Tolerability and efficacy assessment of an oral collagen supplement for the improvement of biophysical and ultrasonographic parameters of skin in middle eastern consumers

Topical skin care products often do not reach the deeper layers of the skin, and oral hydrolyzed collagen is one of the newest and most popular systemic supplementations for skin rejuvenation. However, there are limited information in case of Middle Eastern consumers


| INTRODUC TI ON
The human skin aging phenomenon is a complex and multifactorial process that leads to many functional and aesthetic changes. 1 Internal factors like planned cell destruction, hormonal changes, as well as some diseases such as scleroderma and chronic inflammations affect all organs including the skin. External factors such as exposure to ultraviolet rays or cigarette smoke are other factors which affect skin aging. 2 These factors cause thinning of the epidermis, a decrease in elasticity and an increase in skin lines.
Visible clinical manifestations in the skin aging process include volume reduction, decreased skin elasticity, increased wrinkles, decreased epidermal thickness, irregularity of skin tissue, sagging, and dry skin. 3 Collagen is the most abundant protein in the body and the main component of connective tissue, which makes up about 25-35% of the total protein in the body. Collagen is synthesized in the body, by a combination of several different amino acids, along with the vitamin C as a co factor. Glycine, proline, and hydroxyproline makeup 57% of the total amino acids of collagen. 4 During aging, the disintegration of the skin collagen network to shorter less organized fibers increases; therefore dermis thickness and collagen density decreases. Parallel to this degradation, the synthesis of extracellular matrix components by fibroblasts is also slowed down. 5 Topical skin care products such as creams, lotions, and serums often do not reach the deeper layers of the skin. 6 By the way, injectable collagen can have an effect on skin elasticity not only on healthy skin but also in disorders such as lichen sclerosus. 7,8 Different dietary supplements and systemic antioxidants, such as some marine protein-based macromolecules have been used for skin rejuvenation. However, hydrolyzed collagen is one of the newest and most popular systemic supplementations. 9,10 Different studies have shown that oral hydrolyzed collagen can pose a positive effect on various skin conditions, such as skin moisture content and aging. 11 Bolke et al. in a randomized controlled study showed that the daily consumption of oral supplements containing 2.5 g of hydrolyzed collagen for 12 weeks can significantly improve skin elasticity indices compared to the placebo group. 12 Another study by Asserin et al. in 2015 also reported the efficacy of oral supplementation with collagen peptides to improve hallmarks of skin aging. 13 However, there is limited information on the case of Middle Eastern consumers.
The present study was performed to evaluate the tolerability and effectiveness of an oral supplement sachet containing 5 g of hydrolyzed collagen in Middle Eastern consumers.

| Test product
Collagen Gold supplement sachet (Adrian Salamat Co, Tehran, Iran) containing: • 5000 mg of hydrolyzed collagen types 1 and 3 with a marine source.
• 80 mg of vitamin C.

| Study design & ethical approval
It was a 12-week, single-group, before and after clinical study conducted January to July 2022 in accordance with Declaration of Helsinki. Written informed consent was obtained from all the participants. The patients' data were kept confidential and all measurements were non-invasive and done free of charge.

| Participants
Healthy men and women aged 40-57, with skin type III-IV were recruited for the study. The exclusion criteria were as follows: History of botulinum toxin injection, laser therapy or intense pulsed light therapy during the previous 6 months, cosmetic surgery of the face during the previous 3 years, radiofrequency therapy during the previous year, collagen vascular disease, diabetes mellitus, hypothyroidism, liver or kidney failure, using systemic glucocorticoids, immunomodulators, isotretinoin or systemic hormones during the previous 3 months, using oral supplements containing hyaluronic acid, collagen, and vitamin C during the previous 3 months, using topical corticosteroids and retinoid during the previous 4 weeks, using topical anti-aging products during the previous 4 weeks, being an active smoker (previous 2 years), pregnancy/ breastfeeding, documented allergy to the test product, and a major change in lifestyle (diet, skin care habits, physical activity).

| Objective assessments
Before the first intake of the study product, after 6 and 12 weeks of intake, as well as 4 weeks after stopping the treatment (week 16) objective assessments were performed as below: anti aging, oral collagen, skin elasticity, ultrasound • Skin biophysical parameters including skin hydration, friction (roughness), and elasticity parameters including firmness (R0), gross elasticity (R2), net elasticity (R5), and immediate recovery (R7) on the right cheek by the Corneometer, Frictometer, and Cutometer of the MPA 580 device (Multi Probe Adapter sys-tem® manufactured by Courage + Khazaka electronic GmbH), respectively. Before each measurement, the participants rested for 30 min in a room with climate control, having temperature of 22 ± 2°C and relative humidity of 30-40%.
• Ultrasonography assessment on the right nasolabial fold for measurement of the dermis thickness and density.

| Satisfaction
Other assessments included the participants' overall satisfaction with the treatment, using the Visual Analogues Scale (VAS) from 0 to 10 in such a way that zero was a sign of absolute dissatisfaction and 10 was a sign of maximum satisfaction. Participants' satisfaction also was recorded by answering "yes" or "no" to the standard questionnaire (Table 1).

| Safety and tolerability
The safety and tolerability of the test product were assessed by monitoring adverse effects during the study through information collected in interviews and questionnaires. In case of occurrence of any side effects including a feeling of heaviness, pain, and bloating in the stomach, changes in bowel movements, as well as skin irritation reactions, the severity, and duration were also recorded.

| Statistical analysis
Descriptive and analytical statistics were used to analyze the data.
Quantitative variables were described in terms of mean and standard deviation. To compare the average quantitative results between before, 6 and 12 weeks after the intervention and also 4 weeks after stopping consumption, Paired Sample t Test or its non-parametric equivalent Wilcoxon was used. Data analysis was done using SPSS version 22 software, and p-value <.05 was considered statistically significant.

| RE SULTS
Twenty participants (18 women and 2 men) with an average age of 44.15 years and a standard deviation of 5.36 years (range of 40-57 years) participated in this study. Five participants had skin type III, and 15 of them had type IV.

| Skin elasticity
The primary outcome of this study was skin gross and net elasticity parameters (R2, R5) after 12 weeks of use. A significant improvement was detected in R2 and R5 at week 12 (p-values 0.041 and 0.012, respectively). In case of R5, the improvement was also reported as significant at week six (p-value <0.01). R7 and R0 did not show any significant improvement during the supplement intake.
Four weeks after stopping the use of the supplement, R2 and R5 remained at an increased level and did not show a significant change compared to the end of use (week 12). R7 increased significantly in the week 16 compared to the 12th week ( Figure 1A).

| Ultrasound assessment
A relative, but non-significant increase in dermis density was observed after 12 weeks of treatment. This increasing trend continued until week 16; the increase in dermis density in week 16 was significant compared to the initial values (p-value = 0.03) ( Figure 1B).

| Skin hydration & skin friction
A significant improvement in skin friction was observed after six and 12 weeks of use in the right cheek area (p-value<0.01). Also, no significant difference was observed between the skin friction values at the time of stopping the use and 4 weeks later, which indicates the persistence of the results for up to 4 weeks after stopping the use of the supplement ( Figure 1C). The stratum corneum hydration did not show a significant change in any of the measurement periods.

| Participants' satisfaction
The mean value for overall participants' satisfaction with the treatment at weeks 6, 12, and 16 was 6.9 ± 1.86, 6.78 ± 2.17, and 6.56 ± 1.75 out of 10, respectively, according to the questionnaire, as shown in Figure 2. TA B L E 1 Standard questionnaire for the assessment of participants' satisfaction after using Collagen Gold supplement sachet.

| Tolerability
Adverse effects included a feeling of heaviness, pain, and stomach bloating, which were mild in three female participants and severe in one 57-year-old woman, and in the severe case, they led to withdrawal from the study.

| DISCUSS ION
Hydrolyzed collagen refers to natural bioactive substances used in many oral supplements which provide skin health and beauty. 14 A recent systematic review and meta-analysis, including 19 studies with a total of 1125 participants aged between 20 and 70 years (95% women), showed promising results for 90 days application of hydrolyzed collagen supplementation compared with placebo in terms of skin hydration, elasticity, and wrinkles. 15 However, there is limited information on the case of darker skin prototypes and Middle Eastern consumers.
The purpose of this study was to evaluate the tolerability and effectiveness of an oral supplement sachet containing 5 g of hydrolyzed collagen, vitamin C, and hyaluronic acid in over 40 years old Middle Eastern volunteers with skin types III and IV.

F I G U R E 1 Skin elasticity (A), density (B)
, and friction (C) at baseline and after 6 and 12 weeks of intake of study products, as well as after follow-up (week 16). *singnificant compared to baseline. #significant compared to week 12.

F I G U R E 2
Satisfaction of the participants after 6 and 12-weeks intake of study product, as well as after followup (week 16).
The significant improvement in the main elasticity parameters (R2 and R5) and the maintenance of this improvement for 4 weeks after stopping the intake, indicated the appropriate effectiveness of the tested supplement in improving skin elasticity. R2 measures the total skin stretch including viscous deformation, which represents the perceived stretchiness of hydrated skin. R5 is independent of viscous deformation and is used to characterize the mechanism of skin movement, testing specifically the tensioned nature of the skin. 16,17 Lombardi el al. reported that a type I collagen could induce the collagen synthesis as well as the differentiation in a fibroblast cell line. 18 So it could facilitate the rearrangement and improvement of existing collagen and elastin fibers, accompanying with hyaluronic acid, and lead to advanced elasticity properties (R2 and R5).
In case of skin firmness (R0), no significant improvement was observed during the supplement intake. Kawałkiewicz et al. which determined the firmness and elasticity of facial skin in different age groups, also described that the skin elasticity, and an ability of skin to return to the original shape (R2) is greater in younger patients, while skin firmness is constant in the different age groups and R0 is probably independent of age. 19 A relative increase in dermis density was also observed after Evidence from animal models shows that oral administration of collagen reduces epidermal hyperplasia caused by the sun's ultraviolet rays and increases fibroblasts and extracellular matrix proteins such as hyaluronic acid and hydroxyproline. 21,22 Fibroblasts are the main cells of the dermis that produce collagen and extracellular matrix and play an important role in maintaining and improving the skin's elastic properties. 23 Moreover, oral collagen peptides can reduce the production of metalloproteinase 3 (MMP-3) and IL-1α, and accordingly slow down the process of collagen destruction in the dermis. 24,25 Similar results were reported in clinical trials that administered 2.5 and 5 g collagen peptides, demonstrating that supplementation of 2.5 g collagen peptides for 90 days would be sufficient to obtain beneficial effects. 15 In contrary to the study conducted by Bolke et al, 12 in the current study, no increase was observed in the moisture content of the stratum corneum after 12 weeks of taking the hydrolyzed collagen supplement. This could be due to the different location of the skin moisture measurement in our study. In most of the similar studies, skin hydration was measured in the normally protected areas like the forearm and arm, 15 while in the current study, measurements were performed in the face area (cheek), which is exposed to environmental factors such as sunlight. Therefore, this result would be similar to the real weather condition of middle east with sun exposure, high temperature and low humidity.
It could be also the reason for the delayed improvement in dermis density in the current study, since the skin aging process is probably more accelerated in the exposed area, compared to the protected ones. 12 A significant improvement in skin friction after six and 12 weeks of use and the persistence of these results for up to 4 weeks after stopping use were observed in the present study. This indicates the beneficial effect of the intervention for improving skin roughness in early stages of intake, which is probably due to the improvement of the skin's elasticity parameters. 26 This delayed effectiveness could be due to the delayed effect of In most of the previous studies, the use of liquid and solid forms of oral collagen supplements (capsules, sachets, and syrups) showed good patient acceptability and no adverse effects. 15 Tak et al. assessed the tolerability of oral collagen compared to a placebo in middle-aged women. According to the results, application of oral collagen was well-tolerated, and no notable adverse effect was reported in any of the active and placebo groups. They concluded that oral ingestion of collagen is well-tolerated in middle-aged women. 28 We faced a few gastrointestinal (GI) complications related to the study intervention, which was more severe in one of the elder women with history of mild GI discomfort.
It could be suggested that individuals with a history of GI com-

| CON CLUS ION
According to the results, objective dermatological measurements demonstrated that oral collagen peptides in combination with other dermo-nutrients like vitamin C and hyaluronic acid improved skin elasticity, roughness, and dermis echo density significantly, which could be stable after stopping the treatment for up to 4 weeks. The collagen supplement also proved to be safe and well-tolerated during the entire period of use except for rare gastrointestinal complication.

AUTH O R CO NTR I B UTI O N S
A.S. and A.F. performed the research. A.S. and A.F. and S.AN. designed the research study. M.M. and T.Y. and F.K contributed essential reagents or tools. A.S. analyzed the data. M.M and A.S. and A.F wrote the paper.

ACK N OWLED G M ENTS
This study was funded by Adrian Salamat (Tehran, Iran). This funding was used for lab equipment, personal costs, travel, and transportation and author stipends.

CO N FLI C T O F I NTER E S T S TATEM ENT
One of the authors Faegheh Kazemi is the Regulatory Affairs of sponsor company (Adrian Salamat). Other authors confirmed to have no conflict of interest in this article content.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

E TH I C S S TATEM ENT
Authors declare that the ethics committee, registered in IRCT clinical trial registry and all participants provided written informed consent.