After washing, the fresh porcine placenta underwent two freeze (−20˚C)-thaw (under 10˚C) cycles. The obtained freeze-thaw drip was heated at 65˚C for 6 h and subjected to vacuum-freeze drying, followed by removal of debris and filtration. Powdered PPE was reconstituted in sterilized water to a concentration of 50 mg/mL for in vitro testing. The gel formula containing 1.1 mg/mL PPE was kindly provided by Kasyu Industries Co., Ltd. (Fukuoka, Japan).

The protocol was reviewed and approved by the Ethical Committee for Research in Septem-Soken. The study was designed as an open-labeled, single-blinded and randomized half-side investigation, without a control group. Fifteen female volunteers, recruited from our volunteer pool and aged 40 - 52 years (48.3 ± 3.7), participated in this study after informed consent was obtained from all volunteers. The volunteers were advised to apply a pearl-size amount (approximately 0.28 g) of the PPE gel on the designated half side of the face, twice daily for six weeks. The wrinkle width, water-holding property, and elasticity were assessed using Visia Evolution (Canfield Scientific, Parsippany, NJ), Skicon-200EX (Yayoi, Tokyo, Japan), and Cutometer®MPA580 (Courage + Khazaka electronic GmbH, Cologne, Germany), respectively, at weeks 0 and 6. The laboratory conditions were maintained in the temperature range of 20˚C - 22˚C, with a relative humidity of 50% ± 5%, during measurements. The data are expressed as the ratio (%) to week 0 in the control and treated sites, respectively.

Normal human dermal fibroblasts (NHDFs) were obtained from PromoCell. NHDFs were maintained in fibroblasts growth media 2 (FGM2), supplemented with 20 µL/mL fetal calf serum, 1 ng/mL basic fibroblast growth factor (bFGF), and 5 µg/mL insulin at 37˚C in a 5% CO₂-humidified atmosphere. The cells (F4-F6) were then subjected to experiments. HaCaT cells, an immortal human epidermal keratinocyte cell line, were maintained in Ca²⁺-free Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 5% Ca²⁺-depleted FCS, 4 mM glutamine, 1 mM sodium pyruvate, and 2 mM CaCl₂ at 37˚C in a 5% CO₂-humidified atmosphere.

In brief, HaCaT cells were inoculated into 96-well plates at a cell density of 5 × 10³ cells/well and maintained in a 5% CO₂-humidified atmosphere at 37˚C for 24 h; then, the cells were further incubated in the presence of the indicated PPE concentrations. The neutral red assay was performed according to the manufacturer’s instruction (Sigma-Aldrich, St. Louis, MO).

In brief, HaCaT cells were seeded into 96-well plates at a cell density of 2 × 10³ cells/well in a 5% CO₂-humidified atmosphere at 37˚C for 24 h. After incubation, the medium were removed. Then, 200 µL/well of the medium containing the indicated concentration of PPE and predetermined concentration of BrdU was added to wells, and cells were subjected to further incubation for 48 h. The incorporated BrdU was detected using a BrdU Cell Proliferation Assay kit (Cell Signaling, Boston, MA, USA), according to the manufacturer’s instructions.

To estimate the production of type I collagen from NHDFs and hyaluronic acid in HaCaT cells, NHDFs and HaCaT cells were seeded into 24-well plates at a cell density of 2.5 × 10⁴ cells and maintained in a 5% CO₂-humidified atmosphere at 37˚C for 24 h. The cells were then treated with 5% PPE for 24 h. The amount of type I collagen and hyaluronic acid were measured in the supernatant using a type I collagen ELISA kit and hyaluronic acid ELISA kit (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions.

HaCaT cells were seeded into 6-well plates at a cell density of 3 × 10⁵ cells/well and maintained in a 5% CO₂-humidified atmosphere at 37˚C for 24 h; then, the cells were further cultivated in the presence of KX-1 or its fractions for 24 h. Cells were harvested and total RNA was prepared using an SV RNA isolation kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions, followed by reverse transcription with ReverTra Ace® qPCR RT Master Mix (TOYOBO, Osaka, Japan). PCR amplification and detection were performed on a CFX96 real-time PCR system (BioRad, Hercules, CA, USA) using a KAPA SYBR FAST qPCR master mix (KAPA Biosystems, Woburn, MA, USA). The following primer pairs were used: β-actin, 5’-GATGAGATTGGCATGGCTTT-3’ (sense) and 5’-CACCTTCACCGTTCCAGTTT-3’ (antisense); CERS3, 5’-ACAT TCCACAAGGCAACCATTG-3’ (sense) and 5’-CTCTTGATTCCGCCGACTCC-3’ (antisense); FLG, 5’-TGGCAGCTATGGTAGTGCAG-3’ (sense) and 5’-CCAAA CGCACTTGCTTTACA-3’ (antisense); TGM1, 5’-ACTACGGCCAGTTTGACC AC-3’ (sense) and 5’-TCGGGAGTAATCACCAGACC-3’ (antisense); and KLK7, 5’-ATGGCAAGATCCCTTCTCCT-3’ (sense) and 5’-GGCGCCATCAATAATC TTGT-3’ (antisense). Target mRNA expression was quantified using the comparative threshold cycle (Ct) method for relative quantification (2^−δδCt), normalized to the geometric mean of the reference gene β-actin.

Data are expressed as the mean ± standard deviation (SD). Statistical analysis was performed using Student t-test. A value of p < 0.1 was considered statistically significant forin vivo tests. A value of p < 0.05, was assumed to be significant for in vitro tests.

To evaluate the effects of PPE on skin conditions, various skin properties were assessed. The number of detectable wrinkles was reduced (Figure 1(a)) and the calculated wrinkle index was also significantly decreased to 72.1% of the control (Figure 1(b)). In contrast, conductance was significantly increased to 126.5 % at the PPE-treated site, when compared with the control site (Figure 1(c)); no significant changes in gross elasticity (Figure 1(d)) and elastic recovery (Figure 1(e)) were observed.

The neutral red assay was used to estimate the cytotoxicity of PPE. The results revealed that PPE did not significantly affect cell viability at concentrations ranging between 1 and 5% of PPE (Figure 2(a)). Likewise, no significant effects on cell proliferation were observed at 2% and 5% of PPE (Figure 2(b)). Thus, a concentration of 5% PPE was used for further experiments.

To estimate the effects of PPE on extracellular matrix production, the production of type I collagen and hyaluronic acid was assessed in NHDF. Treatment with PPE induced a marginal but significant enhancement in type I collagen production (Figure 3(a)). In contrast, PPE did not impact hyaluronic acid production (Figure 3(b)).

Next, we evaluated the effects of PPE on the mRNA expression of proteins involved in skin barrier function and water-holding properies. The mRNA expressions levels of ceramide synthase 3 (CERS3), filaggrin (FLG), transglutaminase 1 (TGM1) and kallikrein-7 (KLK7) were significantly upregulated to 1.85, 1.35, 1.76, and 1.62, respectively (Figures 4(a)-(d)).