The LNC wrinkle eye cream (LNC-EC; Japan Bio Products Co) was used.

This randomized, single-blind clinical trial was conducted from January 20 to February 3, 2022, in Kirei Testing Labo Co, Ltd. We established a single group to compare the untreated and treated sites in the same participant. Randomization was performed to reduce bias.

This study was reviewed and approved by the Kirei Testing Laboratory Ethics Committee on January 18, 2022 (ID 22000134) and registered in the University Hospital Medical Information Network Clinical Trials Registry (UMIN000051646). The participants signed an informed consent, which included the study purpose, methods, confidentiality, and the right to withdraw. Confidentiality was ensured. Study procedures were conducted according to the World Medical Association’s Helsinki Declaration and its amendments.

The inclusion criteria were as follows: healthy Japanese women aged ≥20 and <59 years at the time of consent, women with wrinkle grades 1 to 3 at both eye corners, and women with skin classified as Fitzpatrick type III or IV. Exclusion criteria included the following:

If any information rendering them ineligible was identified during the study, they were excluded from the analysis. A total of 24 participants were selected for the study through visual assessment based on the wrinkle grade standard (8 grades: 0-7). Informed consent was obtained from all participants. Furthermore, a consultation service was established at a medical institution to address any health-related enquiries.

Test products were randomly assigned to participants using the half-face method on symmetrical areas of the face, with no apparent bias in the left-right distribution [11]. Participants were instructed to apply the test product (0.5 g) to one-half of the face both in the morning and at night, consistently at the same time, and to continue applying the product until the final measurement date.

Photographic and visual assessments were conducted in accordance with the guidelines for evaluating antiwrinkle products, as authorized by the Japanese Cosmetic Science Society. Trained experts, proficient in wrinkle evaluation, assigned participants an 8-point wrinkle grade score using the wrinkle grade evaluation criteria and photographs provided by the Japanese Society of Cosmetic Science and Technology [12].

Skin replicas of the target site (10 × 10 mm or larger) were made and analyzed using a replica analysis system (ASA-03RXD; ASH, Japan) to calculate the wrinkle area and volume ratios, maximum wrinkle depth and width, average wrinkle depth, and the number of wrinkles [13].

The untreated and treated sides were compared by calculating the change (Δ) at 2 weeks (baseline scores minus 2-week scores, reported as the mean change from baseline for a parameter). Skin replica analysis was performed using a statistical software program built into the replica analysis system (ASA-03RXD; Nihon Ash Co, Ltd). For each clinical parameter, the groups were compared using the Wilcoxon signed rank test and a paired 2-tailed t test (P=.05) for wrinkle and replica analysis, respectively, to determine statistically significant differences in improvement between the untreated and treated sides.

The wrinkle grade on the treated side was 2.25 at baseline but decreased to 2.0 after 2 weeks of use, resulting in less noticeable wrinkles. On the untreated side, the grade remained at 2.0 from baseline to the end of the 2-week period (Figure 1A). A significant difference was observed when subtracting the pretest from the posttest assessment value based on the wrinkle grade standard (Figure 1B).

No significant differences were observed in pre- and posttest results for wrinkle area ratio, wrinkle volume percentage, maximum wrinkle depth, and wrinkle mean depth. Meanwhile, a significant difference was detected in pre- and posttest results for maximum wrinkle width and the number of wrinkles (Figure 2).

In this study, LNC-EC was applied to healthy participants for 2 weeks to verify its cosmetic effects. Both wrinkle grade evaluation and replica analysis demonstrated its antiwrinkle effects at the eye corners. The replica analysis revealed a reduction in the maximum width and the number of wrinkles. These results suggest that horse placental extract has cosmetic benefits.

Wrinkles can result from external factors like dryness due to decreased temperature and humidity as well as internal factors related to reduced moisture retention due to aging. This study was conducted during winter when temperature and humidity levels are at their lowest. Consequently, external factors could potentially compromise the barrier function of each skin layer. The stratum corneum’s barrier function weakens during winter due to smaller corneocytes compared to summer [14]. Epidermal dryness leads to structural changes causing shallow wrinkles, so preventing dryness is crucial in improving wrinkles. Horse placental extract enhances skin barrier function by promoting the growth of Staphylococcus epidermidis and antimicrobial peptide expression in human epidermal keratinocytes [8]. Furthermore, aged human keratinocytes exhibit increased stratifin expression, suppressing filaggrin and serine palmitoyltransferase involved in epidermal moisturization [15]. Horse placenta extract reduces stratifin expression in aged human cultured epidermal cells [9], suggesting that strengthening the skin barrier with this extract may help prevent worsening wrinkles.

Ultraviolet B stimulates epidermal keratinocytes, resulting in the release of inflammatory cytokines (interleukin [IL]-1α, IL-6, and tumor necrosis factor-α), which stimulate dermal fibroblasts and keratinocytes to increase matrix metalloproteinase (MMP)-1, MMP-3, and MMP-9—enzymes that degrade collagen and elastic fibers, causing wrinkles [16]. Horse placenta extract suppresses IL-6 expression, which increases with age, in cultured aged human epidermal cells [9]. This suggests that the extract may indirectly mitigate the worsening of wrinkles by preventing increased MMP expression. Considering these factors, LNC-EC’s antiwrinkle effects in this study likely result from the placental extract’s various biological activities.

LNC-EC appears effective in improving the appearance of eye-corner wrinkles, presumably by reducing their maximum width and number. No adverse events associated with LNC-EC application were observed during the study, indicating its safety for use.