Buchi rotavapor r 144

Schisandrae Fructus were collected around Mungyeong- (Gyeongsangbuk-do, Republic of Korea) and washed three times with tap water before storage at −20°C. The frozen samples were lyophilized and homogenized using a grinder before extraction. The materials were extracted with 20% ethanol (EESF) at room temperature for 24 h, filtered, and concentrated using a rotary vacuum evaporator (Buchi Rotavapor R-144, BÜCHI Labortechnik, Flawil, Switzerland). The extract was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) as a 50 mg/mL stock solution and stored at 4°C until use. In addition, oxymetholone, 17β-hydroxy-2-hydroxymethylidene-17α-methyl-3-androstanone (Celltrion Pharm Inc., Jincheon, Republic of Korea), which is an orally active 17α-alkylated anabolic-androgenic steroid first described by Ringold et al. [38 (link)], was used as the reference drug; it was also stored in a refrigerator at 4°C to protect from light and degeneration, until use.

Reddish brown clear SSeo was prepared by maceration and hydrodistillation methods as follows. Briefly, dried seeds of S. chinensis (Turcz.) Baillon were collected around Mungyeong-city (Gyeongbuk, Republic of Korea) on October 2013 and completely dried at 180°C in a furnace (Daihan Scientific Co., Seoul, Republic of Korea). A voucher specimen (accession number DSSC-1) was deposited at the Medical Research Center for Globalization of Herbal Formulation of Daegu Haany University. The dried seeds were then pulverized and lyophilized in a programmable freeze dryer (Freezone 1; Labconco Co., Kansas City, MO, USA). Lyophilized materials were extracted with 100% ethanol by maceration at room temperature for 24 h, filtered, and then concentrated using a rotary vacuum evaporator (Buchi Rotavapor R-144, BÜCHI Labortechnik, Flawil, Switzerland). Finally, the SSeo (Lot. 2012KuSSeo) was isolated by hydrodistillation using a Clevenger-type apparatus for 3 h according to the method recommended in a previous study [40 (link)]. The oil was stored in a refrigerator at 4°C to protect from light and degeneration. The yield of the oil based on the dried weight of the Schisandrae Semen was 0.66%.

Ecklonia stolonifera samples were collected from Jeju Island, Jeju Province, Korea. Botanical identification was made by Wook Jae Lee (Jeju Technopark, Jeju, Korea), and a sample specimen was deposited at the herbarium of the Jeju Biodiversity Research Institute, Jeju, Korea. The dried Ecklonia stolonifera samples were homogenized into a fine powder using an electric mixer (HMF-3100S, Hanil Electric, Seoul, Korea). The Ecklonia stolonifera extract was prepared by dissolving the powder in 80% ethanol at room temperature. This solution was then filtered and concentrated using a rotary vacuum evaporator (Buchi Rotavapor R-144, Buchi Labortechnik, Flawil, Switzerland).

PT was acquired off the coast of Jeju Island. The collected samples were sterilized and dried in a cool place. PT was purchased from Jeju Technopark Biodiversity Research Institute (gift certificate sample number: JBRI-16041). PT specimens were stored in the Herbarium of the Jeju Institute of Biological Diversity, and the identification of deposited PTs was performed by Dr. Wookjae Lee (Jeju Technopark, Jeju, Korea) [33 (link)]. The samples were dehydrated and ground to a fine powder using an electric mixer (HMF-3100S, Hanil Electric, Seoul, Korea). A 40–50 mesh standard test sieve was used to obtain the smallest possible size of the PT powder. To prepare the PT extract, 10 g of well-ground PT powder and 300 mL of 80% ethanol were mixed, extracted at room temperature for 12 h, and centrifuged at 6000× g for 10 min to obtain the supernatant. The supernatant was filtered and concentrated using a rotary vacuum evaporator (Buchi Rotavapor R-144, Buchi Labortechnik, Flawil, Switzerland). The obtained PT extract was freeze-dried, and the PT powder was preserved at −80 °C for subsequent experiments. The PT powder was dissolved in an aqueous solution at a concentration of 4 mg·mL⁻¹ to use in the PT-AuNS medium. The solution was filter sterilized using a 0.2 μm syringe filter.