Line Bank (KCLB®, Seoul, Republic of Korea), and they were grown in RPMI 1640 containing 10% FBS and 1% penicillin/streptomycin at 37°C with 5% CO2 and 95% humidity. MCF-7 cells, which are ER-positive, are typically utilized to examine estrogen activity.

An anti-oxidant effect was analyzed using EZ-SOD assay kit (DoGenBio Co., Ltd, Republic of Korea). In summary, the samples diluted with various concentrations were put into the well, the WST working solution and the enzyme working solution were added and then mixed carefully. Plates were incubated at 37ºC for 20 min and then absorbance was analyzed at 450 nm with an ELISA reader.

The growth of MCF-7 cells was measured using a colorimetric immunoassay based on the measurement of bromodeoxyuridine (BrdU) incorporated by DNA synthesis according to the manufacturer's protocol (Roche Diagnostics GmbH, Mannheim, Germany).

Using an easy-BLUETM RNA extraction kit, total RNA was extracted from MCF-7 cells in accordance with the manufacturer's instructions (iNtRON Biotech, Republic of Korea). The commercial cDNA synthesis kit (Bioneer Corporation, Daejeon, Republic of Korea) was used to create the cDNA. SYBR Green master mix was used for quantitative real-time PCR, and an ABI StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) was used for mRNA detection analysis. Data were normalized to housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Primers were as follows: GAPDH (5’ TCG ACA GTC AGC CGC ATC TTC TTT 3’; 5’ACC AAA TCC GTT GAC TCC GAC CTT 3’), and ER-β (5’ TTC CCA GCA ATG TCA CTA ACT T3’; 5’TTG AGG TTC CGC ATA CAG A 3’). With a melting curve analysis, the typical profile timings were 95°C for 10 min in the first phase, then 15 s at 95°C in the second step, and 30 s at 60°C for 40 cycles. The ΔΔCT method was used to examine the data.

MTT assay was used to measure cell viability. Briefly, 100 µl of cell suspension (1 × 10⁴ cells/well) was cultured in 96-well plates after treatment with CPS219. After adding the MTT solution (5 mg/ml), the cells were incubated at 37°C for 4 h. The DMSO was used to dissolve the insoluble formazan product. The optical density was then assessed at 590 nm with an ELISA reader.

GraphPad Prism software (GraphPad Software Inc., CA, USA) was used to conduct all statistical analyses and to create figure graphs. Data from at least three independent experiments that were carried out in duplicates or triplicates are presented as the mean ± standard error mean (SEM). Treatment effects were analyzed using one-way ANOVA or the independent t-test. A value of p < 0.05 was regarded to be statistically significant.

Sensory evaluation was conducted on three types of freeze-dried CO (Sumatra, Colombia, and Maxim) and their characteristics were compared. In the evaluation, CO with a good balance of color, taste, and smell and relatively low bitterness and acidity produced good results. As shown in Fig. 1A, Maxim CO was rated highest on a 7-point hedonic scale.

Next, to determine the CO concentration, the preference according to the coffee concentrations (1%, 2%, 3%, and 4%) was investigated. Maxim CO 3% was harmonious in color, taste, and smell. In addition, it was found that bitterness and acidity had a significant effect on the preference results (Fig. 1B). Finally, in order to determine the optimal ratio of the three compounds, CO (3%), PF (0.075, 0.15, and 0.3), and BS (0.025, 0.05, and 0.1%) were variously combined and the preference was investigated through sensory evaluation and blind consumer test. The strongest preference in sensory evaluation was seen in samples 9292, 5223, and 5319 (Fig. 1C, p < 0.05). The most preferred number in the blind consumer test, 5229 (CPS219), consisted of 3% CO, 0.3% PF, and 0.05% BS. According to all of the findings, the optimal ratio of CO, PF, and BS was determined as 1:0.1:0.017.

To evaluate the SOD-like activity of CPS219, we performed the SOD assay. As a result, CP had lower SOD-like activity than control. CO or CPS219 significantly increased the SOD-like activity compared to the control (Fig. 2, p < 0.05). In addition, CPS219 significantly increased the SOD-like activity compared to the CO (Fig. 2, p < 0.05).

We investigated the effect of CPS219 on proliferation of MCF-7 cells (an estrogen-dependent cell line). For this purpose, MCF-7 cells were treated with CO, CP, CPS219, or E2 for 24 h or 48 h. Proliferation of MCF-7 cells was measured using a BrdU incorporation assay. Proliferation of MCF-7 cells was considerably increased by treatment with CO, CP, or CPS219 after 24 h, but only CPS219 significantly boosted the proliferation of MCF-7 cells after 48 h (Fig. 3A-B, p < 0.05). E2 showed a both significantly increased for 24 h and 48 h (Fig. 3A-B, p < 0.05). Microscopic observations showed that cells treated with CPS219 showed a higher proliferation rate compared to cells cultured in blank conditions (Fig. 3C).

Estrogen binds to ER-β to have a biological effect.⁸ We examined the mRNA expression of ER-β in MCF-7 cells to see if the cell proliferation induced by CPS219 was connected to the function of ER-β. When compared to the blank, CPS219 dramatically boosted the mRNA levels of ER-β (Fig. 4, p < 0.05). Additionally, the mRNA levels of ER-β significantly increased in E2-treated cells (Fig. 4, p < 0.05).

Finally, we investigated the effect of CPS219 on the viability of MCF-7 cells. MCF-7 cells were treated with CO, CP, CPS219, or E2 for 24 h. Cell viability was determined using an MTT assay. The viability of cells treated with CO, CP, CPS219, or E2 was not significantly different from blank (Fig. 5).