In vitro cytotoxic activity of ginseng leaf/stem extracts obtained by subcritical water extraction

摘要

Ginseng leaf/stem extract produced by subcritical water extraction at high temperature (190°C) possess higher cytotoxic activity against human cancer cell lines than ethanol extract. Subcritical water extraction can be a great candidate for extraction of functional substance from ginseng leaves/stems. Keywords: cytotoxic activity, flavonoid, leaves and stems, Panax ginseng , subcritical water extraction Ginseng ( Panax ginseng Mayer) is a valuable agricultural product used in many traditional medicinal therapies [1] . Although ginseng root sells at a high price, growing ginseng in a forest environment is not a highly lucrative business strategy, because it requires a minimum growth of 5–8 years prior to harvesting [2] . Unlike ginseng root, it is possible to harvest ginseng leaves annually. Furthermore, ginseng leaves and stems were found to be rich in polysaccharides, phenolics, flavonoids, and ginsenosides [3,4] . Previous studies evaluated the bioactivity of extracts of ginseng leaves and stems; however, these extracts were prepared by traditional methods and using organic solvents such as methanol, n-hexane, chloroform, ethyl acetate, and n-butanol [5] . Also, these techniques involve a long extraction period and produce low yields. Subcritical water (SW) extraction has been utilized extensively in various areas of green engineering and material cycling [6,7] . Under subcritical conditions, the dielectric constant of water can be altered [8] . This study aims to compare the antitumor efficacy of ginseng leaf and stem extracts, prepared by both traditional and SW extraction procedures, in human cancer cell lines. Ginseng leaves and stems were extracted by ethanol, hot water, and SW extraction. For ethanol extraction, ginseng leaves and stems (20?g) were mixed with 200?mL of 70% (v/v) ethanol and heated for 3 hours at 60°C in a water bath. For hot water extraction, the sample (20?g) was dissolved in 200?mL distilled water and heated for 3 hours at 80°C in a water bath. After extraction, the slurry was filtered through filter paper (Whatman No. 2, GE Healthcare UK Limited, Amersham Place, Little Chalfont, Buckinghamshire, UK), and the solid residue was extracted twice more under identical conditions. The solvent was evaporated using a rotary evaporator (N-1000V; Eyela, Tokyo, Japan). After the evaporation was completed, the extract was transferred to a freeze-drying tube and lyophilized. The dried sample was then weighed and stored at??20°C prior to analysis [9] . SW extraction was performed using an SW extraction system (DIONEX ASE 100; Dionex Corporation, Sunnyvale, CA, USA). The extraction cell (34?mL) was filled with a mixture of ginseng powder and diatomaceous earth in the ratio of 1:3, and placed vertically in the extraction apparatus. And then distilled water flows in a Milli-Q system (Millipore, Bedford, MA, USA) into the apparatus. Working temperature and static time were set at 110°C, 165°C, and 190°C for 15 minutes. During extraction, the pressure was maintained at less than 500?psi. After extraction, fresh water was pumped through the entire pathway, including the cell, for washing. The SW extracts were freeze-dried and stored at??20°C until used. Human cancer cell lines were purchased from the Korean Cell Line Bank (Seoul National University, Seoul, Korea). The AGS (human stomach adenocarcinoma), HT-29 (human colorectal adenocarcinoma), and MCF-7 (human breast adenocarcinoma) cell lines were maintained in RPMI 1640 medium (Gibco Laboratories, Grand Island, NY, USA) containing 10% heat-inactivated fetal bovine serum (HyClone, Logan, UT, USA), penicillin (100?U/mL), and streptomycin (100?μg/mL). SK-MES-1 (human lung carcinoma) cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin, and streptomycin. HeLa (human cervical adenocarcinoma) cells were grown in Minimum Essential Medium containing 10% heat-inactivated fetal bovine serum, penicillin, and streptomycin. All cell lines were cultured in a 37°C, 5% CO₂ incubator. For experimentation, adherent cells in the logarithmic growth phase were harvested using 0.25% Trypsin (Life Technologies, Inc., Carlsbad, CA, USA). Cells were counted using a hemocytometer (Hausser Scientific, Horsham, PA, USA). For cytotoxicity testing, cells were seeded in new dishes and grown to 80% confluence prior to treatment. Cytotoxicity was determined by quantifying the relative cell number. In this study, cytotoxic activity was assessed by a modified MTT assay, as described previously [10] . Briefly, 100?μL aliquots of the cell suspension were transferred to 96-well microplates (1.5?×?104 cells/well) and incubated for 24 hours. Ginseng extracts were dissolved in phosphate-buffered saline, and the final concentrations were adjusted to 0.25?mg/well, 0.5?mg/well, 1?mg/well, and 2.5?mg/well. Cells were incubated with the extracts for 44 hours at 37°C. At the end of the incubation, MTT