*2.3. Plasma Growth Hormone and Insulin-Like Growth Factor-1 (IGF-1) Levels*

As aging-associated changes of skin condition are partially mediated by decreased levels of growth hormone [13], we analyzed plasma levels of growth hormone before and 30, 60, 120, 150, 180 and 240 min after supplementation. Growth hormone levels before supplementation were more than two-fold higher in the CPO group compared with the placebo group (0.65 ± 0.49 ng/mL in the placebo group vs. 1.41 ± 1.70 ng/mL in the CPO group, *p* = 0.778), suggesting that comparison of growth hormone levels between CPO and placebo groups was unworthy of evaluation. Indeed, we observed no significant differences of plasma growth hormone levels in the CPO group compared with the placebo group. We also evaluated IGF-1 levels at baseline and after 8 weeks of supplementation because IGF-1 levels reflect growth hormone secretion [15]. No statistically significant difference between CPO and placebo groups was observed. However, a statistically significant increase of IGF-1 levels from the baseline was observed only in the CPO group (Figure 3), suggesting that the improvements of skin conditions by CPO were mediated by the increase of IGF-1 levels, at least in part.

**Figure 3.** Dietary supplementation with collagen peptide and ornithine increased plasma insulin-like growth factor-1 (IGF-1) levels. Comparison of plasma IGF-1 levels before and after 8-week supplementation with collagen peptide and ornithine (CPO) (right) or with placebo (left). \* *p* < 0.05 by paired *t*-test. Error bars indicate SD.

#### *2.4. Clinical Safety*

We observed neither adverse events nor severe changes in scores for general biochemical examination of blood or hematologic tests. Adverse events related to the ingestion of CPO were not observed. Thus, safety concerns were not observed.

#### **3. Discussion**

To our knowledge, our study is the first report to demonstrate the combined effects of CPO on skin conditions, plasma growth hormone and IGF-1 levels. We found that dietary supplementation of CPO improved skin elasticity and TEWL. TEWL was increased in the placebo group, while the CPO group showed no increase, indicating that the seasonal increase of TEWL was prevented by CPO (Table 2). As TEWL is linked to the barrier function of skin [1], this result suggests a protective effect of CPO for skin barrier function. In addition to TEWL, we observed a statistically significant difference between placebo and CPO groups in the changes of elasticity from baseline. Elasticity in the placebo group was decreased, while the CPO group showed increased elasticity, suggesting that the seasonal decrease in elasticity was prevented by CPO (Figure 2A). Furthermore, we observed increased IGF-1 levels only in the CPO group, suggesting that the improvements of skin conditions were mediated, at least in part, by increased IGF-1 levels (Figure 3).

The effects of collagen peptide on the improvements of skin moisture, elasticity, wrinkles, ultraviolet-induced erythema and ultraviolet-induced pigmented spots were previously revealed by several groups [28,29,45,47–49]. Furthermore, it has been previously reported that oral administration of marine collagen peptide derived from the skin of Nile Tilapia enhanced the process of wound healing [32]. Marine collagen peptide derived from Chum Salmon also promoted cutaneous wound healing [50,51]. Our study reinforced the beneficial effects of fish-derived collagen peptide to maintain or improve skin conditions such as elasticity and TEWL. Furthermore, we performed combined administration of collagen peptide and ornithine. Similar to collagen peptide, a previous study showed that ornithine enhanced wound healing effects by upregulating collagen synthesis in mice [52], suggesting that both collagen peptide and ornithine contribute to the improvements of skin conditions. In light of the independent effects of collagen peptide and ornithine, we hypothesized that skin conditions would be improved by the synergistic effects of CPO to increase growth hormone and/or IGF-1 levels, as described in the Introduction. Indeed, we observed the increased IGF-1 levels only in the CPO group. Between-group differences for TEWL and elasticity reinforced our hypothesis. However, because the effects of ornithine on skin conditions have not been investigated in humans, we could not conclude that the improvements of skin conditions observed in this study were derived from either the sole effects of collagen peptide or ornithine, or the synergistic effects of CPO. Furthermore, because we did not evaluate the sole effects of collagen peptide in this study, we could not conclude that the previously observed improvements of skin conditions including moisture and TEWL by collagen peptide [28,29,45,47–49] was enhanced by co-administration of ornithine. A comparison of the sole effects of ornithine, collagen peptide, and CPO is required to evaluate the synergist effects of CPO in the future.

As described in the Materials and Methods section, we employed a cutometer with a 6-mm diameter probe, suggesting that the improvement of skin elasticity reflected the state of relatively deep skin areas, such as the dermis. Generally, improvement of skin barrier function leads to the attenuation of TEWL, which results in subsequent improvement of the dermis environment [53,54]. In addition to this general understanding, increased IGF-1 levels in the CPO group suggested that the attenuation of TEWL occurred through the improvement of the dermal environment, which can result in the activation of dermal fibroblasts. Increased collagen scores in the CPO group, as measured by the DermaLab test, support the notion of CPO improving the dermal environment. Furthermore, previous studies have shown that the increased IGF-1 levels or treatment with collagen peptides leads to the activation of dermal fibroblasts [55,56]. Activated dermal fibroblasts construct the firm structure of the basement membrane, which is required for stable adherence of epidermal cells to the basement membrane. This stable adherence maintains an adequate balance between proliferation and differentiation of epidermal cells, leading to the enhancement of barrier function and subsequent attenuation of TEWL [57]. However, further study is required to determine how CPO improved skin elasticity and TEWL, as well as the relationship between improvements of elasticity and TEWL. Furthermore, we focused on the improvement of skin elasticity exclusively in the neck because only a thin muscle is present under neck skin [58]. However, as the effects of IGF-1 would not be restricted only to neck skin, we suspect that the positive effects observed in this study would be applicable to other areas of skin.

In this study, we hypothesized that CPO improved skin condition by increasing the secretion of growth hormone and/or IGF-1. Indeed, we observed increased plasma IGF-1 levels, which reflect increased secretion of growth hormone in the CPO group [15]. However, we did not observe an apparent increase of growth hormone levels immediately after CPO supplementation. One possibility for this result is that CPO enhanced secretion of growth hormone levels during the night, as we required participants to take CPO before bed time and growth hormone is secreted during non-rapid eye movement sleep. Thus, analysis for the effect of CPO on growth hormone secretion during sleep merits future investigation to potentially explain increased IGF-1 levels elicited by CPO supplementation.

Our study has several limitations. Even though the sample size was limited, which rated this study as a pilot trial, we observed improvement of skin elasticity, TEWL and increase of IGF-1 levels by CPO, suggesting the strong effects of CPO. Ornithine is found in freshwater clams, a traditional food for Japanese people. Furthermore, fish dishes are favored by Japanese people. We prohibited participants from continuously ingesting a functional food with identical or similar effects as the active ingredient of the test food. However, we did not estimate the exact dietary intake of CPO by participants; thus, the effects of CPO were potentially underestimated or overestimated. We hypothesized that combined supplementation of CPO elicited increased growth hormone and/or IGF-1 levels, which was followed by the improvement of the skin condition. In fact, we observed improvements of both elasticity and TEWL. However, the only intra-group difference was observed for increased plasma IGF-1 levels in the CPO group. Thus, precise mechanisms underlying how CPO improved the condition of skin, the specific contributions of CPO, and potential synergistic effects of CPO were not elucidated. Further analysis or a large-scale study is required to examine how CPO influences skin conditions.
