Open AccessArticle

Enhancement of Skin Anti-Wrinkling Effects of Arthrospira maxima Phycocynobilin by Combining with Wheat Bran Extract

Eun-Jeong Koh

Taeho Kim

¹,

Yong-Kyun Ryu

Won-Kyu Lee

In-Yung Sunwoo

Hyang Seon Ro

³,

Gibeom Jeon

⁴,

Gyu Rae Kim

⁴,

Hyeon Yong Lee

^4,5,*

and

Woon-Yong Choi

^1,2,*

Jeju Bio Research Center, Korea Institute of Ocean Science and Technology (KIOST), Jeju 63349, Republic of Korea

Department of Marine Technology & Convergence Engineering (Marine Biotechnology), University of Science and Technology, Daejeon 34113, Republic of Korea

R&D Center, NOWCOS, Ltd., A-1004 BYC Highcity, Gasandigital 1, Seoul 08506, Republic of Korea

⁴

R&DB Center, Beautyence Corp., 220 Gasangil, Sejong 30003, Republic of Korea

⁵

Department of Medical Biomaterials Engineering, College of Biomedical Science, Kangwon National University, Kangwon University Road 1, Chuncheon 24341, Republic of Korea

Authors to whom correspondence should be addressed.

Appl. Sci. 2024, 14(22), 10216; https://doi.org/10.3390/app142210216

Submission received: 11 October 2024 / Revised: 5 November 2024 / Accepted: 6 November 2024 / Published: 7 November 2024

(This article belongs to the Section Applied Biosciences and Bioengineering)

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Figure 1
PCB structure and comparison of HPLC chromatogram of standard PCB and SP. (a) PCB structure (b) HPLC chromatogram of standard PCB and SP. Blue line: PCB standard; Black line: SP, the PCB extract from Arthrospira maxima, prepared by water extraction using ultrasonication at room temperature followed by ethanolic extraction at 70 °C. "> Figure 2
Cytotoxicity of the extracts against human skin fibroblasts. PCB, PCB standard; SP, the PCB extract from Arthrospira maxima, prepared by water extraction using ultrasonication at room temperature followed by ethanolic extraction at 70 °C; WB, the wheat bran extract prepared by water extraction at 4 °C for eight hours; SPWB, a mixture of SP and WB containing the same amounts of PCB and SP. Cytotoxicity of PCB standard (a) SP (b), WB (c), and SPWB (d). The mean ± SD values for triplicate experiments are shown; error bars represent the SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the non-treated group. "> Figure 3
DPPH free radical-scavenging activity of the extracts. Human fibroblasts were subjected to different treatments, as indicated. PCB, PCB standard; SP, the PCB extract from Arthrospira maxima, prepared by water extraction using ultrasonication at room temperature followed by ethanolic extraction at 70 °C; WB, the wheat bran extract prepared by water extraction at 4 °C for eight hours; SPWB, a mixture of SP and WB containing the same amounts of PCB and SP. Trolox (50 µg/mL) was used as a positive control. The mean ± SD values for triplicate experiments are shown; error bars represent the SD. *** p < 0.001 compared with the Trolox group. "> Figure 4
Comparison of intracellular ROS production in UVB-irradiated human skin fibroblasts treated with the different extracts. PCB, PCB standard; SP, the PCB extract from Arthrospira maxima, prepared by water extraction using ultrasonication at room temperature followed by ethanolic extraction at 70 °C; WB, the wheat bran extract prepared by water extraction at 4 °C for eight hours; SPWB, a mixture of SP and WB containing the same amounts of PCB and SP. The mean ± SD values for triplicate experiments are shown; error bars represent the SD. * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with the control (UV+) group. "> Figure 5
Collagen levels in UVB-irradiated human skin fibroblasts subjected to different treatments. PCB, PCB standard; SP, the PCB extract from Arthrospira maxima, prepared by water extraction using ultrasonication at room temperature followed by ethanolic extraction at 70 °C; WB, the wheat bran extract prepared by water extraction at 4 °C for eight hours; SPWB, a mixture of SP and WB containing the same amounts of PCB and SP. TGF-β1 (0.1 ng/mL) was used as a positive control. The mean ± SD values for triplicate experiments are shown; error bars represent the SD. *** p < 0.001 compared with the control (UV+) group. "> Figure 6
Inhibition of MMP-1 expression in UVB-irradiated human skin fibroblasts subjected to different treatments. PCB, PCB standard; SP, the PCB extract from Arthrospira maxima, prepared by water extraction using ultrasonication at room temperature followed by ethanolic extraction at 70 °C; WB, the wheat bran extract prepared by water extraction at 4 °C for eight hours; SPWB, a mixture of SP and WB containing the same amounts of PCB and SP. Retinol (0.1% w/v) was used as a positive control. The mean ± SD values for separate triplicate experiments are shown; error bars represent the SD. *** p < 0.001 compared with the control (UV+) group. "> Figure 7
Up- and downregulation of mRNA levels of collagen (Col1A1) and MMP-1, respectively (a) in UVB-irradiated human skin fibroblasts, CCD-986sk. The relative densitometric intensities (b,c) of the bands, normalized against the mRNA levels of the housekeeping gene, GAPDH, in the different treatments, are shown. PCB, PCB standard; SP: the PCB extract from Arthrospira maxima, prepared by water extraction using ultrasonication at room temperature followed by ethanolic extraction at 70 °C; WB: the wheat bran extract prepared by water extraction at 4 °C for eight hours; SPWB: a mixture of SP and WB containing the same amounts of PCB and SP. TGF-β1 (0.1 ng/mL) was used as a positive control. The mean ± SD values for triplicate experiments are shown; error bars indicate the SD. ** p < 0.01, *** p < 0.001 compared with the control (UV+) group. ">

Versions Notes

Abstract

Despite the many beneficial effects of phycocyanobilin (PCB) on human skin, its cosmetic applications have not been extensively investigated owing to its light and temperature sensitivity. This is the first report of PCB extract (SP) derived from marine Arthrospira maxima having skin anti-wrinkling effects associated with antioxidant efficacy and reduction of intracellular reactive oxygen species (ROS) production. We obtained 46.63 ± 1.72 mg PCB/g dry weight of A. maxima in SP through an ethanol extraction process. PCB extracts showed strong effects in increasing collagen synthesis and decreasing matrix metalloproteinases (MMP-1) production. Interestingly, skin anti-wrinkling effects of the PCB extracts were significantly increased by the addition of wheat bran extracts (WB), up to 20–30% of the effects of PCB at all concentrations, possibly due to the synergistic effects of soluble globulins and other active substances in WB. Moreover, the mixture of SP and WB (SPWB) greatly reduced cell cytotoxicity to approximately 15% of that of PCB. SPWB upregulated and downregulated the expression of collagen type I α1 (Col1A1) and MMP-1, respectively, although the downregulation of MMP-1 was higher than that of Col1A1. The optimal SPWB concentration for maintaining the highest skin anti-wrinkling effects was 0.5 mg/mL. We show that SPWB holds promise as a vegan cosmaceutical.

Keywords:

Arthrospira maxima; wheat bran extracts (WB); phycocyanobilin (PCB); Matrix metalloproteinases (MMP-1); collagen type I α1 (Col1A1); skin anti-wrinkling

1. Introduction

Skin aging is a gradual process influenced by prolonged exposure to sunlight, pollution, smoking, stress, and other environmental factors, all of which accelerate the aging process [1]. As skin ages, it generally becomes less elastic and more fragile and experiences persistent barrier dysfunction, resulting in wrinkles, fine lines, and hyperpigmentation or age spots [2]. Wrinkle formation is closely linked to increased production of matrix metalloproteinases (MMPs), particularly MMP-1, which play a role in breaking down the extracellular matrix (ECM) through collagen degradation [3]. This breakdown of collagen leads to a loss of skin elasticity and firmness, contributing to the development of fine lines and wrinkles [4,5]. Environmental factors, especially UV light, further upregulate MMP-1 expression, promoting processes like photoaging [6]. Additionally, oxidative stress from sources such as UV exposure, pollution, and inflammation increases free radical production, which can degrade ECM components like collagen and elastin and stimulate MMP-1 expression, accelerating the skin aging process [6].

The most common approach to counteracting skin aging is to simultaneously inhibit MMP-1 expression and increase collagen production in skin fibroblasts by reducing oxidative stress using antioxidants. Many natural resources with high antioxidant efficacy have been considered for this purpose because their use in cosmetics is considered safe and more eco-friendly compared with that of synthetic chemicals [7].

Spirulina (Arthrospira species) is a prime candidate for use in antiaging approaches, considering its long history of use in treating various diseases and its reputation as a “superfood,” suitable even for consumption in outer space [8]. It is rich in proteins and contains many biologically active compounds, such as c-phycocyanin (cPC), beta-carotene, and chlorophylls [9].

cPC, a two-subunit complex comprising a blue chromophore and apoprotein, is the most abundant bioactive component in spirulina with versatile cosmeceutical activities, such as reducing oxidative stress, photoprotection, anti-skin wrinkling, anti-inflammation, and skin hydration effects [10,11]. However, the potential skin health benefits of cPC have not been realized because of its light and temperature sensitivity [12]. Owing to this critical limitation, cPC is only used in value-added products distributed under low-temperature conditions, such as ice cream and chocolate colorants, and it is rarely used in cosmetics as an active ingredient [12]. To overcome this limitation, cPC can be further broken down into its constituent chromophore and protein subunits [12,13]. This chromophore is known as phycocyanobilin (PCB), which is a royal blue tetrapyrrole pigment mainly responsible for the health benefits of cPC, such as effective reduction of oxidative stress, anti-inflammatory, immunomodulatory, anticancer, and anti-diabetic nephropathy effects, and protection against neural tissue degeneration [14,15,16]. PCB is more stable than cPC as it can simultaneously attach to any protein (for example, bovine and human serum albumin) with free SH groups, such as those provided by cysteine and ethanethiol, without loss of its biological activities [16,17]. Furthermore, whey proteins and polysaccharides such as carrageenan, with high antioxidant effects, have been used to improve the stability of PCB from spirulina [18,19]. Although PCB, derived from spirulina, shows excellent blue color and exerts many beneficial effects on human skin, its cosmetic applications have not been extensively investigated [20,21].

In recent times, trends in cosmeceutical development have been shifting toward vegan cosmetics, driven by the increasing number of consumers seeking plant-based ingredients. For promoting the use of PCB in vegan cosmetics, the development of plant-derived resources that stabilize this pigment is essential. In this study, we investigated the potential cosmetic applications of PCB derived from spirulina in combination with wheat bran extract, focusing on enhancing its anti-wrinkle effects.

2. Materials and Methods

2.1. Materials

In this study, all experiments were conducted with reference to previous research [22]. The PCB standard was purchased from Santa Cruz Biotechnology (sc-396921B, Santa Cruz, CA, USA). Also, Wheat bran powder was acquired by Red Mill Natural Foods (Red Mill Natural Foods, Milwaukie, OR, USA). Dulbecco Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were obtained from GIBCO (GIBCO, Bend, OR, USA). Other materials include 1% gentamycin sulfate, HEPES buffer, the 3-(4,5-dimethythiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was acquired by Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Unless noted, all chemicals were purchased from Sigma-Aldrich, St. Louis, MO, USA.

2.2. Preparation of Samples

PCB was extracted from the marine cyanobacterium Arthrospira maxima (formerly Spirulina maxima) [23], grown and provided by the Jeju Bio Research Center, Korea Institute of Ocean Science and Technology (Jeju, Republic of Korea). First, dried A. maxima powder was suspended in 10 mL of 0.1 M sodium phosphate buffer (pH 7.0) at a 1:100 w/v ratio. The suspension was homogenized for 15 min in a sonicator and subjected to three freeze-thaw cycles. The crude extract was collected by centrifugation at 2322× g for 20 min. The phycocyanins were purified according to the method described by Kumar et al. [24]. The crude extract was subjected to single-step precipitation with 65% (NH₄)₂SO₄ and kept overnight at 4 °C. The pellet was recovered by centrifugation of the extract at 4000× g and 4 °C for 20 min and dissolved in 10 mL of the same extraction buffer. Next, 10 mL of the extract was dialyzed twice against 1000 mL of the extraction buffer in the dark at 4 °C for 24 h. The resulting extract was recovered from the dialysis membrane and filtered through a 0.45 µm syringe filter. To extract PCBs from phycocyanins, 30 g of phycocyanin extract powder was suspended in 1.5 L of ethanol at a ratio of 1:50 (w/v). The extraction was performed at 70 °C for 15 h in the dark. The suspension was centrifuged at 2322× g to separate the crude extract. The supernatant was then filtered through a 0.45 μm filter. After filtration, the solvent was evaporated, and the extract was concentrated to obtain the final PCB powder (SP). SP was stored at −20 °C in a dark place before use. For preparing wheat bran water extract (WB), 20 g of wheat bran powder (Red Mill Natural Foods, Milwaukie, OR, USA) was extracted in 200 mL distilled water at 4 °C by shaking at 50 rpm for 8 h. Then, the extract was centrifuged at 2322× g for 20 min at 4 °C. The supernatant was dialyzed using a 100 kD MW cut-off membrane for 24 h at 4 °C, concentrated using a rotary vacuum evaporator, and finally lyophilized into a powder. For preparing a mixture of SP and WB (SPWB), SP was dissolved in an aqueous solution of WB to the same concentration for further experiments.

2.3. Measurement of PCB Concentration in the Extract

The PCB concentration in the extract was determined using high-performance liquid chromatography (HPLC) on a Waters New Alliance E2695 system (Waters, Co., Milford, MA, USA) equipped with a Waters 2998 PDA detector and COSMOSIL C18 column (5 μm, 150 × 4.6 mm). The mobile phase consisted of two solvents: A (deionized H₂O containing 0.1% trifluoroacetic acid) and B (acetonitrile (ACN) containing 0.1% trifluoroacetic acid). A linear gradient of 40–55% (v/v) aqueous ACN was applied for 15 min at a flow rate of 0.8 mL/min. The injection volume was 10 μL, and the detection wavelength was 666 nm.

2.4. Evaluation of the Cytotoxicity of Extracts

The cytotoxicity of the PCB extract, WB, SPWB, and PCB standard (purity 95%) was evaluated using the 3-(4,5-dimethythiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Briefly, human skin fibroblasts (CCD-986sk, ATCC, Manassas, VA, USA) were seeded in 96 well plates at a density of 1.0 × 10⁴ cells/well and cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% (v/v) fetal bovine serum (FBS), 1% gentamycin sulfate, and HEPES in a 5% CO₂ incubator at 37 °C for 24 h. Then, 200 μL of different concentrations of the extracts or pure PCB were added to the wells, and the incubation was continued for 24 h. Thereafter, 5 μg/mL of MTT solution was added to each well, and the supernatant was removed after 4 h. Finally, 10 µL of acid-isopropanol (0.04 N HCl in isopropanol) was added to each well, and the absorbance at 565 nm was measured using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

2.5. Measurement of the Antioxidant Activities of the Extracts

To analyze the antioxidant activities of SP, WB, and SPWB, 2,2 diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging activity and production of reactive oxygen species (ROS) in skin fibroblasts were evaluated. For measuring the DPPH free radical-scavenging activity, 80 μL of different concentrations of the extracts, pure PCB, and 50 µg/mL Trolox (Sigma-Aldrich), used as a positive control, were added to 200 μL of 0.1 mM DPPH solution in a 96-well plate and further incubated at 25 °C for 30 min in the dark. The absorbance was measured at 525 nm using a microplate reader. The DPPH free radical-scavenging activity (%) of the extracts was estimated using Equation (1).

DPPH (%) = ((Control O.D. − Sample O.D.)/(Control O.D.)) × 100

(1)

Intracellular ROS production in UVB-irradiated human skin fibroblasts was estimated using 2′,7′-dichlorofluorescein diacetate (H2-DCF-DA). CCD986sk cells (4.0 × 10⁴ cells/well) were seeded in a 48 well-plate and cultured in DMEM, supplemented with 10% FBS at 37 °C for 24 h. The culture medium was replaced with phosphate-buffered saline (PBS) (pH 7.4) (Sigma-Aldrich), and the cells were exposed to UVB at a wavelength of 312 nm and intensity of 60 mJ/cm² using an ultraviolet lamp (model VL-6 M, Vilber Lourmat, Marine, France) for 150 s at room temperature [UV(+)] or unexposed [UV(−)]. Thereafter, 100 μL of different concentrations of the extracts and pure PCB were added to each well, and the plates were further incubated for 24 h. The cells were washed with PBS and treated with 10 μM DCF-DA in Hank’s balanced salt solution in the dark for 40 min. The cells were then washed twice with PBS, suspended in 1% Triton X-100, and the fluorescence of the cells suspension was measured using a fluorescence spectrophotometer (UTX-20M, Biostep Co., Burkhardsdorf, Germany) at excitation and emission wavelengths of 490 and 525 nm, respectively.

2.6. Evaluation of Collagen Synthesis in Skin Fibroblasts Treated with the Extracts

Collagen production in UVB-irradiated skin fibroblasts was determined using the following method with a Procollagen Type 95 I C-peptide (PIP) EIA Kit (Takara, Otsu, Japan). First, 100 μL of the antibody-POD conjugate solution was added to a 96-well plate, and 20 μL of different concentrations of SP, WB, and SPWB and pure PCB was added to the plate in which 1.0 × 10⁴ CCD986sk cells/well were cultured with DMEM, supplemented with 10% FBS for 24 h. Thereafter, 0.1 ng/mL of transforming growth factor-β1 (TGF-β1, Sigma, St. Louis, MO, USA) was added as a positive control. The cells were further cultured for 3 h at 37 °C, and then the medium was discarded and washed three times with washing buffer. Thereafter, substrate solution was added to each well, and the plate was incubated for 15 min at room temperature. The reaction was stopped by adding 100 μL of 1 N H₂SO₄ to the wells. After shaking the plate for one minute, the optical density of the solution in each well was measured at 450 nm using a microplate reader, and the concentration of collagen produced by the cells was estimated using the standard curve provided with the kit.

2.7. Evaluation of the Inhibition of MMP-1 Expression by the Extracts

MMP-1 levels in UVB-irradiated human skin fibroblasts were measured using an ELISA Kit. CCD-986sk cells (1.0 × 10⁴ cells/well) were cultured with DMEM, supplemented with 10% FBS at 37 °C for 24 h. Thereafter, different concentrations of SP, WB, SPWB, pure PCB, and 0.1% (w/v) retinol (trans-retinol, Sigma-Aldrich) as a positive control were added to each well, and the plate was further incubated for 48 h. Then, 100 μL of the medium was transferred to the plate provided with the human MMP-1 ELISA Kit (RayBiotech, Norcross, GA, USA) and reacted for two hours at room temperature. The medium was removed from the wells, which were washed three times with PBS buffer, and 100 μL of MMP-1 detection antibody was added to each well. After one hour of incubation at room temperature, the medium was removed, and the cells were washed with PBS. A streptavidin solution (10 μL) was added to each well, and the plates were shaken for 45 min at room temperature. After removing the solution, the cells were washed twice with PBS. Thereafter, 100 μL of substrate solution was added to each well, the plate was further incubated for 30 min in the dark, and then 50 μL of stop solution was added to stop the reaction. The levels of MMP-1 produced in the cells were estimated by comparing the absorbance of the samples in each well at 450 nm with a linear regression curve for the standard provided with the kit.

2.8. Reverse Transcription Polymerase Chain Reaction (RT-PCR)

The expression levels of collagen and MMP-1 in UVB-irradiated human skin fibroblasts were measured using RT-PCR. First, total RNA was extracted from 5.0 × 10⁴ CCD986 cells/well treated with SPWB, and 0.1 ng/mL TGF-β1 or 0.1% (w/v) retinol using the TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). cDNA was prepared from 1 mg of RNA using the reverse transcriptase provided with the cDNA kit (First Strand cDNA Synthesis Kit, Fermentas, Hanover, MD, USA) at 37 °C for one hour. Then, 1–2 mL of cDNA template and a ReddyMixTM PCR master mix (Abgene, Surrey, UK) were mixed, and PCR was performed on a thermocycler (XP Thermal Cycler, TC-XP, BIOER Tech. Co., Hangzhou, China). The optimal conditions for obtaining PCR products of collagen and MMP-1 genes were 298 and 35 amplification cycles of 95 °C for one minute, 60 °C for one minute, and 73 °C for one minute. The sequences of the oligonucleotide primers for collagen type I α1 (Col1A1), MMP-1, and GAPDH (a housekeeping gene) were as follows (Biogenia, Seoul, Republic of Korea): Col1A1 (forward: primer 5′-CTC GAG GTG GAC ACC ACC CT-3′; reverse: 5′-CAG CTG GAT GGC CAC ATC GG-3′), MMP-1 (forward: 5′-TGG TCT GAA AGC AGT TTG AA-3′; reverse: 5′-CCA CTC GTC GAC TGC TGA GT-3′), and GAPDH (forward, 5′-ATT GTT GCC ATC AAT GAC CC-3′; reverse, 5′-AGT AGA GGC AGG GAT GAT GT-3′). PCR products were electrophoresed on 2% agarose gels and stained with ethidium bromide. The intensity of the bands was quantified using a densitometry program (Bio ID; Vilber Lourmat, Torcy Z.I., France).

2.9. Statistical Analysis

All statistical analyses were performed using the Statistical Package for GraphPad Software (version 5.0; GraphPad Software, Inc., San Diego, CA, USA). One-way analysis of variance (ANOVA) was used to compare the groups. Significant differences between the mean values were assessed using Tukey’s test. The p-value in the multiple comparison results (*, **, and ***) indicates significant differences between the groups (p < 0.05, p < 0.01, and p < 0.001, respectively).

3. Results and Discussion

3.1. Concentration of PCB in SP and Cytotoxicity of the Extracts

First, the amount of PCB present in SP was determined by comparing the HPLC chromatograms with those of the standard PCB (Figure 1). The concentration of PCB in SP was estimated to be 46.63 ± 1.72 mg PCB/g dry weight of A. maxima.

As shown in Figure 2a, the viability of cells treated with pure PCB was lower than that of cells treated with the extracts, with a significantly reduced cell viability of 83.37 ± 3.39% at concentrations of pure PCB greater than 0.5 mg/mL compared with that in the non-treated group (p < 0.01). Notably, at 0.5 mg/mL, SP, which contained approximately 23.3 µg/mL of pure PCB, exhibited 86.19 ± 2.98% cell viability. A similar pattern was observed for all other concentrations. This indicated that other components of SP may have played a protective role against the cytotoxicity of human skin fibroblasts, whereas this was not the case for fibroblasts treated with pure PCB. At the highest concentration (1 mg/mL), SP showed 78.03 ± 6.58% cell viability, whereas no cytotoxicity was observed at 0.1 mg/mL. In contrast, WB was nontoxic across all treatment concentrations, with 103.25 ± 7.16% cell viability at the highest concentration (1 mg/mL). At the same concentration, SPWB showed 82.47 ± 1.84% cell viability, whereas SP (78.03 ± 6.58%, p < 0.01) and PCB (70.80 ± 2.43%, p < 0.001) demonstrated significant toxicity compared with the non-treated group. Therefore, we used concentrations up to 0.1 mg/mL for PCB and SP, 1 mg/mL for WB, and 0.5 mg/mL for SPWB in this study. These results indicate that the addition of WB reduced the cytotoxicity of PCB.

3.2. Antioxidant Effects of PCB, SP, WB, and SPWB

We evaluated the antioxidant effects of SP, WB, SPWB, and PCB by measuring the DPPH free-radical scavenging activities and compared them to the activity of 50 µg/mL Trolox, which was used as a positive control (Figure 3). Treatment with 23.3 µg/mL PCB, which corresponded to the concentration present in 0.5 mg/mL of SPWB, resulted in 68.46 ± 2.02% of free radical-scavenging activity, and a similar effect of 59.02 ± 1.59% antioxidant activity was also observed for 0.1 mg/mL SP.

Both WB and SPWB exhibited much higher effects than the same concentration of SP, which implied that WB had a synergistic effect on the antioxidant activity of SP. Indeed, the antioxidant effects of wheat bran and its extracts have been demonstrated previously [25]. Therefore, these findings indicate that WB may increase the antioxidant effects of SP, which was expected because the addition of WB also lowered cytotoxicity (Figure 2). These promising results could be due to the higher amounts of phenolic substances in WB and, more probably, because of several types of proteins, such as soluble globulins, which are composed of multiple polypeptide chains and have molecular weights in the 10–100 kDa range. Wheat albumin in the WB could stabilize PCB by preventing or decreasing the breakage of its bonds with soluble albumin in WB [26,27]. This hypothesis is supported by other studies showing that various proteins, such as bovine serum albumin, beta-lactoglobulin, and whey proteins, prevent the digestion of PCB by protecting the cysteine bonding between them, and bilirubin, which has a very similar structure to PCB, is known to be stabilized by the complex of human serum albumin in human blood [18,19,28]. Therefore, the addition of WB enhanced the antioxidant effects of SP and protected against the death of human skin fibroblasts, possibly due to the synergistic effects of various phenolic compounds and binding with soluble globulin-like wheat albumins present in WB.

Various factors, such as visible light, environmental pollution, and solar UV light, increase ROS generation, leading to skin aging. Continuous accumulation of intracellular ROS in skin cells leads to oxidative stress, which ultimately causes cell death and induces cellular senescence [29]. To evaluate the inhibitory effects of the extracts on intracellular ROS production, we measured ROS levels using DCF-DA staining of UVB-irradiated human skin fibroblast cells treated with the extracts (Figure 4).

As expected, SPWB substantially inhibited ROS production. Considering the ROS levels in the UV(−) group to be 100%, the levels were increased to 187.23 ± 9.71% in the UV(+) group but only to 107.46 ± 5.06%, 149.61 ± 5.27%, and 121.58 ± 7.25% in the SPWB, 0.1 mg/mL SP, and 1 mg/mL WB treatments, respectively. Notably, the reduction in ROS production by WB and SPWB was concentration-dependent, except for a similar decrease at the highest concentrations of 1 and 0.5 mg/mL, respectively. These results indicated that the optimal concentration of SPWB was approximately 0.5 mg/mL for high antioxidant activities and inhibition of ROS generation. Moreover, SPWB showed synergistic ROS reduction activities of SP and WB, which corroborated with the DPPH-scavenging effects of SPWB and supported the hypothesis that WB could enhance the activity of SP. These findings suggest that the strong antioxidant effects of SPWB could be due to the protection against a decrease in the amounts of PCB in SP and increased antioxidant activities of WB.

3.3. Skin Anti-Wrinkling Effects of SP

UV-induced ROS production is associated with skin wrinkling and hyperpigmentation, leading to skin aging. Reduction in collagen levels and increased secretion of matrix-degrading enzymes such as MMPs in old skin compared with that in young skin are considered the major causes of human skin wrinkling [30]. Collagen comprises approximately 25% of all proteins in the human body and provides a foundation for connective tissues, including the skin. Many modern aesthetic methods aim to improve or stimulate the synthesis of collagen fibers in the skin [31]. UV irradiation is the main external factor that damages collagen fibers. It penetrates deep into the skin, affecting cells, collagen, elastic fibers, and other extracellular proteins. UV rays are absorbed more easily by hydrophobic amino acids, such as tryptophan, tyrosine, and phenylalanine, which are exposed during protein denaturation caused by sunburn, leading to collagen damage. In addition, UV rays impair the chaperone proteins responsible for proper collagen assembly, resulting in abnormal collagen formation [29].

In view of the results obtained for free radical scavenging and ROS production (Figure 3 and Figure 4), we assessed the improvement in photoaging by evaluating the collagen levels in cells exposed to UV irradiation and treated with the extracts.

As shown in Figure 5, 0.1 mg/mL SP (115.46 ± 3.39, p < 0.001), 1 mg/mL WB (109.17 ± 1.86, p < 0.001), and SPWB (138.52 ± 1.77, p < 0.001) increased the collagen levels in human skin fibroblasts compared with those in the UV(+) group (44.92 ± 6.26). We considered the effects of the extracts on collagen levels in the UV-irradiated cells because of their high antioxidant activities associated with the inhibition of ROS production. In particular, the results of collagen production were similar to those of the antioxidant activities of SP and SPWB, as shown in Figure 3, and the enhanced effects of SPWB could be attributed to the synergism of components in SP and the protective effects of WB. Therefore, these findings suggest that PCB and SP, containing the same amount of PCB in the extract, could increase collagen synthesis in human skin fibroblasts and that the efficacy of SP was mostly due to PCB.

UV irradiation increases the synthesis and expression of MMP-1 in dermal fibroblasts, a process driven by the excessive generation of ROS, which plays a critical role in photoaging [30]. The levels of MMP-1 in UVB-irradiated human skin fibroblast cells were determined (Figure 6) to confirm the skin anti-wrinkling effects of the PCB extracts from A. maxima. The MMP-1 levels were significantly decreased upon treatment with the extracts (PCB, 99.70 ± 6.85; SP, 92.60% ± 0.95; WB, 109.35 ± 0.66) compared with that in the UV(+) group (148.94 ± 8.68, p < 0.001). Notably, SPWB suppressed the MMP-1 levels to a greater extent (77.36% ± 0.68) than the other samples.

The composition of collagen fibers differs across organs based on their functions. In the skin, which endures mechanical stress, fibrillar collagens predominate, with higher amounts of type I collagen and smaller amounts of types III and V. Type I collagen is found in various tissues, including the skin, bone, cornea, sclera, and blood vessel walls. In addition to its mechanical role, it has a signaling function and contributes to the organization of the ECM, influencing the structure of the epidermis and dermis. COL1A1 synthesizes the alpha-1 chain of collagen type I [31,32]. The reduction in dermal collagen levels in UVB-irradiated skin is a key factor in photoaging, as UVB exposure decreases COL1A1 expression and suppresses collagen synthesis [32].

To determine the effects of the SPWB on the expression levels of collagen type I α1 (Col1A1), we analyzed its mRNA levels in UVB-irradiated cells. As shown in Figure 7, the mRNA levels of Col1A1 decreased following UVB irradiation, whereas they increased in the treatment with the positive control, TGFβ1. However, SPWB treatment upregulated the expression of Col1A1 compared with that in the UV(+) group (p < 0.001). PCB increased collagen expression to a level similar to that of the SPWB treatment (p < 0.001). UVB-induced generation of ROS leads to the collapse of dermal fibroblasts via collagen fragmentation. The resulting fibroblast collapse produces more ROS and activates the c-Jun/AP-1 complex, which subsequently upregulates MMPs and downregulates TGF-β signaling. Additionally, MMP-1 suppresses collagen synthesis by degrading type I collagen and negatively affecting COL1A1 expression [28,31]. Thus, we analyzed the MMP-1 mRNA levels in UVB-irradiated skin fibroblasts treated with the extracts. SPWB and PCB decreased the mRNA levels of MMP-1 compared with those in the UV(+) group (p < 0.01). The MMP-1 mRNA levels in fibroblasts treated with 0.1 g/mL SPWB were lower than in those treated with 0.1 mg/mL PCB. We believe that the effect of PCB may be enhanced upon WB addition because of the stabilization of PCB and the synergistic effects of various substances, such as soluble globulin and polyphenols, present in WB. Our findings suggest that various types of albumin and other substances in WB help stabilize PCB in the extracts and attenuate its cytotoxicity. Additionally, SPWB improved skin wrinkling and exhibited synergistic effects of PCB and SP. However, this hypothesis was not fully explored in the present study. Future research should elucidate the interaction between PCB in SP and WB and investigate the involvement of the c-Jun/AP-1 and TGF-β signaling pathways in mediating the observed effects.

4. Conclusions

In this study, PCB, SP, and SPWB effectively suppressed ROS generation in UVB-irradiated CCD986SK cells. Additionally, these samples promoted collagen synthesis by reducing MMP-1 and stimulating COL1A1. These findings suggest that the observed anti-wrinkle effects may be attributed to the antioxidant properties of the samples, as indicated by DPPH analysis, which attenuate UV-induced oxidative stress and regulate collagen synthesis-related factors. Notably, the combination of WB and SP (SPWB) improved skin wrinkling more effectively than either SP or WB alone, likely due to the stabilization of PCB and the synergistic effects of active compounds in WB, such as soluble globulin and polyphenols. Further research is needed to clarify the mechanisms underlying these anti-wrinkle effects. These findings highlight the potential for developing high-value vegan cosmeceuticals using plant-derived byproducts.

Author Contributions

Conceptualization and writing—original draft, E.-J.K., W.-Y.C., and H.Y.L.; methodology, T.K.; software and visualization, W.-K.L. and Y.-K.R.; methodology and validation, H.S.R. and I.-Y.S.; data curation, G.R.K. and G.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Korea Institute of Marine Science & Technology Promotion (KIMST), funded by the Ministry of Oceans and Fisheries, Korea (RS-2023-00241852). This research was supported by the Korea Institute of Marine Science & Technology Promotion (KIMST), funded by the Ministry of Oceans and Fisheries (20220380).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors.

Conflicts of Interest

Author Hyang Seon Ro was employed by the company R&D Center, NOWCOS, Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. PCB structure and comparison of HPLC chromatogram of standard PCB and SP. (a) PCB structure (b) HPLC chromatogram of standard PCB and SP. Blue line: PCB standard; Black line: SP, the PCB extract from Arthrospira maxima, prepared by water extraction using ultrasonication at room temperature followed by ethanolic extraction at 70 °C.

Figure 2. Cytotoxicity of the extracts against human skin fibroblasts. PCB, PCB standard; SP, the PCB extract from Arthrospira maxima, prepared by water extraction using ultrasonication at room temperature followed by ethanolic extraction at 70 °C; WB, the wheat bran extract prepared by water extraction at 4 °C for eight hours; SPWB, a mixture of SP and WB containing the same amounts of PCB and SP. Cytotoxicity of PCB standard (a) SP (b), WB (c), and SPWB (d). The mean ± SD values for triplicate experiments are shown; error bars represent the SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the non-treated group.

Figure 3. DPPH free radical-scavenging activity of the extracts. Human fibroblasts were subjected to different treatments, as indicated. PCB, PCB standard; SP, the PCB extract from Arthrospira maxima, prepared by water extraction using ultrasonication at room temperature followed by ethanolic extraction at 70 °C; WB, the wheat bran extract prepared by water extraction at 4 °C for eight hours; SPWB, a mixture of SP and WB containing the same amounts of PCB and SP. Trolox (50 µg/mL) was used as a positive control. The mean ± SD values for triplicate experiments are shown; error bars represent the SD. *** p < 0.001 compared with the Trolox group.

Figure 4. Comparison of intracellular ROS production in UVB-irradiated human skin fibroblasts treated with the different extracts. PCB, PCB standard; SP, the PCB extract from Arthrospira maxima, prepared by water extraction using ultrasonication at room temperature followed by ethanolic extraction at 70 °C; WB, the wheat bran extract prepared by water extraction at 4 °C for eight hours; SPWB, a mixture of SP and WB containing the same amounts of PCB and SP. The mean ± SD values for triplicate experiments are shown; error bars represent the SD. * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with the control (UV+) group.

Figure 5. Collagen levels in UVB-irradiated human skin fibroblasts subjected to different treatments. PCB, PCB standard; SP, the PCB extract from Arthrospira maxima, prepared by water extraction using ultrasonication at room temperature followed by ethanolic extraction at 70 °C; WB, the wheat bran extract prepared by water extraction at 4 °C for eight hours; SPWB, a mixture of SP and WB containing the same amounts of PCB and SP. TGF-β1 (0.1 ng/mL) was used as a positive control. The mean ± SD values for triplicate experiments are shown; error bars represent the SD. *** p < 0.001 compared with the control (UV+) group.

Figure 6. Inhibition of MMP-1 expression in UVB-irradiated human skin fibroblasts subjected to different treatments. PCB, PCB standard; SP, the PCB extract from Arthrospira maxima, prepared by water extraction using ultrasonication at room temperature followed by ethanolic extraction at 70 °C; WB, the wheat bran extract prepared by water extraction at 4 °C for eight hours; SPWB, a mixture of SP and WB containing the same amounts of PCB and SP. Retinol (0.1% w/v) was used as a positive control. The mean ± SD values for separate triplicate experiments are shown; error bars represent the SD. *** p < 0.001 compared with the control (UV+) group.

Figure 7. Up- and downregulation of mRNA levels of collagen (Col1A1) and MMP-1, respectively (a) in UVB-irradiated human skin fibroblasts, CCD-986sk. The relative densitometric intensities (b,c) of the bands, normalized against the mRNA levels of the housekeeping gene, GAPDH, in the different treatments, are shown. PCB, PCB standard; SP: the PCB extract from Arthrospira maxima, prepared by water extraction using ultrasonication at room temperature followed by ethanolic extraction at 70 °C; WB: the wheat bran extract prepared by water extraction at 4 °C for eight hours; SPWB: a mixture of SP and WB containing the same amounts of PCB and SP. TGF-β1 (0.1 ng/mL) was used as a positive control. The mean ± SD values for triplicate experiments are shown; error bars indicate the SD. ** p < 0.01, *** p < 0.001 compared with the control (UV+) group.

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MDPI and ACS Style

Koh, E.-J.; Kim, T.; Ryu, Y.-K.; Lee, W.-K.; Sunwoo, I.-Y.; Ro, H.S.; Jeon, G.; Kim, G.R.; Lee, H.Y.; Choi, W.-Y. Enhancement of Skin Anti-Wrinkling Effects of Arthrospira maxima Phycocynobilin by Combining with Wheat Bran Extract. Appl. Sci. 2024, 14, 10216. https://doi.org/10.3390/app142210216

AMA Style

Koh E-J, Kim T, Ryu Y-K, Lee W-K, Sunwoo I-Y, Ro HS, Jeon G, Kim GR, Lee HY, Choi W-Y. Enhancement of Skin Anti-Wrinkling Effects of Arthrospira maxima Phycocynobilin by Combining with Wheat Bran Extract. Applied Sciences. 2024; 14(22):10216. https://doi.org/10.3390/app142210216

Chicago/Turabian Style

Koh, Eun-Jeong, Taeho Kim, Yong-Kyun Ryu, Won-Kyu Lee, In-Yung Sunwoo, Hyang Seon Ro, Gibeom Jeon, Gyu Rae Kim, Hyeon Yong Lee, and Woon-Yong Choi. 2024. "Enhancement of Skin Anti-Wrinkling Effects of Arthrospira maxima Phycocynobilin by Combining with Wheat Bran Extract" Applied Sciences 14, no. 22: 10216. https://doi.org/10.3390/app142210216

APA Style

Koh, E. -J., Kim, T., Ryu, Y. -K., Lee, W. -K., Sunwoo, I. -Y., Ro, H. S., Jeon, G., Kim, G. R., Lee, H. Y., & Choi, W. -Y. (2024). Enhancement of Skin Anti-Wrinkling Effects of Arthrospira maxima Phycocynobilin by Combining with Wheat Bran Extract. Applied Sciences, 14(22), 10216. https://doi.org/10.3390/app142210216

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