Nutr Res Pract.  2014 Jun;8(3):257-266.

Anti-carcinogenic effects of non-polar components containing licochalcone A in roasted licorice root

• Park SY
• Kim EJ
• Choi HJ
• Seon MR
• Lim SS
• Kang YH
• Choi MS
• Lee KW
• Yoon Park

Abstract

BACKGROUND
/OBJECTIVE: Licorice has been shown to possess cancer chemopreventive effects. However, glycyrrhizin, a major component in licorice, was found to interfere with steroid metabolism and cause edema and hypertension. The roasting process of licorice modifies the chemical composition and converts glycyrrhizin to glycyrrhetinic acid. The purpose of this study was to examine the anti-carcinogenic effects of the ethanol extract of roasted licorice (EERL) and to identify the active compound in EERL.
MATERIALS/METHODS
Ethanol and aqueous extracts of roasted and un-roasted licorice were prepared. The active fraction was separated from the methylene chloride (MC)-soluble fraction of EERL and the structure of the purified compound was determined by nuclear magnetic resonance spectroscopy. The anti-carcinogenic effects of licorice extracts and licochalcone A was evaluated using a MTT assay, Western blot, flow cytometry, and two-stage skin carcinogenesis model.
RESULTS
EERL was determined to be more potent and efficacious than the ethanol extract of un-roasted licorice in inhibiting the growth of DU145 and MLL prostate cancer cells, as well as HT-29 colon cancer cells. The aqueous extracts of un-roasted and roasted licorice showed minimal effects on cell growth. EERL potently inhibited growth of MCF-7 and MDA-MB-231 breast, B16-F10 melanoma, and A375 and A2058 skin cancer cells, whereas EERL slightly stimulated the growth of normal IEC-6 intestinal epithelial cells and CCD118SK fibroblasts. The MC-soluble fraction was more efficacious than EERL in inhibiting DU145 cell growth. Licochalcone A was isolated from the MC fraction and identified as the active compound of EERL. Both EERL and licochalcone A induced apoptosis of DU145 cells. EERL potently inhibited chemically-induced skin papilloma formation in mice.
CONCLUSIONS
Non-polar compounds in EERL exert potent anti-carcinogenic effects, and that roasted rather than un-roasted licorice should be favored as a cancer preventive agent, whether being used as an additive to food or medicine preparations.

Keyword

Roasted licorice roots; apoptosis; licochalcone A; cancer

MeSH Terms

Animals
Anticarcinogenic Agents*
Apoptosis
Blotting, Western
Breast
Carcinogenesis
Colonic Neoplasms
Edema
Epithelial Cells
Ethanol
Fibroblasts
Flow Cytometry
Glycyrrhetinic Acid
Glycyrrhiza*
Glycyrrhizic Acid
Hypertension
Magnetic Resonance Spectroscopy
Melanoma
Metabolism
Methylene Chloride
Mice
Papilloma
Prostatic Neoplasms
Skin
Skin Neoplasms
Spectrum Analysis
Anticarcinogenic Agents
Ethanol
Glycyrrhetinic Acid
Glycyrrhizic Acid
Methylene Chloride

Figure

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  Fig. 1 Ethanol extract of roasted licorice (EERL) is more potent and efficacious in inhibiting cancer cell growth than ethanol extract of un-roasted licorice (EEUL). Tumor cells were serum-deprived or starved with their respective media and were incubated with the indicated extract as described in the Methods section. The viable cell numbers were estimated by MTT assay. Each bar represents the mean ± SEM. Means with different letters differ significantly, P < 0.05.

• 

  Fig. 2 EERL induces apoptosis in DU145 cells. Serum-deprived cells were treated with 0, 5, 10 or 15 µg/mL EERL for 24 h. (A) Cells were trypsinized, loaded with 7-aminoactinomycin D and Annexin V, and then analyzed by flow cytometry. The number of living cells and apoptotic cells is expressed as a percentage of total cell number. (B) Cells were stained with propidium iodide, and the cell cycle was analyzed via flow cytometry. Each bar represents the mean ± SEM (n = 6). (C,D,F) Total cell lysates (C,F) and cytosolic and mitochondrial fractions (D) were prepared and analyzed by Western blotting with the indicated antibodies. Photographs of the chemiluminescent detection of the blots, which were representative of three independent experiments, are shown. The relative abundance of each band to their own b-actin, α-tubulin, or heat shock protein (HSP) 60 was quantified, and the control levels were set at 1. The adjusted mean ± SEM (n = 3) of each band is shown above each blot. (E) Cells were loaded with JC-1 and then analyzed by flow cytometry. The number of cells with normally polarized mitochondrial membranes (red) and cells with depolarized mitochondrial membranes (green) was expressed as a percentage of total cell number. Each bar represents the mean ± SEM (n = 6). Means without a common letter differ, P < 0.05.

• 

  Fig. 3 Non-polar components of EERL inhibit the growth of DU145 cells. (A) Fractionation plan for the identification of the active compound in EERL. (B) Cells were treated with 0, 1, 2 or 3 mg/mL of the MC fraction. The viable cell numbers were determined by MTT assay and represent means ± SEM (n = 6). Means without a common letter differ, P < 0.05. (C) Cells were treated with 3 mg/mL of the individual MPLC fractions for 48 h. Cell numbers were estimated by the MTT assay. Each bar represents the mean ± SEM (n = 6). *Significant difference from the MC fraction, P < 0.01.

• 

  Fig. 4 The active compound of EERL licochalcone A induces apoptosis in DU145 cells. (A) Structure of Licochalcone A. (B) Serum-deprived cells were treated with 0-8 mmol/L licochalcone A, and cell numbers were estimated by MTT assay. Each bar represents the mean ± SEM (n = 6). (C) Serum-deprived cells were treated with licochalcone A for 24 h. Cells were stained with 7-aminoactinomycin D and Annexin V, and were then analyzed by flow cytometry. The number of living cells and apoptotic cells is expressed as a percentage of total cell number. Each bar represents the mean ± SEM (n = 6). (D) Cell lysates were analyzed by Western blotting with the indicated antibodies. Photographs of the chemiluminescent detection of the blots, which were representative of three independent experiments, are shown. The relative abundance of each band to their own β-actin was quantified and the control levels were set at 1. The adjusted mean ± SEM (n = 3) of each band is shown above each blot. (E) Cells were treated with or without 8 µmol/L licochalcone A for 24 h and stained with propidium iodide, and the cell cycle was analyzed via flow cytometry. Each bar represents the mean ± SEM (n = 6). Means with different letters differ significantly, P < 0.05.

• 

  Fig. 5 EERL inhibits skin papilloma formation. Tumor formation in mice was initiated with the topical application of DMBA (0.2 µmol/mouse) and promoted with TPA (10 nmol/mouse) twice weekly, starting one week after the initiation. The control group received vehicle only. EERL was applied topically 30 min prior to each TPA treatment. (A) Photographs of mice skin. (B) Percentage of mice with papillomas. (C) Average number of papillomas/mouse. Means without a common letter differ, P < 0.05.

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