Skh 1

Twelve-month-old and eight-week-old young female albino hairless mice (Skh-1) were purchased from Orient Bio (Seongnam-si, Korea). The animals were allowed to feed ad libitum and were acclimated for one week prior to the study. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the Center for Phenogenomics Animal Research, Woojung BSC (Suwon, Korea, accredited by the Association for Assessment and Accreditation of Laboratory Animal Care). The aged mice were randomly divided into two equivalent groups, one administered vehicle and the other TEE. The animals were orally fed with a dose of 400 mg/kg using a feeding needle once daily for six weeks and were sacrificed six hours after the last administration. For vehicle-treated young and aged mice, the same volume (i.e., 0.2 mL) of 0.5% carboxymethyl cellulose-sodium solution was administered once daily for six weeks (Figure 1A). Each group composed of nine mice and vehicle-treated young mice were used as positive control.

Five-week-old female albino hairless mice (Skh-1) were provided by Orient Bio (Seongnam, Korea). Animals were acclimated for 1 week prior to the study and had free access to water and food. The Institutional Animal Care and Use Committee (No. 2014-0057, 19 May 2014) of the Biomedical Research Institute, Kyongpook National University approved all experimental protocols. Six to eight mice were allocated into seven groups.
The test compounds and vehicle (0.5% sodium carboxymethylcellulose) were orally administered for nine weeks. Body weight and food intake were monitored on a weekly basis. A UVB-induced photoaging procedure was undertaken, as described previously. A UVB irradiation device that included a TL20W/12RS UV lamp (Philips, Eindhoven, The Netherlands) with an emission spectrum between 275 and 380 nm (peak, 310–315 nm) served as the UV source. Initially, the minimal UVB dose on the dorsal skin of mice as was measured as the minimal edema dose (MEdD) comparable with the minimal erythema dose in human skin. In contrast to human skin, the mouse skin showed peak responses to UVB primarily as edema, an increased thickness of the dorsal skin at 48 h post-UVB irradiation. The irradiation dose was increased weekly by 0.5 MEdD (1 MEdD = 100 mJ/cm²) up to 2 MEdD and then maintained at 2 MEdD. UVB irradiation was stopped after nine weeks (Figure 1A).

Eight-week-old female hairless mice (SKH-1: Orient Bio Inc., Seongnam, Korea) were housed in temperature- (23 ± 2°C), humidity- (55 ± 10%), and light- (12 h day/12 h night) controlled conditions for 24 weeks at Yonsei Laboratory Animal Research Center (YLARC; Seoul, Korea). All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of YLARC (Permit number is 2013-0113). Mice were randomly assigned into two groups: (1) middle-aged (MA) group and (2) MA administered with KPE group. Mice in the KPE administered group orally received 200 mg/kg/day KPE for 24 weeks. For the young group, 7-week-old female hairless mice were purchased from Orient Bio Inc. (Seongnam, Korea) at 1 week before they were killed and were allowed to acclimatize for 1 week. After 24 weeks, the mice were killed under anesthesia by using an intraperitoneal injection of a mixture of zoletil (Virbac, Carros, France) and rompun (Bayer Korea Ltd., Seoul, Korea). Sample from the dorsal skin were rapidly frozen in liquid nitrogen and stored at −70°C. Skin biopsy samples were fixed in 10% buffered formalin for optical microscopy in order to analyze histological changes. All efforts were made to minimize the suffering of mice.

The ethanol-extracted V. uliginosum samples used in the experiments were obtained from Chong Kun Dang Co., (Seoul, Korea). Hairless mice (SKH-1, 4-week-old male) were purchased from Orient Bio Inc. (Seungnam, Korea). The mice were housed in controlled rooms (21 ± 1 °C, 50% relative humidity) with light (12:12 h light:dark cycle). All experiments were approved by the Korea University Institutional Animal Care & Use Committee (KUIACUC-2019-0062). After adaptation to the housing conditions for 1 week, six mice were allocated to each group, with a total of seven groups.

Six-week-old female hairless mice (SKH-1; Orientbio Inc., Seoul, Korea) were used to study the antiphotoaging effects of WESBD and EESBD. After a 1-week adaptation period under controlled conditions (temperature: 23 ± 2 °C, humidity: 50 ± 10%, 12 h light/dark cycle), the mice were randomly divided into six groups (n = 7 per group): no treatment (control), UVB irradiation (UVB), UVB irradiation and pretreatment with WESBD 25 mg/kg/bw (UVB + WESBD 25), WESBD 50 mg/kg/bw (UVB + WESBD 50), EESBD 5 mg/kg/bw (UVB+ EESBD 5), and EESBD 10 mg/kg/bw (UVB+ EESBD 10). The SKH-1 hairless mice were treated with WESBD and EESBD dissolved in working solution (propylene glycol:ethanol = 7:3) and then exposed to UVB (50–200 mJ/cm; 1 minimal erythematous dose (MED): 50 mJ/cm²) every other day for 10 weeks using a microprocessor-controlled UV irradiation system (Bio-Link 312; Vilber Co., Suebia, Germany) The total amount of UVB irradiation was 78 MED (3900 mJ/cm²). The MED was defined as the dose of UVB radiation required to produce minimal erythema after 24 h.