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. 2022 Dec 12;17(12):e0277188. doi: 10.1371/journal.pone.0277188

The antiaging effects of a product containing collagen and ascorbic acid: In vitro, ex vivo, and pre-post intervention clinical trial

Tae Kyeong Ryu 1,2,3,#, Hanna Lee 1,2,3,#, Dong Keon Yon 4, Da Yeong Nam 1,2,3, Soo Yun Lee 1,2,3, Byung Ho Shin 1,2,3, Go Woon Choi 1,2,3, Da Som Jeon 1,2,3, Bo Bae Oh 1,2,3, Ji Hyun Kim 1,2,3, Young Yoon 1,2,3, Hyun Jeong Kim 1,2,3, Luc Duteil 5, Christelle Bruno-Bonnet 6, Chan Yeong Heo 1,2,3,*, So Min Kang 1,2,3,*
Editor: Parasuraman Padmanabhan7
PMCID: PMC9744321  PMID: 36508415

Abstract

Various substances, including collagen (Naticol®) and ascorbic acid, that inhibit and prevent skin aging have been studied. Collagen prevents skin aging, has anti-inflammatory effects, and assists in normal wound healing. Ascorbic acid is a representative antioxidant that plays a role in collagen synthesis. To achieve a synergistic effect of collagen and ascorbic acid on all skin types, we prepared a product named “TEENIALL.” In addition, we used a container to separate ascorbic acid and collagen to prevent the oxidation of ascorbic acid. To confirm the effects of TEENIALL, we first confirmed its penetrability in fibroblasts, keratinocytes, melanocyte, and human skin tissues. Thereafter, we confirmed the collagen synthesis ability in normal human fibroblasts. Based on the results of in vitro tests, we conducted a clinical trial (KCT0006916) on female volunteers, aged 40 to 59 years, with skin wrinkles and hyperpigmentation, to evaluate the effects of the product in improving skin wrinkles, skin lifting, and pigmentation areas before using the product, and after 2 and 4 weeks of using the product. The values of nine wrinkle parameters that were evaluated decreased and those for skin sagging, pigmentation, dermal density, and mechanical imprint (pressure) relief were improved. Skin wrinkle and pigmentation were evaluated to ensure that the improvement effect was maintained even after 1 week of discontinuing the product use. The evaluation confirmed that the effects were sustained compared to those after 4 weeks of using the product. Additionally, skin wrinkles, skin lifting, radiance, and moisture content in the skin improved immediately after using the product once. Based on the results of in vitro and ex vivo experiments and the clinical trial, we show that the product containing ascorbic acid and collagen was effective in alleviating skin aging.

Introduction

The skin undergoes a wide variety of physiological and pathological changes over time [1]. In view of the increasing interest in beauty attributes in recent times, besides issues of physiological and pathological changes in the skin, studies are being conducted to decipher the mechanisms underlying skin aging, and to find the associated biological changes, which might help in devising strategies for inhibition of the skin aging [2,3].

Skin aging can be largely categorized as intrinsic aging, which is inevitable over time, and extrinsic aging (photoaging), which is caused due to a variety of reasons, such as exposure to sunlight, stress, and smoking; the phenotypes of the two aging types differ [4]. Intrinsic aging is relatively mild, and is manifested as fine lines, dry skin, and decreased elasticity [5]. However, typical photoaging results in thick and deep wrinkles, pigmentation, very rough texture, and sagging [6]. With the accumulation of damage, there is an increase in the levels of reactive oxygen species (ROS) production [7]. Moreover, there are changes in the properties and amounts of matrix proteins, as the signaling system is regulated by changes in the levels of cytokines [8,9].

Wrinkles are distinct clinical features of aging that appear with the decrease in collagen and elasticity due to an increase in matrix metalloproteinases (MMPs), such as collagenase and elastase; histologically, water loss, epidermal contraction, and flattening are seen [10]. In the dermis, collagen accounts for more than 90%, and type I and type III collagen account for 85–90% and 10–15% of the total collagen present, respectively [11,12]. Another clinical feature—pigmentation—is caused due to the increase in melanin content through activation of melanocytes [13]. Various substances with potential to inhibit and prevent such skin aging are being studied. Among these are collagen and ascorbic acid. Ingestion or topical application of collagen prevents skin aging, has anti-inflammatory effects, and assists in normal wound healing [2,3]. Weishardt, a world-renowned collagen manufacturer, has a variety of fish collagen peptides, named Naticol®, with different molecular weights and formulations; these peptides are widely used in cosmetics, medicines, and food [14]. In particular, Naticol® BPMG is composed of collagen type 1 and type 3, which are the main components of the dermis. It is produced from high quality raw materials (fish skin) and through specific enzymatic hydrolysis [15].

Ascorbic acid is a representative water-soluble antioxidant. Two different mechanisms are described for the antioxidant properties of ascorbic acid [16]. First, it reacts with free radicals in aqueous components of the body, such as cytoplasm, plasma, and extracellular fluid, and inactivates them [17]. It lowers the activity of lipoxygenase, an enzyme that catalyzed the peroxidation of lipids. Second, it regenerates the oxidized form of vitamin E, which prevents lipid peroxidation very effectively [18]. It also plays a role in collagen synthesis [19]. For the synthesis of collagen, proline and lysine must be converted to hydroxyproline and hydroxylysine to enable stronger hydrogen bonding between collagen chains. Three enzymes (prolyl-3-hydroxylases, prolyl-4-hydroxylases, and lysyl hydroxylases) use ascorbic acid as a coenzyme [20]. The antiaging effects of ascorbic acid in clinical practice have been shown in many previous studies [21].

This study was conducted to evaluate the effects of applying TEENIALL (TEENIALL Double Concentrate Vitamin C Youth Ampoule, WELBORN, Korea) containing Naticol® (Weishardt, France) and ascorbic acid (Quali®-C, United Kingdom) together as an antiaging compound. First, experiments were conducted using cells and tissues to confirm the permeability and collagen synthesis ability of Naticol® and ascorbic acid, and thereafter, clinical studies were conducted on human subjects. There is a limit for consumption of ascorbic acid owing to the stability problem associated with its oxidation. To overcome this, an effort was made by using a special container. Before opening the product, ascorbic acid and collagen were separated, and the two substances could be mixed immediately before use. Moreover, the container was designed to block light. We evaluated the synergistic effect of Naticol® and ascorbic acid and the benefits of using the special container on the antiaging effect. We also assessed whether the effect was maintained even after the discontinuation of the product. The effect was verified through various clinical tests on 21 Korean female volunteers, aged 40–59 years, with skin folds (eye corners, forehead, and nasolabial folds) and pigmentation on the face (Figs 1 and 2A).

Fig 1. CONSORT 2010 flow diagram.

Fig 1

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Fig 2.

Fig 2

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(A) Collagen and ascorbic acid have beneficial effect on skin aging. (B) Fluorescence imaging of fibroblasts, keratinocytes, and melanocytes in the control and test groups at ×400 (Scale bar = 20 μm) and ×1000 (Scale bar = 5 μm) magnification. (C) fluorescence imaging of a cross-section of human skin tissue in the control and test groups (×400, Scale bar = 50 μm, n = 20). (D) Analysis of collagen synthesis depending on the concentration of the test product (n = 4, ***p < 0.001).

Materials and methods

Test product

TEENIALL (TEENIALL Double Concentrate Vitamin C Youth Ampoule, WELBORN, Korea) is composed of powder type ascorbic acid and liquid type collagen (Naticol BPMG). Ascorbic acid contained in this product is in the form of fine particles, with a diameter of 37 μm, and the molecular weight of collagen is 1.74 Mw/Mn. In clinical trial, this product was divided into powder and liquid components, so that both formulations could be mixed and applied immediately before use.

In vitro and ex vivo analyses

Cell penetration assay

The cell penetration assay was performed using human fibroblasts (Hs68 cell line), human keratinocytes (HaCaT cell line), and mouse melanocytes (B16F10 cell line). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco Invitrogen, Carlsbad, USA), containing the TEENIALL powder. Thereafter, the cells were stained with DAPI (VECTASHIELD® Antifade Mounting Medium with DAPI). Fluorescence of cells was measured using a fluorescence microscope (Zeiss Axio Observer 7, Germany).

Measurement of collagen synthesis

Human normal fibroblasts (neonatal; Gibco, C0045C) were treated with DMEM containing TEENIALL at a concentration of 0.01, 0.02, or 0.05%. Collagen synthesis was measured using an ELISA kit (Procollagen Type I C-peptide [PIP] EIA Kit, TaKaRa, Tokyo, Japan).

Penetration assay

The penetration assay was performed using Franz diffusion cells with a diffusion area of 3 cm2, and a 7 mL receptor chamber volume. The receptor medium contained saline solution. TEENIALL (0.4 mL), with labeled protein (Alexa Fluor™ 488, Invitrogen, USA) and control solution (PBS), was applied to the surface of the human skin tissue (Derma:Lab™, Korea). Thereafter, tissue slides were made and the sections were stained with DAPI. Fluorescence from skin sections was measured using a fluorescence microscope.

Clinical trial

Study procedure

Sample size calculation. Although there has been no study on the antiaging potential of TEENIALL (ascorbic acid and collagen), prior to conducting clinical research, we calculated the sample size based on a our in vivo study on the relationship between collagen synthesis and TEENIALL [22].

We originally calculated that for each group to have a 95% power and 0.05 alpha (sample size calculation) to report a 44.5%–83.1% (18.7% in certain experiments) improvement in collagen synthesis (229.1 [SD 31.5] ng/mL for control group versus 269 ng/mL in certain experiments for treatment group), at a 5% significance level, we would need to enroll 16 patients in each group. Considering the various exclusion conditions (30%), finally, we included 21 participants [23,24].

This study was conducted in accordance with Good Clinical Practice (GCP), Ministry of Food and Drug Safety (MFDS), and Standard Operating Procedures (SOP) of KSRC CO., LTD. The study was approved by the Institutional Review Board at H&BIO KSRC CO. (IRB: HBABN01-210315-HR-0205-01, CRIS number: KCT0006916). Over 20 subjects meeting the inclusion and exclusion criteria were enrolled. For 4 weeks, the product was applied to subjects on a daily basis around the whole face after washing the face twice a day. In this study, treatment was applied for 4 weeks; the study was conducted as a single test and the measurements were made immediately after the application of the product once, and 2 and 4 weeks later. The study investigator explained the purpose, procedures, and anticipated adverse reactions or side effects to the subjects. The investigator received an informed consent form (ICF) and case report form (CRF) from the subjects who agreed to participate in the study prior to it. The study was conducted from March to June 2021.

Over 20 subjects were recruited according to the MFDS guidelines. For 4 weeks, the product was applied to subjects on a daily basis over the entire face after washing the face twice a day. The application was discontinued for 1 week. The inclusion and exclusion criteria were set as follows:

Inclusion criteria

  • Korean female volunteers, aged 40–59 years, with skin folds (eye corners, forehead, and nasolabial folds) and pigmentation on the face.

  • Subjects who were not hypersensitive to vitamin C.

  • Healthy subjects, free from acute and chronic diseases, including skin conditions.

  • Subjects who had voluntarily signed the informed consent form after understanding the complete explanation of the purpose and protocol of this study.

  • Subjects who were available for follow up during the study period.

Exclusion criteria

  • Women who were pregnant, lactating, or planning to become pregnant within 6 months.

  • Subjects who had skin diseases, including active atopic dermatitis, psoriasis, eczema, and active seasonal allergies on the test region.

  • Subjects who had used antibacterial agents, immunosuppressants, or external skin preparations containing steroids and treatments for chronic skin conditions for more than 1 month to treat skin conditions on the test region.

  • Subjects who had not passed 1 month since participating in the same study.

  • Subjects who used the same or similar efficacy cosmetics and medicines on the test site within 3 months prior to the start of the study.

  • Subjects with chronic diseases (asthma, diabetes, hypertension, etc.).

  • Subjects on contraceptives, antihistamines, and anti-inflammatory drugs.

  • Subjects who were employed in this clinical research institute.

  • Subjects who were considered inappropriate according to the judgment of the investigator.

Twenty-one subjects (females; mean age, 50.95 ± 5.54 years; age, 40–59 years) participated in the 4-weeks treatment test (two of the twenty-three subjects dropped out [follow-up failure: #03 and #09]), and twenty subjects (mean age, 51.40 ± 5.28 years; age, 40–59 years) participated in the discontinuation 1 week test after 4-weeks treatment (one more subject dropped out [follow-up failure: #11]). Subjects who met the inclusion and exclusion criteria completed the study according to the protocol. The information and characteristics of the subjects were investigated using questionnaires.

Measurement of wrinkles

The Primos-CR (Canfield Scientific, Parsippany-Troy Hills, New Jersey) was used to evaluate the three-dimensional surface of the skin. Data files captured using the Primos-CR were analyzed using the Primos software.

Measurement of skin sagging

F-RAY (BEYOUNG, Korea) images of facial contour curves of the selected cheek area were taken before and immediately after the use of the product. The angle of the contour line was analyzed for the captured contour image using an analysis program, Image Pro® 10 (Media Cybernetics, USA).

Measurement of pigmentation

The skin color of the cheek of volunteers was measured using a CM-26dG spectrophotometer (Konica Minolta, INC., Osaka, Japan). The L* value expresses the relative brightness following the Commission Internationale de l’Eclairage (CIE) L*a*b* system.

Measurement of dermal density

The skin dermal density of the cheek of volunteers was measured using the Ultrasound Probe of DermaLab® Series SkinLab Combo (Cortex Technology, Denmark). Low density was displayed as a dark color and high density was displayed as a bright color based on the signal strength.

Measurement of mechanical imprint (pressure) relief

The skin pressure impression of the cheek of volunteers made by applying physical force to uneven cotton (8room, Korea). The Primos-CR was used to evaluate the surface of the skin. The image captured using the Primos-CR was analyzed using the Primos software.

Measurement of skin gloss (radiance)

Mark-Vu (Mark-Vu®; PSI PLUS Co., Ltd., Suwon, Korea), a skin diagnostic imaging system, was used to analyze the skin gloss. The analysis was performed in the designated area on the polarized image using the Image-Pro® 10 (Media Cybernetics, USA) program.

Measurement of hydration in the stratum corneum

Dermal hydration was measured dielectrically with an open-ended coaxial probe that was layered in structure (Moisture Meter-D; Delphin Technologies Ltd.).

Statistical analysis

Statistical analysis was performed using the SPSS® software program (IBM, USA). The normality was verified using the Shapiro–Wilk test and by examining the kurtosis and skewness. Statistical analysis of variables for parametric values was performed using the paired t-test and RM-ANOVA. Two-side p-values <0.05 were considered significant(p-values <0.05; * and p-values <0.001; ***).

Results

In vitro and ex vivo analyses

Cell penetration assay

Significant fluorescence from many small granules in the cytoplasm was observed in the test group of fibroblasts, keratinocytes, and melanocytes indicating the penetration of the product (TEENIALL) into these cells (Fig 2B, S1 Table).

Skin penetration assay

We conducted a skin stratum corneum permeability test to confirm the effectiveness of collagen and ascorbic acid permeation in the stratum corneum (n = 20). Skin layer fluorescence was observed in 50% of the randomly photographed areas in the experimental group unlike in the control group. Fluorescence of the test substance was observed in the stratum corneum and human skin tissue, which indicates that the test substance penetrated the stratum corneum (Fig 2C).

Measurement of collagen synthesis

We investigated whether collagen and ascorbic acid affect the biosynthesis of collagen. As evident from the results of the collagen production test in normal human fibroblasts, the expression of collagen decreased by about 54.2% upon UV irradiation. In addition, comparing the change in the amount of collagen in the case of test product and that in the group without UV irradiation (BLANK; No UV irradiation and treatment), the loss in collagen expression due to UV radiation was completely recovered to 144.3% for 0.01% treatment, 171.1% for 0.02% treatment, and 183.1% for 0.05% treatment (Fig 2D, S1 Table).

Analyses after treatment for 4 weeks

The results of the clinical trial showed changes in wrinkles around the eyes, nasolabial folds, and forehead; skin lift; reduction in skin pigmentation, dermal density, and mechanical imprint (pressure) relief after 4 weeks of using the product.

Analysis of wrinkles (Crow’s feet, nasolabial fold, and forehead)

We conducted a test on 21 subjects to confirm the antiwrinkle effect of the product. When compared with the baseline, all parameters of skin wrinkles (Average depth of wrinkles, Mean depth biggest wrinkle, Max. depth biggest wrinkle, Total wrinkle count, Total wrinkle volume, Total wrinkle area, Total length of wrinkles, Ra [Arithmetic average], and Rz [Average maximum height of the profile]) improved significantly at 2 and 4 weeks after treatment (p < 0.05); for Crow’s feet, the rate of change in the abovementioned nine all parameters was 8.90%, 9.36%, 11.01%, 10.43%, 11.62%, 4.39%, 4.24%, 8.76%, and 7.34%, respectively, after 4 weeks compared with the baseline; for nasolabial fold, the rate of change in the parameters was 10.06%, 10.25%, 8.26%, 16.63%, 10.70%, 1.85%, 5.34%, 9.26%, and 7.30%, respectively, after 4 weeks compared with the baseline; for forehead, the rate of change in the parameters was 10.56%, 10.22%, 11.74, 13.72%, 10.36%, 4.32%, 4.95%, 10.25, and 8.69%, respectively, after 4 weeks compared with the baseline (Fig 3A and 3B, S1 Table).

Fig 3.

Fig 3

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(A) Measurement of Average depth of wrinkles, Mean depth biggest wrinkles, Max. depth biggest wrinkles, Total wrinkle count, Total wrinkle volume, Total wrinkle area, Total length of wrinkles, Ra, and Rz parameters. (B) Polarized image and 3D image of Crow’s feet, nasolabial folds, forehead wrinkles. (C) F-RAY, the facial contour line image, and graph of the left cheek area. (D) Measurement of skin pigmentation. Cross-polarized image of the left cheek area. (E) Measurement of dermal density and ultrasonographic image of dermal density. (F) Effect for mechanical imprint (pressure) to skin and 3D image mechanical imprint (pressure) relief of the right cheek area.

Analysis of skin sagging

We conducted a test on 21 subjects to confirm the skin-lifting effect of the product. When compared with the baseline, skin sagging was significantly improved (p < 0.05) at 4 weeks after the treatment, with a 3.21% change (Fig 3C, S1 Table).

Analysis of skin pigmentation

We investigated the effect of the product in improving skin pigmentation in 21 subjects. When compared with the baseline, skin pigmentation was significantly improved (p < 0.05) at 4 weeks after the treatment, with a 0.55% change (Fig 3D, S1 Table).

Analysis of dermal density

We conducted a study on 21 subjects to evaluate the effect of the product in improving dermal density. When compared with the baseline, dermal density was significantly improved (p < 0.05) at 4 weeks after the treatment, with a 17.60% change (Fig 3E, S1 Table).

Analysis of mechanical imprint (pressure) relief

We conducted a test on 21 subjects to evaluate the effect of the product on the mechanical imprint (pressure) relief. When compared with the baseline, the value of the parameter before the treatment was 51.7% and that after 4 weeks treatment was 29.54%. Thus, after 4-weeks treatment the parameter improved by 22.16% compared with that before the treatment (Fig 3F, S1 Table).

Analysis after 1 week of discontinuation of the product

After observing the improvement effects after 4 weeks of the product use, we further checked the sustenance of the effects after 1 week of discontinuation of the product use.

Analysis of Crow’s feet

We conducted a study on 20 subjects to evaluate the improvement in Crow’s feet and found it to be significantly improved (p < 0.05) at 4 weeks after using the product compared with that before using the product (p < 0.05). Significant improvement continued even after 1 week of discontinuation of the product (p < 0.05). There was no significant difference in Crow’s feet after 4 weeks of product use and 1 week after discontinuation of the product (Fig 4A and 4B, S1 Table).

Fig 4.

Fig 4

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(A) Measurement of Ra, Rmax, Rz, Rp, and Rv parameters. (B) Polarized and 3D images of Crow’s feet after 4 weeks of product use and 1 week after discontinuation. (C) Effect on skin color improvement after 4 weeks of product use and 1 week after discontinuation and cross-polarized image of skin color in hyperpigmented areas.

Analysis of skin pigmentation

We conducted a study on 20 subjects to evaluate the improvement in pigmentation upon using the product. The skin color (L* value) of the pigmented area was significantly improved (p < 0.05) at 4 weeks after using the product compared with that before using the product (p < 0.05). Significant improvement continued even after 1 week of discontinuation of the product (p < 0.05). There was no significant difference in the pigmentation after 4 weeks of product use and 1 week after discontinuation of the product use (Fig 4C, S1 Table).

Analysis of the effects after single application of the product

The results of the clinical trial indicated the changes in fine lines in the eye area, skin sagging, gloss, and hydration immediately after the application of the product once.

Analysis of fine lines in the eye area

We confirmed the effect of the product use in improving the fine wrinkles in 20 subjects. When compared with the baseline, all the parameters of fine wrinkles (Ra [Arithmetic average], Rmax [Maximum peak to valley roughness height], Rz [Average maximum height of the profile], Rp [Largest positive deviation], and Rv [Largest negative deviation]) improved significantly immediately after the test product was applied, with a decrease of 8.70%, 7.72%, 7.94%, 8.48%, and 7.25%, respectively (Fig 5A and 5B, S1 Table).

Fig 5.

Fig 5

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(A) Measurement of Ra, Rmax, Rz, Rp, and Rv parameters for fine wrinkles around the eyes. (B) Polarized and 3D images of wrinkles around the eyes after one use of the product. (C) F-RAY the facial contour line image of the right cheek area after using the product once. (D) Effect of a single use of the product in improving the facial gloss and polarized image of skin gloss after a single use of the product. (E) Facial moisturizing effect after using the product once.

Analysis of skin sagging

We checked the facial lifting effects of the product in 20 subjects. When compared with the baseline, skin sagging was significantly improved (p < 0.05) immediately after the test product was applied, with a 0.88% change (Fig 5C, S1 Table).

Analysis of skin gloss

We confirmed the skin radiance improvement effect of the product in 20 subjects. When compared with the baseline, the skin gloss improved significantly after the application of the product (p < 0.05), with an increase of 2.28% (Fig 5D, S1 Table).

Analysis of skin surface to a depth of 2.5 mm hydration

We evaluated the effect of the product in improving the skin surface to a depth of 2.5 mm hydration in 20 subjects. When compared with the baseline, the skin surface to a depth of 2.5 mm hydration was significantly improved (p < 0.05) at immediately after the application of the test product, with a 7.11% change (Fig 5E, S1 Table).

Safety evaluation

None of the subjects showed any adverse skin reaction during the study period. We performed a 24-hours occlusive patch test. After removing the patch, each test region was observed after 30 minutes and 24 hours, according to the PCPC guidelines. With regard to the primary irritation potential for human skin, the test product was found to be in the “none to slight” range (range of response = 0.00, S1 Text, S2 Table).

Discussion

The purpose of this study was to evaluate the antiaging potential of a mixture of collagen and ascorbic acid. We investigated whether the synergistic effect of these two substances could be achieved while maintaining the absorption of collagen (Naticol®) and the stability of ascorbic acid. We found that the product was effective in alleviating skin aging through ex vivo and clinical trials.

First, in the evaluation of the permeability of the product across skin stratum corneum in ex vivo human skin tissue, fluorescence was evident as a result of penetration, confirming the penetrability of the product down to the stratum corneum of the skin.

In the clinical trial, values for all the parameters for the eye area, nasolabial folds, and forehead wrinkles were significantly improved after 2 and 4 weeks of using the product, and facial contours and skin color were also significantly improved. Moreover, there was a significant improvement in the skin density and mechanical imprint relief 4 weeks after using the product. Mechanical imprint relief is a new clinical method devised on its own. With the decrease in skin elasticity, marks left on the skin do not disappear easily. It was designed mainly from the traces of the duvet or pillow that occur while sleeping. The fact that the marks left upon waking up disappear in a short time can be interpreted as an improvement in the elasticity of the skin. We evaluated the degree of improvement in skin elasticity by creating marks with the application of constant pressure for a certain period of time.

Skin wrinkles and skin color were measured again after discontinuation of the product use following 1 week of its application. Based on the results, it was confirmed that the effect of the product was sustained even after discontinuation. Moreover, it was confirmed that the skin wrinkles, skin lifting, radiance and moisture content in the skin were significantly improved after a single use of the product.

Collagen, which occupies most of the dermal extracellular matrix and plays an important role in maintaining the strength and viscoelasticity of the skin, is a major structural protein accounting for 75% of the dry weight of the skin and maintains its elasticity [25]. Therefore, when collagen is insufficient, the skin loses elasticity and moisture and wrinkles appear [26]. The synthesis of collagen types I and III is affected by ascorbic acid, iron, and silicon, and proline and lysine, the major constituent amino acids of collagen, are through to be involved in the process [20]. In cosmetics, delivery of collagen to the skin is an important challenge. Considering this issue, we selected Weishardt’s fish collagen. This collagen, which has low molecular weight (Mean Mw: 2kDa) and high dispersability in a microgranulated form, is a component of TEENIALL [2].

Ascorbic acid, also called vitamin C, is the most powerful and broad-spectrum antioxidant among the antioxidant vitamins present in cells [27,28]. It may not be efficiently delivered to the skin due to its unstable state in cosmetics. In particular, natural ascorbic acid is easily decomposed by oxygen and light [29]. To overcome these shortcomings, we used containers that allowed physical separation of ascorbic acid and collagen. The two substances were mixed immediately before using the product.

Conclusions

The combined use of ascorbic acid and collagen resulted in skin whitening and wrinkle improvement, as well as in the improvement of skin elasticity, radiance, and moisturizing effects. In addition, the effects of ascorbic acid and collagen were synergistic and were maintained even after discontinuation of the product. Although the effects of the combination of collagen and ascorbic acid were clinically confirmed, the molecular mechanisms underlying the effects of each were not confirmed. Therefore, it would be good to check the effects of each component separately to elucidate the exact mechanism. Moreover, it may be necessary to conduct a study comparing the data for each. We believe that there will be better results if the improvement effect is measured when collagen and ascorbic acid are used over a long time.

Supporting information

S1 Checklist. TREND statement checklist.

(PDF)

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S1 Table. P-Values for the graphs included in the figures.

(DOCX)

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S2 Table. Formula for safety evaluation.

(DOCX)

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S1 Text. Skin primary irritation procedure.

(DOCX)

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S1 File

(DOCX)

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S2 File

(DOCX)

Click here for additional data file. (1.6MB, docx)

Data Availability

All relevant data are within the article and its Supporting information files.

Funding Statement

The author(s) received no specific funding for this work.

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Decision Letter 0

Parasuraman Padmanabhan

10 May 2022

PONE-D-22-07546The anti-aging effects of the product including both collagen and ascorbic acid: in vitro, ex vivo and pre-post intervention clinical trialsPLOS ONE

Dear Dr. Kang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Kind regards,

Parasuraman Padmanabhan, Ph.D

Academic Editor

PLOS ONE

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Upon resubmission, please provide the following:

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4. Please amend the manuscript submission data (via Edit Submission) to include authors Tae Kyeong Ryu, Han Na Lee, Da Yeong Nam, Soo Yun Lee, Byung Ho Shin, Go Woon Choi, Da Som Jeon, Bo Bae Oh, Ji Hyun Kim, Young Yoon, Hyun Jeong Kim, Luc Duteil, Christelle Bruno-Bonnet and Chan Yeong Heo.

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Additional Editor Comments:

Reproducibility of In vitro & Ex vivo experiments will be repeated and new results may be added.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Partly

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: A clinical trial was conducted which aimed to study the effect of TEENIALL on improving skin wrinkles, skin lifting, and pigmentation areas after 2 and 4 weeks of using the product. Skin wrinkling, sagging, and pigmentation decreased after TEENIALL usage.

Major revisions:

Provide summary tables of results. Include precise p-values instead of “p<0.05” or “p< 0.001.”

Minor revisions:

1- The standard statistical term for average is mean.

2- Study procedure: State precisely the total number of subjects enrolled and analyzed instead of vaguely indicating “over 20 subjects.”

3- Safety Evaluation: Provide an interpretation for “Range of response = 0.00.”

4- State and justify the study’s target sample size with a pre-study statistical power calculation. The power calculation should include: sample size, alpha level (indicating one or two-sided), minimal detectable difference and statistical testing method.

5- To assist in the review process, add line numbering to the document.

Reviewer #2: Reproducibility of In vitro & Ex vivo is not easy as the given details of the methods used/adopted is not sufficient for a scientific journal and its readers.

The source of the cell lines are not given.

Use of generic names for the evaluated products/ details of components could make it of better use for the scientific community to advance science.

Reviewer #3: The manuscript “The anti-aging effects of the product including both collagen and ascorbic acid: in vitro, ex vivo and pre-post intervention clinical trials” by Ryu et.al. elaborates the use of TEENIALL to enhance the synergetic effect between collagen and ascorbic acid, tested to decrease skin wrinkles and hyperpigmentation in humans within 40 to 59 years. The research had focused on use of TEENIALL in reducing aging effects, with visible results after the first use. These findings should thus motivate further research and would deserve publication. However, I do not think that this paper is publishable in its present form and that at least one round of minor revision is needed.

In the introduction, page 2, the sentence starting with “Ascorbic acid is a representative….” can be given as a separate paragraph.

The products Naticol ® and Quali®-C, are mentioned as “raw materials” in most of the places. Please change that to “products” or any other word of similar meaning.

In the study procedure the sentences” In this study, treatment was applied once, 2 and 4 weeks, the study was conducted as a single test. Over 20 subjects meeting the inclusion and exclusion criteria were enrolled” and “For 4 weeks, the product was applied to subjects on a daily basis around the whole face after washing the face twice a day” are unclear and misleading.

Please correct the grammatical errors in the manuscript. There are several grammatical errors found.

The English language is generally unsatisfactory.

I suggest rewriting the conclusion. The line starting with “In summary….can be corrected”

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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Attachment

Submitted filename: PONE-D-22-07546.docx

Click here for additional data file. (12.5KB, docx)
PLoS One. 2022 Dec 12;17(12):e0277188. doi: 10.1371/journal.pone.0277188.r002

Author response to Decision Letter 0

30 Jun 2022

June 13, 2022

Dr. Parasuraman Padmanabhan

Academic Editor

PLOS ONE

Dear Dr. Padmanabhan:

It is with great pleasure that we resubmit the revised version of our manuscript, presently titled “The antiaging effects of a product containing collagen and ascorbic acid: In vitro, ex vivo, and pre-post intervention clinical trial,” by Tae Kyeong Ryu et al. to PLOS ONE. The manuscript ID is PONE-D-22-07546R1.

We appreciate the decision of the Editors to reconsider our manuscript and are grateful to the reviewers for their meticulous comments. We have sincerely endeavored to address each comment and have made appropriate modifications to our manuscript. We acknowledge that our manuscript has benefited from the insightful suggestions. We hope that the revised manuscript addresses all the concerns and would be considered suitable for publication in PLOS ONE. If you have any further queries, please do not hesitate to contact me.

Thank you for your kind consideration of our manuscript. We look forward to a favorable decision.

Sincerely yours,

So Min Kang, Ph.D.

Major changes and additions in the revised manuscript

We have revised the manuscript and included a number of additional values in supplemental tables.

We marked the edited part of the manuscript with yellow highlight.

<Reviewer #1>

[1] Revision (Major)

Provide summary tables of results. Include precise p-values instead of “p<0.05” or “p<0.001.”

Response: We would like to thank the reviewer for this comment. As suggested, we have provided the exact p-values for the data presented in the graphs as supporting information (S1 Table).

[2] Revision (Minor)

The standard statistical term for average is mean.

Response: We agree with the reviewer’s contention that “mean” rather than “average” is the standard statistical term. Therefore, as suggested, we have replaced “average” with “mean” throughout the revised manuscript.

[3] Revision (Minor)

Study procedure: State precisely the total number of subjects enrolled and analyzed instead of vaguely indicating “over 20 subjects.”

Response: Thank you for your comment. We would like to submit that we used “over 20 subjects” in accordance with the cosmetic clinical trial method guidelines followed in the Republic of Korea (Ministry of Food and Drug Safety MFDS, Guidelines for Cosmetics Human Application Test and Efficacy Test).

The number of people who participated in the actual test has been mentioned for the each evaluation item in the Results section. (The number of subjects participating in the trial is underlined.) The number of participating subjects for all evaluations was 21. However, in the evaluation of “after 1 week of discontinuation of product use,” one subject dropped out due to withdrawal of consent, and finally, 20 participants participated. We performed analysis on those 20 subjects.

[4] Revision (Minor)

Safety Evaluation: Provide an interpretation for “Range of response = 0.00.”

Response: Safety evaluation aimed at evaluating the primary skin irritation potential of the test materials using a primary patch test for the human skin. We performed a 24-hours occlusive patch test. After removing the patch, each test region was observed after 30 minutes and 24 hours, according to the PCPC guidelines. Over 30 subjects were selected according to the recommendations in the MFDS guidelines. We derived the results with reference to Tables 1 and 2 below. Therefore, using this test procedure for the primary irritation potential for human skin, the test product was found to be in the “none to slight” range (range of response = 0.00). We have added this information the Results section (Line 353 on page 15 of the manuscript) and attached Supporting Text file.

[5] Revision (Minor)

State and justify the study’s target sample size with a pre-study statistical power calculation. The power calculation should include: sample size, alpha level (indicating one or two-sided), minimal detectable difference and statistical testing method.

Response: We apologize for insufficient application of statistical techniques for justifying the target sample size and for not introducing our study methodology in detail. We have added information to reinforce our main results (Line 142 on page 6 of the manuscript). We hope that the changes would address the reviewer’s concern.

[6] Revision (Minor)

To assist in the review process, add line numbering to the document.

Response: We would like to thank the reviewer for the comments. We have added line numbers to the Word document.

<Reviewer #2>

[1] Revision

The source of the cell lines are not given.

Response: We would like to thank the reviewer for the comments. We have added the source of the cell lines in the revised manuscript.

[2] Revision

Use of generic names for the evaluated products/ details of components could make it of better use for the scientific community to advance science.

Response: We would like to thank the reviewer for the pertinent suggestion. We have used generic names instead of “TEENIALL” and “Naticol®.” Moreover, we have added a description about the ascorbic acid and collagen mixed component, which we refer to as TEENIALL, in the revised manuscript.

<Reviewer #3>

[1] Revision

In the introduction, page 2, the sentence starting with “Ascorbic acid is a representative….” can be given as a separate paragraph.

Response: We appreciate this suggestion. We have presented the text mentioned by the reviewer in a separate paragraph the Introduction section (Line 80 on page 4 of the manuscript).

[2] Revision

The products Naticol ® and Quali®-C, are mentioned as “raw materials” in most of the places. Please change that to “products” or any other word of similar meaning.

Response: We would like to thank the reviewer for the comment. We have changed “raw materials” to specific terms (“Naticol® and ascorbic acid”).

[3] Revision

In the study procedure the sentences” In this study, treatment was applied once, 2 and 4 weeks, the study was conducted as a single test. Over 20 subjects meeting the inclusion and exclusion criteria were enrolled” and “For 4 weeks, the product was applied to subjects on a daily basis around the whole face after washing the face twice a day” are unclear and misleading.

Response: We apologize for the lack to clarity. We have revised the description and hope that it conveys the intended meaning.

[4] Revision

Please correct the grammatical errors in the manuscript. There are several grammatical errors found. The English language is generally unsatisfactory. I suggest rewriting the conclusion. The line starting with “In summary….can be corrected”

Response: We acknowledge that there were language issues in the manuscript. In accordance with the reviewer’s comment, we have employed a professional scientific editing service to ensure that the language of the revised manuscript meets the publishable standards.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (77.3KB, docx)

Decision Letter 1

Parasuraman Padmanabhan

24 Oct 2022

The antiaging effects of a product containing collagen and ascorbic acid: In vitro, ex vivo, and pre-post intervention clinical trial

PONE-D-22-07546R1

Dear Dr. Kang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Parasuraman Padmanabhan, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Please addressed by the reviewer raised comments. Follow the journal guidelines.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Minor revisions: (Page numbers refer to those in the tracked changes version of revision 1.)

1- Line 165: Indicate the statistical testing method which attains 95% power.

2- In line 176, "product" is misspelled.

3- Line 187: Consider replacing "around" with "over."

4- Line 254: Drop the word "The" from the beginning of the sentence. Replace "using kurtosis and skewness" with "by examining the kurtoisis and skewness."

5- Line 256: Distinguish between the results in which p<0.05 and p<0.001 were considered statistically significant.

6. Within the text, replace "p<0.05” or “p<0.001” with more precise p-values.

Reviewer #2: Line 65-66 - rephrase to avoid redundant use of word `increase'.

Line 156 - `Product' spelling incorrect

Line 250-251 - Verify if the group without UV irradiation is the control instead of BLANK

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

Acceptance letter

Parasuraman Padmanabhan

1 Dec 2022

PONE-D-22-07546R1

The antiaging effects of a product containing collagen and ascorbic acid: In vitro, ex vivo, and pre-post intervention clinical trial

Dear Dr. Kang:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Parasuraman Padmanabhan

Academic Editor

PLOS ONE

Associated Data

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    Supplementary Materials

    S1 Checklist. TREND statement checklist.

    (PDF)

    Click here for additional data file. (572.1KB, pdf)
    S1 Table. P-Values for the graphs included in the figures.

    (DOCX)

    Click here for additional data file. (23.2KB, docx)
    S2 Table. Formula for safety evaluation.

    (DOCX)

    Click here for additional data file. (16.6KB, docx)
    S1 Text. Skin primary irritation procedure.

    (DOCX)

    Click here for additional data file. (16.9KB, docx)
    S1 File

    (DOCX)

    Click here for additional data file. (1.7MB, docx)
    S2 File

    (DOCX)

    Click here for additional data file. (1.6MB, docx)
    Attachment

    Submitted filename: PONE-D-22-07546.docx

    Click here for additional data file. (12.5KB, docx)
    Attachment

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    Click here for additional data file. (77.3KB, docx)

    Data Availability Statement

    All relevant data are within the article and its Supporting information files.

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