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The effect of isoflavonoid contents in SH003 and its subfractions on breast cancer

Article information

J Korean Med. 2022;43(3):79-93
Publication date (electronic) : 2022 September 1
doi : https://doi.org/10.13048/jkm.22032
Yu-Jeong Choi1,#orcid_icon, Won-Geun Choi1,#orcid_icon, Kangwook Lee4orcid_icon, Miso Jeong1orcid_icon, Sang Cheol Park2orcid_icon, Young Pyo Jang3orcid_icon, Seong-Gyu Ko4,orcid_icon
1Department of Science in Korean Medicine, Graduate School, Kyung Hee University
2Department of Life and Nanopharmaceutical Sciences, Graduate School, Kyung Hee University
3Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University
4Department of Preventive Medicine, College of Korean Medicine, Kyung Hee University
Correspondence to: Seong-Gyu Ko, Department of Preventive Medicine, College of Korean Medicine, Kyung Hee University, 26 Kyungheedae Rd., Dongdaemun-gu, Seoul 02447, Korea, Tel: +82-2-961-0329, E-mail: epiko@khu.ac.kr
#

These authors contributed equally to this work.

Received 2022 June 10; Revised 2022 July 8; Accepted 2022 August 16.

Abstract

Objectives

We investigated the isoflavone contained in SH003 and its fractions, and the effect of these components on the inhibition of breast cancer.

Methods

The isoflavones in solvent fractions of SH003 extract were identified by UPLC-MS and its contents were quantified using HPLC analysis. The estrogenic activity of SH003 or fractions was assessed by ERE luciferase assay in estrogen receptor (ER)-positive MCF-7 cells. To test the breast cancer inhibitory effect, the cell viability was measured using an MTT assay.

Results

In this study, we demonstrated that SH003 and fractions contain 4 isoflavones which are calycosin-7-β -D-glucoside, formononetin-7-β-D-glucoside, calycosin, and formononetin. Despite containing isoflavones, estrogen-dependent transcription activity was not altered by both SH003 and fractions. On the other hand, SH003 and fractions inhibited the cell viability of breast cancer. In addition, its isoflavone components also showed reduced cell viability in various breast cancer cells.

Conclusions

Overall, the phytoestrogen included in SH003 and fractions did not influence the estrogenic activity, emphasizing the safety of SH003 and fractions in breast cancer treatment.

Keywords: SH003; fractions; isoflavone; phytoestrogen; anti-cancer effect; breast cancer
Fig. 1

Scheme for preparation of fractions from SH003

Fig. 2

UPLC-MS chromatograms of SH003

(A) UPLC-MS TIC (top) and UPLC-UV (bottom) analysis. (B) The identification of major isoflavones. 1: calycosin 7-O-β-D-glucoside, 2: formononetin 7-O-β-D-glucoside, 3: calycosin, 4: formononetin.

Fig. 3

HPLC Standard curve of isoflavone

The standard curve was determined by peak areas calculated following the concentration (μg/mL) of the injected isoflavone standards.

Fig. 4

HPLC chromatograms of isoflavone standards and SH003 crude extract

(A) SH003 extract. (B) Isoflavone standards.

Fig. 5

A comparison of HPLC chromatograms between the batch number of SH003 extract

(A) SH003 Lot1. (B) SH003 Lot2. (C) SH003 Lot3.

Fig. 6

The analysis of ERE luciferase activity by SH003 and fractions

MCF-7 cells were transfected with ERE reporter plasmid for 24 h. Transfected cells were treated with SH003, fractions, and E2 for 24 h. Luciferase activity was measured using Fluoroskan FL Microplate Luminometer (ThermoScientific, USA). The Renilla luciferase is used to normalization.

Fig. 7

The effect of SH003 and fractions on breast cancer cell death

Breast cancer cells were treated with SH003 and fractions for 48h at indicated dose. The cell viability was detected by MTT assay. Data represent the mean ±SD. * P < 0.05. (A) ER+ breast cancer, (B) HER2+ breast cancer, (C) Triple-negative breast cancer (TNBC).

Fig. 8

The effect of isoflavone components on breast cancer cell death

Breast cancer cells were treated with 4 isoflavone for 48 h. The cell viability was measured by MTT assay. * P < 0.05.

Extraction yield of SH003 fractions

The quantification of isoflavones in SH003 and SH003 fractions

Acknowledgement

This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2020R1A5A2019413).

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Article information Continued

Fig. 1

Fig. 1

Scheme for preparation of fractions from SH003

Fig. 2

Fig. 2

UPLC-MS chromatograms of SH003

(A) UPLC-MS TIC (top) and UPLC-UV (bottom) analysis. (B) The identification of major isoflavones. 1: calycosin 7-O-β-D-glucoside, 2: formononetin 7-O-β-D-glucoside, 3: calycosin, 4: formononetin.

Fig. 3

Fig. 3

HPLC Standard curve of isoflavone

The standard curve was determined by peak areas calculated following the concentration (μg/mL) of the injected isoflavone standards.

Fig. 4

Fig. 4

HPLC chromatograms of isoflavone standards and SH003 crude extract

(A) SH003 extract. (B) Isoflavone standards.

Fig. 5

Fig. 5

A comparison of HPLC chromatograms between the batch number of SH003 extract

(A) SH003 Lot1. (B) SH003 Lot2. (C) SH003 Lot3.

Fig. 6

Fig. 6

The analysis of ERE luciferase activity by SH003 and fractions

MCF-7 cells were transfected with ERE reporter plasmid for 24 h. Transfected cells were treated with SH003, fractions, and E2 for 24 h. Luciferase activity was measured using Fluoroskan FL Microplate Luminometer (ThermoScientific, USA). The Renilla luciferase is used to normalization.

Fig. 7

Fig. 7

The effect of SH003 and fractions on breast cancer cell death

Breast cancer cells were treated with SH003 and fractions for 48h at indicated dose. The cell viability was detected by MTT assay. Data represent the mean ±SD. * P < 0.05. (A) ER+ breast cancer, (B) HER2+ breast cancer, (C) Triple-negative breast cancer (TNBC).

Fig. 8

Fig. 8

The effect of isoflavone components on breast cancer cell death

Breast cancer cells were treated with 4 isoflavone for 48 h. The cell viability was measured by MTT assay. * P < 0.05.

Table 1

Extraction yield of SH003 fractions

Lot Powder (g) Extraction yield (%)
H2O layer 1 177.4 88.7
2 175.6 87.8
3 165.1 82.55
n-Hexane layer 1 6.2 3.1
2 6.2 3.1
3 6.1 3.05
Chloroform layer 1 3.8 1.9
2 3.26 1.63
3 6.2 4.1
Ethyl acetate layer 1 1.68 0.84
2 1 0.5
3 0.25 0.13
Butanol layer 1 5.18 2.59
2 3.47 1.74
3 1.93 0.97

Table 2

The quantification of isoflavones in SH003 and SH003 fractions

Content (%)
Lot 1 SH003 Water Butanol Ethyl acetate Chloroform Hexane
 Calycosin-7-O-β-D-glucoside 0.1 0.05 0.95 1.5 0.05 0
 Formononetin-7-O-β-D-glucoside 0.04 0.01 0.29 1 0.09 0
 Calycosin 0.05 0.01 0.18 0.45 0.62 0
 Formononetin 0.06 0.01 0.11 0.18 0.46 0.09
Lot 2
 Calycosin-7-O-β-D-glucoside 0.1 0.05 0.95 1.49 0.05 0
 Formononetin-7-O-β-D-glucoside 0.04 0.01 0.3 1 0.09 0
 Calycosin 0.06 0.01 0.18 0.47 0.63 0
 Formononetin 0.07 0.01 0.12 0.2 0.53 0.09
Lot 3
 Calycosin-7-O-β-D-glucoside 0.11 0.05 0.94 1.68 0.05 0
 Formononetin-7-O-β-D-glucoside 0.04 0.01 0.29 1.06 0.09 0
 Calycosin 0.05 0.01 0.18 0.46 0.62 0
 Formononetin 0.06 0.01 0.11 0.18 0.51 0.1
MEAN+SD (%) SH003 Water Butanol Ethyl acetate Chloroform Hexane
 Calycosin-7-O-β-D-glucoside 0.10±0 0.05±0 0.95±0 1.56±0.05 0.05±0 0
 Formononetin-7-O-β-D-glucoside 0.04±0 0.01±0 0.29±0 1.02±0.02 0.09±0 0
 Calycosin 0.05±0 0.01±0 0.18±0 0.46±0 0.62±0 0
 Formononetin 0.06±0 0.01±0 0.11±0 0.19±0 0.5±0.02 0.09±0