**1. Introduction**

The major role of skin is the protection of our body from external stimuli. Skin also plays an important role in maintaining homeostasis, including protection against loss of moisture and adjustment of body temperature [1]. Furthermore, aging-associated changes of skin conditions, such as the increased wrinkles and decreased skin elasticity, are a major concern for maintaining quality of life. Skin is composed of epidermis, dermis, and subcutaneous tissue. Collagen and elastin in the dermis maintain the structure of skin and create its elasticity [2,3]. Notably, the fibrous protein collagen, which plays a major role in maintaining the mechanical strength of skin, constitutes the majority of the dermis. The collagen molecule is formed by the three polypeptides, named alpha chains. The alpha chains are composed of high levels of glycine, hydroxyproline, proline and alanine [4]. Thus, collagen has unique amino acid composition. These collagen molecules assemble to form collagen fibrils by cross linking. Then, collagen fibrils assemble to form large collagen fiber. There are several types of collagen, and type I and type III collagen are the most abundant collagen in the skin [5]. Because collagen synthesis decreases with aging, decreased collagen content is one of the major causes of aging-associated changes of skin conditions [6,7]. As skin aging and nutrition states are linked [8], the improvement of skin conditions by dietary supplementation is a topic of increasing public interest [9].

The effects of growth hormone, a peptide hormone secreted from the pituitary gland (especially during the first few hours of sleep), cover a broad range of biological phenomena including cell growth, proliferation, regeneration, and metabolism [10–12]. For skin, severe growth hormone deficiency results in early aging, such as wrinkling and dryness [13]. In addition to the direct effects of growth hormone to several tissues, it also elicits indirect effects mediated by insulin-like growth factor-1 (IGF-1) [12]. Plasma IGF-1 levels correlate with plasma growth hormone levels, as production of IGF-1 by the liver is stimulated by growth hormone [14,15]. Similar to growth hormone, IGF-1 activates cell growth in several tissues including bone [16], muscle [17], and skin [18,19], whereby it contributes to both epidermal or dermal skin development and maintenance. Secretion of growth hormone is decreased with aging, suggesting that aging-associated changes of skin conditions are mediated, at least in part, by decreased levels of growth hormone, or its associated decrease of IGF-1 [20]. Thus, these previous studies suggest that aging-associated changes of skin conditions might be improved by increasing growth hormone and/or IGF-1 levels.

In recent years, many products containing collagen or a denatured form of collagen have been used for a wide variety of purposes, including cosmetics and food. With regard to food or supplements, the effects of collagen have been controversial, as orally ingested native collagen or its partially hydrolyzed form, gelatin, are not efficiently absorbed [21]. However, several lines of evidence revealed the beneficial role of collagen-derived small peptides, which exhibit high absorbability compared with native collagen [21,22], for a wide variety of tissues including bone [23], joint [24], muscle [25], tendon [26], and skin [27–29] in humans. Collagen has been isolated from many marine, brackish water and freshwater sources such as fishes [30–32] and mollusks [33–35]. Compared with collagen peptide derived from land animals, collagen peptide derived from these aquatic sources has unique molecular and biological properties for amino acid composition, antioxidant activity, neuroprotective activity and anti-skin aging activity, because of low temperature and/or high salt condition in the surrounding environment [36–39]. Furthermore, a previous study showed that collagen derived from sea- and freshwater-rainbow trout had quite similar amino acid composition and molecular weight properties [40]. Collagen derived from two marine demosponges, *Axinella cannabina* and *Suberites carnosus*, collected from the Aegean and the Ionian Seas, respectively, had low imino acid content, and showed lower or similar denaturation temperatures compared with collagen derived from other marine organisms such as tropical fish [41], suggesting the universal properties of collagen derived from aquatic sources. In addition to collagen peptide, ornithine is a non-essential, non-protein amino acid contained in various foods such as freshwater clams. Recent studies have highlighted ornithine as a functional food for improving sleep quality [42] and recovery from fatigue [43,44] in human.

The beneficial effects of collagen peptide for skin conditions have been analyzed by several groups both in rodents [39] and humans [27–29,45]. However, to our knowledge, the effects of collagen peptide, ornithine or the combined effects of collagen peptide and ornithine (CPO) on the increase of growth hormone and/or IGF-1 levels, and the subsequent improvements of skin conditions have not been investigated. A previous study showed the increase of plasma growth hormone levels after ingestion of ornithine [46], though its relationship to the improvements of skin conditions have not been investigated. In this study, we anticipated the combinational effects of fish-derived collagen peptide and ornithine on skin conditions, and plasma growth hormone and/or IGF-1 levels in healthy Japanese people. We hypothesized that orally administered CPO induced an increase of plasma growth hormone and/or IGF-1 levels, which exerted subsequent improvements of skin conditions including elasticity, moisture and transepidermal water loss (TEWL).

#### **2. Results**

#### *2.1. Participants*

Forty participants were recruited from the Osaka area, of whom 22 participants (aged from 31 to 48 years, 18 females and 4 males) exhibiting low skin moisture and elasticity were enrolled. Participants were recruited from September to October 2017. Included participants were assigned to either the CPO group (*n* = 11) or placebo group (*n* = 11). All participants completed the study. One participant in the CPO group was excluded from analysis because of aberrant blood ureic acid levels both before and after supplementation (Figure 1).

**Figure 1.** Flow diagram of participants.

Finally, 21 participants (aged from 31 to 48 years, 17 females and 4 males) were analyzed. Thus, per protocol set analysis was performed. Statistically significant differences between originally included participants and finally analyzed participants were not observed for baseline scores including age, skin elasticity, and moisture. This study consisted of an 8-week administration period from October to December 2017. CPO and placebo groups were matched according to age, gender, body mass index (BMI), skin elasticity, and moisture at baseline (Tables 1 and 2).


**Table 1.** Baseline characteristics of participants who completed 8-week test.

Average ingestion rate was 100 ± 0% and 99.8 ± 0.5% in CPO and placebo groups, respectively. All subjects had a more than 98% ingestion rate. No statistically significant differences were observed for ingestion rate between CPO group and placebo groups.
