Bioactivity of Fermented Green Coffee Bean Extract Containing High Chlorogenic Acid and Surfactin

Abstract

Chlorogenic acid (CGA) is a major component of green coffee beans. Surfactin, a cyclic lipopeptide, is produced and secreted by Bacillus subtilis strains. In this study, bioactivities of fermented green coffee bean extract (FGCBE) and the individual compounds, CGA and surfactin. were compared in HepG2 cells. The concentration of surfactin and CGA in the FGCBE and non-fermented green coffee bean extract (NFGCBE) were determined to be 9.2 and 7.33 and 0.72 and 0.53 mg·mL⁻¹, respectively. The FGCBE contained about 20% and 26% more CGA and surfactin than the NFGCBE. Although CGA and surfactin exhibited cytotoxicity at concentrations more than 100 and 20 μg respectively, the FGCBE 50 containing CGA (460 μg·mL⁻¹) and surfactin (720 μg·mL⁻¹) effectively prevented cell death by oxidative stress and also strongly activated the proliferation of cells incubated with under 50 μM H₂O₂. The CGA and surfactin in FGCBE were 9.2 and 72 times higher than the CGA and surfactin compounds (50 and 10 μg·mL⁻¹). The relative proliferation of the FGCBE-treated cells also was 3.3 and 8.8 times higher than the CGA and surfactin compounds treated the oxidative stressed cells with 50 μM H₂O₂. These results suggest that the single compounds such as CGA and surfactin generally have cytotoxicity at low concentration of them but FGCBE contained them acted as strong antioxidants, activators of cell proliferation, inhibitors of cell apoptosis. Various bioactive compounds in fermented coffee bean also seem to help cell proliferation and decreasing of cytotoxicity by CGA and surfactin in coffee bean.

Introduction

Chlorogenic acid (CGA) is one of the most abundant polyphenol compounds in the human diet. It is a major phenolic compound that can be isolated from the leaves and fruits of plants.¹ Green coffee beans contain the largest amounts of CGA found in plants, ranging from 6% to 12%.² CGA, a known antioxidant, has been reported to have anti-inflammatory properties, anticarcinogenic activity, and protective effects on neuronal cells.^3

–6 CGA is used in various additives for beverages, cosmetics, tea products, and foods, as well as medical substances.⁷ Although there are many biological benefits for CGA, CGA has side effects including increasing of blood pressure, plasma homocysteine and cytotoxicity.^8
–10

It consists of the ester of caffeic acid and quinic acid. The main subgroups of CGAs are 3-O-caffeoylquinic acid (n-chlorogenic acid), 5-O-caffeoylquinic acid (neo-chlorogenic acid), and 4-O-caffeoylquinic acid (crypto-chlorogenic acid). n-Chlorogenic acid is the most abundant compound in green coffee bean.¹

Surfactin is a prominent surfactant and is used as an antibiotic. The lipopeptide, surfactin, is synthesized by a bacterial surfactin synthase and aids in the activation of plasminogen in blood. The subtypes of surfactin are characterized by the same peptide length, but different residues.^11,12 In various studies, surfactin has been found to exhibit beneficial functions, including antibacterial, antiviral, antifungal, and anti-mycoplasmal activities.^13,14 Unfortunately, surfactin is not able to be used as a medicine because it has cytotoxicity such as hemolysis in normal cells.¹⁵

Reactive oxygen species (ROS) are produced by the normal metabolism of oxygen and play important roles in cell signaling and homeostasis.¹⁶ ROS are also responsible for the induction of apoptosis, that is, programmed cell death.¹⁷ ROS are chemically reactive molecules containing oxygen. There are various ROS compounds, including the superoxide anion (O₂ ⁻), hydroxyl radical (·OH), and hydrogen peroxide (H₂O₂), which cause oxidative deterioration of deoxyribonucleic acid (DNA), proteins, and lipids, and accelerate aging and mitochondrial death in cells.¹⁸

In this study, green coffee bean was fermented by Bacillus subtilis, and concentrations of CGAs and surfactin of the fermented green coffee bean extract (FGCBE) were compared with non-fermented green coffee bean (NFGCBE). These major compounds and the extract were applied to liver hepatocellular carcinoma (HepG2) cells to evaluate their cytotoxicity, viability and proliferation on oxidative stress. This study also documents the effectiveness of FGCBE for treating oxidatively stressed HepG2 cells through the comparison of medicinal efficacy between the compounds and FGCBE.

Materials and Methods

Materials

CGA (>95%; HPLC grade; p-no. C3878-1G, catalog no. 327-97-9), neo-chlorogenic acid (>98%; HPLC grade; p-no. 94419-10 MG), and surfactin (>98%; HPLC grade; p-no. s3523-10 mg) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile (HPLC grade) was obtained from Merck KGaA (Darmstadt, Germany) and analytical grade phosphoric acid (49–51%; HPLC grade) was procured from Sigma-Aldrich. Trifluoroacetic acid (99%; p-no. T6508-500ML, catalog no. 76-05-1) was obtained from Sigma-Aldrich and methanol was from obtained from Merck KGaA (Darmstadt, Germany). The solvents were filtered through a 0.45 μM membrane filter (Agilent Technologies; Santana Clara, USA). The fermented (Coffea arabica) and non-fermented GCBE were manufactured by Leader Green (Kyeong-gi, Korea). The ground green coffee beans (120 mesh) were diluted with distilled water and fermented by Bacillius megaterium (ATCC 14945) for 30 days at 37°C.

Apparatus and operation conditions

Liquid chromatography

Chromatographic analysis was performed on a Thermo Scientific Dionex Ultimate 3000 system (Thermo Fisher Scientific, Inc., Germering, German) consisting of a binary pump solvent management system and an auto-sampler. Hydrosphere C18 (150 × 4.6 mm, I.D. S-5 μm, 12 nm (p/n HS12S05-1546WT)) and YMC-triart C18 (250 × 4.6 mm I.D. S-5 μm, 12 nm (p/n TA12S05-2546WT)) columns were employed and the column temperature was maintained at 40°C. The UV detector was set at 324 nm for CGA and 205 nm or 210 nm for surfactin. The mobile phase was prepared daily, filtered under vacuum through a 0.45 μm membrane filter, and degassed before use. The flow rate was set at 1.0 mL·min⁻¹.

Standard solution and sample preparation

The respective CGA standards (n-chlorogenic acid and neo-chlorogenic acid; 10 mg·mL⁻¹) were dissolved in deionized distilled water and working standards were prepared at concentrations of 0.1565, 0.3125, 0.625, 1.25, 2.5, 5, 10, and 20 mg·mL⁻¹. The identification and quantitation of cypto-chlorogenic acid were performed in reference to another study.¹⁹ The surfactin standard (10 mg·mL⁻¹) was dissolved in methanol and working standards of surfactin were prepared at concentrations of 0.3125, 0.625, 1.25, and 2.5 mg·mL⁻¹. Each standard was injected (10 μL) by an auto-sampler. FGCBE and NFGCBE were dissolved in deionized distilled water. The respective solutions were filtered through a 0.45 μm membrane filter paper and the clear filtrate (diluted in ratios of 1:100 and 1:500) was used for high performance liquid chromatography (HPLC) analysis.

Cell culture

A human liver carcinoma cell line (HepG2 cells) was cultured in RPMI1640 medium supplemented with penicillin (100 U·mL⁻¹), streptomycin (100 μg·mL⁻¹), and 10% FBS (Invitrogen, USA). The HepG2 Cells were maintained in a humidified atmosphere of 5% CO₂ at 37°C. The medium was then replaced and the cells were cultured for an additional 2 days in RPMI. The cells were seeded at a density of 2 × 10⁶ cells/well in a 100pi disk or 1 × 10⁴ cells/well in a 96-well plate for 24 h before each treatment.

Cell viability assay

The cell viability was determined using the MTT assay (Promega cell proliferation assay; p-no. G3580, Madison, USA). HepG2 cells were treated with CGA, surfactin, FGCBE, NFGCBE, and H₂O₂ and seeded at a density of 1 × 10⁴ cells in a 96-well culture plate and incubated at 37°C with 5% (v/v) CO₂ for 24 h. A 20 μL aliquot of MTT was added to each well and incubated for 3 h. After 3 h of incubation, the absorbance was measured at a wavelength of 490 nm using a microplate spectrometer (Chromate, Awareness Technology, Inc., the Netherlands). All experiments were performed in triplicate.

Statistical analysis

All data were analyzed by ANOVA using SPSS software. When the P-values were found to be significant (P < .05), the means were compared using Tukey's method.

Results

Contents of CGAs and surfactin in fermented green coffee bean

The CGA and surfactin in the FGCBE were analyzed by UHPLC (Fig. 1). The retention times of neochlorogenic acid and CGA were 5.93 and 9.19 min, respectively. The seven peaks of the surfactin isomers were detected by UHPLC, and the corresponding peak numbers and retention times were 1–10.36, 2–12.47, 3–16.18, 4–17.27, 5–18.67, 6–20.81, and 7–22.35 min (Fig. 1b). The standard plots of CGA and surfactin are displayed based on analysis of the standards by UHPLC (Fig. 2a, b). The concentrations of surfactin and CGA in the FGCBE and NFGCBE were measured to be 9.2 and 7.33 and 0.72 and 0.53 mg·mL⁻¹, respectively. The FGCBE contained about 20% and 26% more of the respective compounds than NFGCBE (Fig. 2c).

FIG. 1.

Analysis of chlorogenic acid and surfactin in the extract by UV-UHPLC. (a)The peaks of n-chlorogenic acid and new-chlorogenic acid are displayed at 9.19 and 5.93 min, respectively. (b) The peaks of surfactin are displayed at 10.36, 12.47, 16.18, 17.27, 18.67, 20.81, and 22.36 min. UV-UHPLC, UV-ultra-high-performance liquid chromatographic.

FIG. 2.

Standard curves and measurement of chlorogenic acid and surfactin in fermented GCBE and non-fermented GCBE. (a, b) Standard curves of neo-chlorogenic acid, n-chlorogenic acid, and surfactin, respectively, from UV-UHPLC analysis. (c) Content of CGA and surfactin in FGCBE and NFGCBE.The values are expressed as the average ± SD of triplicate experiments (P < .05). CGA, chlorogenic acid; GCBE, green coffee bean extract; FGCBE, fermented GCBE; NFGCBE, non-fermented GCBE.

Viability, proliferation, protection activity and cytotoxicity of CGA and FGCBE for oxidatively stressed HepG2 cells

Based on the cell viability assay (Figs. 3 and 4), the treatment dosages were selected as H₂O₂ (10–100 μM), surfactin (10 μg·mL⁻¹), CGA (50 μg·mL⁻¹), and FGCBE (50 μg·mL⁻¹). The proliferation and inhibition of the HepG2 cells were measured by MTT assay after 24 h incubation of the cells treated with CGA, surfactin, or FGCBE.

FIG. 3.

Cell viability determined by MTT assay. The cells were treated with CGA, surfactin, NFGCBE, and FGCBE for 24 h. (a) Standard curve for MTT assay. (b, c) Cell viability in the presence of CGA and surfactin. (d) Cell viability after treatment with FGCBE and NFGCBE. The values are expressed as the average ± SD of triplicate experiments (P < .05).

FIG. 4.

Cytotoxicity of H₂O₂ toward HepG2 cells. The cells were incubated with different dosages (0–100 μM) of H₂O₂ for 24 h. The cell viability was evaluated by the MTT assay method. The values are expressed as the average ± SD of triplicate experiments (P < .05).

CGA activated proliferation of the cells and prevented cell death, both in non-stressed and oxidatively stressed environments under 20 μM H₂O₂ (Fig. 5a). Although the viability of the oxidatively stressed HepG2 cells without CGA declined dramatically, the proliferation of the stressed cells treated with CGA increased dramatically (Fig. 5a, b). In 10–50 μM H₂O₂, the relative value of the proliferation of the stressed cells was measured at 3.62, 6.47, 3.94 and 2.1, and the peak value was obtained with 20 μM H₂O₂ (Fig. 5b). Notably, although the cells had cytotoxic damage at over 100 μg·mL⁻¹ CGA (Fig. 3b) the oxidatively stressed HepG2 cells exposed to the low concentrations of CGA increased their viability and proliferation (Figs. 3 and 5).

FIG. 5.

Effect of CGA on oxidatively stressed HepG2 cells. The cells were incubated with H₂O₂ and CGA for 24 h. (a) Cell viability was measured by MTT assay method. (b) Plot of relative value of cell proliferation of control and CGA-treated cells. The values are expressed as the average ± SD of triplicate experiments (P < .05).

The best efficacy for activation of cellular proliferation as well as prevention of cell death was achieved with 50 μg·mL⁻¹ FGCBE (Fig. 6). CGA in FGCBE 50 were calculated at 460 μg·mL⁻¹ and the HepG2 cells exposed to the FGCBE50 exhibited high cell viability under 50 μM H₂O₂ (Fig. 6). In 10–100 μM H₂O₂, the relative value of the proliferation of the stressed cells was measured at 2.9, 7.4, 13.1 and 1.8, and the peak value was obtained with 50 μM H₂O₂ (Fig. 6b). FGCBE effectively prevented cell death due to oxidative stress and also strongly activated cell proliferation with the use of 50 μM H₂O₂ (Fig. 6b). Especially, the CGA concentration in FGCBE 50 was 72 times higher in CGA 50 and the value of relative cell viability in FGCBE 50 were highest at 50 μM H₂O₂ (Fig. 7).

FIG. 6.

Effect of FGCBE on oxidatively stressed HepG2 cells. (a) HepG2 cells treated with FGCBE were cultured in H₂O₂ (0–100 μM). (b) Plot of relative proliferation rate of control and FGCBE-treated cells. The values are expressed as the average ± SD of triplicate experiments (P < .05).

FIG. 7.

Comparison of cell viability by treating of CGA and FGCBE on oxidatively stressed HepG2 cells. (a) The Concentration of CGA in FGCBE (50 μg) were calculated at 460 μg·mL⁻¹. FGCBE contained CGA about 9.2 times more than CGA 50 μg·mL⁻¹. (b) Comparison of cell viability between CGA 50 and FGCBE 50 on the cells (c) The plot of relative cell viability rate between CGA and FGCBE.

Cytotoxicity and viability between surfactin and FGCBE for oxidatively stressed HepG2 cells

Surfactin (10 μg·mL⁻¹) synergistically activated cytotoxicity against oxidatively stressed HepG2 cells (Fig. 8a). Notably, 10 μM H₂O₂ induced strong cytotoxicity, which was 2.7 times higher than that observed with the control when the HepG2 cells exposed to H₂O₂ were treated with surfactin (Fig. 8b). The concentration of surfactin in FGCBE 50 were measured at 720 μg·mL⁻¹. This value was 72 times higher than the single compound, 10 μg·mL⁻¹ surfactin (Fig. 9a). The more surprising observation was that FGCBE treated cells showed higher viability than the surfactin 10 treated cells under oxidative stress. The relative values for viability were 7.9, 9.8, 8.9 and 1.2 at 10–50 μM H₂O₂ (Fig. 9b, c).

FIG. 8.

Cytotoxicity of surfactin for oxidatively stressed HepG2 cells. (a) HepG2 cells incubated with various dosages of H₂O₂ were treated with surfactin (10 μM). After 24 h of incubation, the cell viability was measured by MTT assay. (b) Plot of relative value of cytotoxicity of control and surfactin-treated cells. The values are expressed as the average ± SD of triplicate experiments (P < .05).

FIG. 9.

Comparison of cell viability by treating of surfactin and FGCBE on oxidatively stressed HepG2 cells. (a) The Concentration of surfactin in FGCBE (50 μg) were calculated at 720 μg·mL⁻¹. FGCBE contained surfactin about 72 times more than surfactin 10 μg·mL⁻¹. (b) Comparison of cell viability between surfactin and FGCBE on the cells (c) The plot for relative cell viability rate by treating of surfactin and FGCBE.

Discussion

CGA is usually extracted from green coffee bean using hot water, methanol, or ethanol.²⁰ In contrast with other studies, this study used an extract from green coffee bean fermented by B. subtilis. The efficiency of this fermentation method was determined by quantitation of the CGA content in the green coffee bean extract. Quantitation of CGA was performed with HPLC, ultra high performance liquid chromatography (UHPLC), and liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS).^21,22 UHPLC with UV detection is a sensitive, precise, accurate, and linear method for the routine analysis of GCA. Herein, we analyzed the content of CGA (9.2 mg·mL⁻¹) and surfactin (0.72 mg·mL⁻¹) in the FGCBE. Previous studies that quantified CGA documented nine peaks of CGA isomers in the HPLC spectrum.²³ Three of these isomers, that is, n-chlorogenicacid (3-CQA), neo-chlorogenic acid (5-CQA), and crypto-chlorogenic acid (4-CGA), accounted for over 90%.²³ In this study, two CGA isomers standards were used for calibration, and the total concentration of these isomers in FGCBE and NFGCBE was about 9.2 and 7.33 mg·mL⁻¹, respectively (P < .05). The concentration of CGA in FGCBE was 20% higher than that in NFGCBE. This suggests that bacterial fermentation increases the concentration of CGA in FGCBE via bacterial metabolism. According to other studies, various independent variables, including the extraction temperature and fermentation and extraction time, significantly affected the determined content of medicinal compounds in an extract.^24
–26 Therefore, the processing conditions must be carefully optimized to obtain an excellent extract. H₂O₂ is known to induce apoptosis through the ROS pathway.²⁷ Previous reports have demonstrated the cytotoxic effects of CGA on several human cell types, including oral squamous cell carcinoma (HSC-2), salivary gland tumor (HSG), and chronic myeloid leukemia cell lines.^24,25 CGA also induced cell death at concentrations exceeding 200 μM.²⁶ In this study, HepG2 cell exposed to CGA over 100 mg·mL⁻¹ were increased dramatically their apoptosis. In contrast, HepG2 cell exposed to FGCBE 50 containing high concentration of CGA (460 mg·mL⁻¹) prevented cell apoptosis (Fig. 7). Also, the relative viability of the FGCBE 50-treated cells was higher than that of the CGA treated cells in an oxidative-stress environment (Fig. 7b, c). These results suggest that CGA possesses cytotoxic activity at higher concentrations, but metabolites in FGCBE increase cell viability and decreases the cytotoxicity of high CGA in the cells.

Surfactin is composed of a heptapeptide cycle (L-Glu–L-Leu–D-Leu–L-Val–L-Asp–D-Leu–L-Leu) closed by a C13–C15 hydroxy fatty acid to form a lactone ring system.²⁸ The C15-surfactin is a major isoform in Bacillus subtilisnatto.²⁹ In this study, we determined the quantity of C15-surfactin in FGCBE and NFGCBE. Analysis of the surfactin standard by UHPLC showed seven peaks, and the concentration of surfactin in FGCBE and NFGCBE was measured to be 0.73 and 0.53 mg·mL⁻¹, respectively (Fig. 2). The concentration of surfactin in FGCBE was 26% higher than that in NFGCBE. Surfactin has bioactive characteristics, including antibacterial, antifungal, antiviral, anti-mycoplasma, and hemolytic activity.³⁰ Nevertheless, surfactin has critical drawbacks, such as non-specific cytotoxicity, and causes hemolysis at concentrations of 40–60 μM. Fortunately, the toxic effects of surfactin are not significant at concentrations below 25 μM.²⁶ In various reports,^31,32 surfactin has been shown to induce apoptosis, but little is known about how the combination of surfactin and other metabolites produced by the bacterial fermentation affect cell viability and cytotoxicity.

This study compares the cytotoxicity and cell viability between surfactin and FGCBE containing surfactin at high concentration in HepG2 cells. Although the surfactin (10 μg·mL⁻¹)-treated cells did display an increasing of cytotoxicity in HepG2 cells (Fig. 8), the cells treated with FGCBE 50 displayed a dramatic reduction in the cytotoxicity (Fig. 9). These results indicate that the single compounds, surfactin has strong cytotoxicity for cancer cells but the strong cytotoxicity also has a side effect of cytoxicity to normal cells. In contrast, metabolites in FGCBE containing high surfactin reduces the cytotoxicity in normal cells as well as increasing of anti-oxidant stress.

Generally, fermented extracts contain more medicinal compounds than the non-fermented congeners.^33,34 In this study, FGCBE was also found to contain more CGA and surfactin than NFCBE (Fig. 2c). The relative value of the proliferation for the FGCBE-treated cells was higher than that achieved with the control. Moreover, the FGCBE-treated cells displayed a dramatic increase in the cell proliferation with the use of 20 and 50 μM H₂O₂. In a recent study,³⁵ the NFGCBE (100 μg·mL⁻¹) induced cytotoxicity was examined in various cell types including human lung, colorectal, oesophageal and urinary bladder carcinomal cells. However, treatment with FGCBE (100 μg·mL⁻¹) activated cell viability and proliferation. This result means that FGCBE has potentially positive effects as antioxidant for various organs in human body. Consequently, these results indicate that FGCBE contains effective antioxidants, such as CGA, that are generated by fermentation, and the extract was also effective for preventing cell senescence, death, and apoptosis.

In conclusion, this study demonstrates the improved function, including anti-oxidative, anti-cancer, and activation of cell proliferation, of FGCBE. CGA and surfactin in FGCBE functioned as effective antioxidants and FGCBE exhibited improved anti-cancer efficacy, At adequate concentrations, CGA also activated the proliferation of HepG2 cells.

Footnotes

Acknowledgment

This research was supported by Korea University (K1808591) and the Agriculture, Food, and Rural Affairs Research Center Support Program (714001-07), Ministry of Agriculture, Food, and Rural Affairs.

Author Disclosure Statement

No competing financial interests exist.

References

Clifford

, Johnston

, Knight

, Kuhnert

: Hierarchical scheme for LC-MSn identification of chlorogenic acids. J Agric Food Chem, 2003; 51:2900–2911.

Farah

, de Paulis

, Trugo

, Martin

: Effect of roasting on the formation of chlorogenic acid lactones in coffee. J Agric Food Chem, 2005; 53:1505–1513.

Kono

, Kashine

, Yoneyama

, Sakamoto

, Matsui

, Shibata

: Iron chelation by chlorogenic acid as a natural antioxidant. Biosci Biotechnol Biochem, 1998; 62:22–27.

Krakauer

: The polyphenol chlorogenic acid inhibits staphylococcal exotoxin-induced inflammatory cytokines and chemokines. Immunopharmacol Immunotoxicol, 2002; 24:113–119.

Tanaka

, Nishikawa

, Shima

, et al.: Inhibitory effects of chlorogenic acid, reserpine, polyprenoic acid (E-5166), or coffee on hepatocarcinogenesis in rats and hamsters. Basic Life Sci, 1990; 52:429–440.

Kim

, Heo

, Kim

, Yang

, Lee

: Sweet and sour cherry phenolics and their protective effects on neuronal cells. J Agric Food Chem, 2005; 53:9921–9927.

Jiang

, Satoh

, Kusama

, Watanabe

, Sakagami

: Interaction between chlorogenic acid and antioxidants. Anticancer Res, 2000; 20:2473–2476.

Jiang

, Satoh

, Watanabe

, Kusama

, Sakagami

: Inhibition of chlorogenic acid-induced cytotoxicity by CoCl₂ . Anticancer Res, 2001; 21:3349–3353.

Olthof

, Hollman

, Zock

, Katan

: Consumption of high doses of chlorogenic acid, present in coffee, or of black tea increases plasma total homocysteine concentrations in humans. Am J Clin Nutr, 2001; 73:532–538.

10.

Higdon

, Frei

: Coffee and health: A review of recent human research. Crit Rev Food Sci Nutr, 2006; 46:101–123.

11.

Peypoux

, Bonmatin

, Wallach

: Recent trends in the biochemistry of surfactin. Appl Microbiol Biotechnol, 1999; 51:553–563.

12.

Ongena

, Ja

: Bacillus lipopeptides: Versatile weapons for plant disease biocontrol. Trends Microbiol, 2008; 16:115–125.

13.

Heerklotz

, Seelig

: Detergent-like action of the antibiotic peptide surfactin on lipid membranes. Biophys J, 2001; 81:1547–1554.

14.

Jung

, Lee

, Kim

: Recent studies on natural products as anti-HIV agents. Curr Med Chem, 2000; 7:649–661.

15.

Kracht

, Rokos

, Ozel

, Kowall

, Pauli

, Vater

: Antiviral and hemolytic activities of surfactin isoforms and their methyl ester derivatives. J Antibiot (Tokyo), 1999; 52:613–619.

16.

Devasagayam

, Tilak

, Boloor

, Sane

, Ghaskadbi

, Lele

: Free radicals and antioxidants in human health: Current status and future prospects. J Assoc Physicians India, 2004; 52:794–804.

17.

Wang

, Lee

, Mau

: ROS generation, apoptosis and autophagy play important roles in silver nanoparticles triggered cytotoxicity in Beas2B cells. Toxicol Lett, 2014; 229:S200.

18.

Wardman

: The importance of radiation chemistry to radiation and free radical biology (The 2008 Silvanus Thompson Memorial Lecture). Br J Radiol, 2009; 82:89–104.

19.

Naegele

: Determination of Chlorogenic Acid in Coffee Products According to DIN 10767. Food Testing & Agriculture–Food Authenticity. Aligent Technologies, Inc., Santa Clara, CA, 2013.

20.

Budryn

, Nebesny

, Podsedek

, et al.: Effect of different extraction methods on the recovery of chlorogenic acids, caffeine and Maillard reaction products in coffee beans. Eur Food Res Technol, 2009; 228:913–922.

21.

Chen

, Yu

QMJ

, Li

, Luo

, Liu

: Extraction and HPLC characterization of chlorogenic acid from tobacco residuals. Sep Sci Technol, 2007; 42:3481–3492.

22.

Gruz

, Novak

, Strnad

: Rapid analysis of phenolic acids in beverages by UPLC-MS/MS. Food Chem, 2008; 111:789–794.

23.

Clifford

, Zheng

, Kuhnert

: Profiling the chlorogenic acids of aster by HPLC-MSn. Phytochem Anal, 2006; 17:384–393.

24.

Nadi

: Bioactive compound retention in Echiumamoenum Fisch. & C. A. Mey. petals: Effect of fluidized bed drying conditions. Int J Food Properties, 2017; 20:2249–2260.

25.

Kalt

, Mcdonald

, Donner

: Anthocyanins, phenolics, and antioxidant capacity of processed lowbush blueberry products. J Food Sci, 2000; 65:390–393.

26.

Skrede

, Wrolstad

, Durst

: Changes in anthocyanins and polyphenolics during juice processing of highbush blueberries (Vaccinium corymbosum L.). J Food Sci, 2000; 65:357–364.

27.

Singh

, Sharma

, Singh

: Hydrogen peroxide induces apoptosis in HeLa cells through mitochondrial pathway. Mitochondrion, 2007; 7:367–373.

28.

Jiang

, Kusama

, Satoh

, Takayama

, Watanabe

, Sakagami

: Induction of cytotoxicity by chlorogenic acid in human oral tumor cell lines. Phytomedicine, 2000; 7:483–491.

29.

Rakshit

, Mandal

, Pal

, et al.: Involvement of ROS in chlorogenic acid-induced apoptosis of Bcr-Abl+ CML cells. Biochem Pharmacol, 2010; 80:1662–1675.

30.

Ramos

, Alia

, Bravo

, Goya

: Comparative effects of food-derived polyphenols on the viability and apoptosis of a human hepatoma cell line (HepG2). J Agric Food Chem, 2005; 53:1271–1280.

31.

Cao

, Wang

, et al.: Surfactin induces apoptosis in human breast cancer MCF-7 cells through a ROS/JNK-mediated mitochondrial/caspase pathway. Chem Biol Interact, 2010; 183:357–362.

32.

Kim

, Kim

, et al.: Surfactin from Bacillus subtilis displays anti-proliferative effect via apoptosis induction, cell cycle arrest and survival signaling suppression. FEBS Lett, 2007; 581:865–871.

33.

Hayes

, Stanton

, Fitzgerald

, Ross

: Putting microbes to work: Dairy fermentation, cell factories and bioactive peptides. Part II: Bioactive peptide functions. Biotech J, 2007; 2:435–449.

34.

Van Boekel

, Fogliano

, Pellegrini

, et al.: A review on the beneficial aspects of food processing. Mol Nutr Food Res, 2010; 54:1215–1247.

35.

Amigo-Benavent

, Wang

, Mateos

, Sarria

, Bravo

: Antiproliferative and cytotoxic effects of green coffee and yerba mate extracts, their main hydroxycinnamic acids, methylxanthine and metabolites in different human cell lines. Food Chem Toxicol, 2017; 106(Pt A): 125–138.