Sample preparation

Corn silk was collected from the corn named ‘Kwangpyeongok’ cultivated by the Korean National Institute of Rural Development. The corn silk was treated with a silk bag before corn silk got pollen on them. The unpollinated corn silks were collected after 3 days of silking, the corn silk was cut into approximately 5 to 10 cm size, and extracted at room temperature with ethanol (Sigma, St. Louis, MO, USA). The process for preparing corn silk extract with high content of maysin was described previously (6). Briefly, corn silk was cut into about 5~10 cm size immediately after harvesting. Then, 2 L of 99% ethanol was added to 1 kg of corn silk and incubated at room temperature for 15 days. The extract was filtered twice and concentrated under reduced pressure at 40°C to obtain corn silk extract. Chlorophyll was removed by adding methylene chloride (CH₂Cl₂) 1:1.5 (v:v) to the corn silk extract. The pulverized corn silk extract was again concentrated under reduced pressure. The concentrated corn silk extract was then loaded onto a C18 column to remove carbohydrate and polyphenol materials. As the mobile phase, distilled water was used for carbohydrate removal while 100% ethanol was used for polyphenol removal. From the corn silk extract, active substances including maysin were extracted with distilled water and ethanol as the mobile phase solvent at a flow rate of 20 mL/min for 70 min. The maysin content in the final corn silk extract was 35%. The sample was dried and used in the form of solid powder. The test substance was weighed with an electronic scale on the day of use. It was then added with an excipient to prepare different concentrations (0~50 mg/mL). Corn silk extract, the test substance, was supplied by the Rural Development Administration and National Institute of Crop Science, Republic of Korea. In general, the maysin content in the corn silk extract ranged from 5.2 to 230.5 mg/100 g. However, the content of maysin in the corn silk extract used in this study was 2,783 mg/100 g.