Peptides are widely used in cosmetic formulations to stimulate extracellular matrix (ECM) synthesis, while silybin (a flavonolignan from Silybum marianum) offers retinol-like benefits through antioxidant and photoprotective activity. This study evaluated a novel anti-aging cream combining seven bioactive peptides with silybin to assess synergistic effects on ECM regeneration and clinical skin rejuvenation. In vitro assays in human dermal fibroblasts and keratinocytes revealed that the formulation rapidly upregulated gene and protein expression of collagen types I, III, IV, and XVII and lysyl oxidase (LOX) within 4–16 h. Ex-vivo, ultraviolet (UV)-damaged skin explants treated with the peptide–silybin complex showed enhanced recovery of collagen, elastic fibers, and LOX versus untreated controls. A 56-day clinical study (n = 31) demonstrated significant improvements in wrinkle area and volume, elasticity (+12.5%), firmness (+20.7%), and dermal density (+78%, all p< 0.001). No adverse effects were reported, and over 80% of participants noted improved skin texture and firmness. These findings highlight a novel synergy between peptides and silybin, with rapid ECM activation and clinical efficacy. To our knowledge, this is the first evidence of a cosmetic peptide formulation significantly upregulating LOX expression, suggesting a new mechanism for strengthening dermal architecture and improving skin resilience. Future studies should elucidate the mechanisms underlying these effects and assess whether other botanicals confer complementary benefits when combined with peptide blends.
Skin aging occurs via intrinsic chronologic processes and extrinsic factors (primarily photoaging due to ultraviolet (UV) exposure. While intrinsic aging leads to gradual, inevitable changes, extrinsic aging—especially from cumulative UV radiation—accelerates structural degradation of the skin, manifesting as wrinkles, sagging, laxity, and uneven texture. Photoaged skin shows heightened matrix metalloproteinase activity and increased collagen degradation compared to naturally aged skin. At the molecular level, these visible aging signs are underpinned by changes in the extracellular matrix (ECM). Collagen fibers and elastin networks become fragmented and depleted with age and UV exposure, and non-enzymatic crosslinks such as advanced glycation end products (AGEs) accumulate, causing the ECM to become stiffer and less compliant. The net result is a loss of dermal structural integrity and elasticity.
Among the enzymes crucial for ECM stability is lysyl oxidase (LOX). LOX catalyzes covalent cross-linking of collagen and elastin fibers, thereby enhancing tensile strength and limiting the dermal matrix’s susceptibility to protease-mediated degradation. A decline in LOX expression and activity has been observed in adulthood, which contributes to weaker, more fragile collagen bundles and reduced skin elasticity. Indeed, inadequate collagen/elastin cross-linking resulting from low LOX is thought to accelerate dermal matrix degeneration and promote wrinkle formation in aged skin. Restoration of collagen, elastin, and LOX levels in the skin is therefore an important strategy for effective anti-aging interventions.
Recent advances in cosmetic science suggest that synergistic combinations of peptides can activate skin repair pathways more effectively than single peptides alone. Signal peptides are short bioactive sequences that can mimic fragments of functional proteins, thereby stimulating cells to produce ECM components or modulate cellular signaling relevant to skin structure and functions. For instance, topically applied peptide formula has been shown to increase dermal collagen content and reduce wrinkles in clinical studies. In this study, we formulated a unique blend of seven peptides, each chosen for distinct but complementary anti-aging mechanisms: two cyclic peptides and five signal peptides. Cyclic peptides (including Linum usitatissimum seed extract and synthetic cyclopeptide-161) are known for their conformational stability and enhanced skin penetration. The linseed-derived cyclic peptide has been reported to stimulate elastin expression in fibroblasts, improving elasticity, while cyclopeptide-161 is a neurotransmitter-inhibiting peptide aimed at reducing expression lines (unpublished data). The formula also contains tetradecyl aminobutyroylvalylaminobutyric urea trifluoroacetate, which has been shown to promote dermal firming by increasing collagen I production (even under UV-stress conditions) and boosting the production of decorin and lumican, thereby strengthening collagen fibril architecture and reducing skin sagging. Palmitoyl tripeptide-5 is a well-known signal peptide that can activate transforming growth factor beta (TGF-β) pathways similar to thrombospondin-1, leading to increased collagen types I and III synthesis and reduced matrix metalloproteinase activity. Acetyl tetrapeptide-11 promotes keratinocyte proliferation and has been found to upregulate collagen XVII, a key component of the dermal–epidermal junction (DEJ), thereby improving epidermal–dermal anchoring. Acetyl tetrapeptide-9 stimulates the production of collagen I as well as lumican (a proteoglycan involved in collagen fibril organization), supporting dermal matrix density. Finally, hexapeptide-9 is believed to stimulate fibroblasts to increase production of collagens and elastic fibers. By combining these seven peptides, our formulation targets multiple biological pathways in parallel, including collagen synthesis, elastin production, and dermal–epidermal junction reinforcement, for a multifaceted anti-aging effect.
Peptides primarily improve structural wrinkles via dermal ECM support; however, they have limited effects on epidermal renewal and lack intrinsic free-radical–scavenging activity, which may constrain their standalone efficacy against photoaging-related oxidative stress. Retinol—particularly when combined with antioxidants such as ferulic acid—has been shown to attenuate ROS levels and oxidative injury in UVB-induced photoaging models, thereby complementing the limitations of peptides and providing broader anti-aging benefits. In parallel, retinoid-like botanical antioxidants have gained attention as gentle yet effective anti-aging agents. Silybin, a major flavonolignan isolated from Silybum marianum, has demonstrated notable antioxidant, anti-inflammatory, and photoprotective properties. Treatment with silybin induced morphological changes analogous to those triggered by retinoic acid, inhibited the differentiation process of keratinocytes, reduced the expression levels of keratinocyte-specific terminal differentiation markers, and promoted the expression of proteins composing the basement membrane. Silybin has been reported to exhibit superior anti-aging efficacy compared to the well-known retinoid-like compound, bakuchiol. Unlike retinoids, however, silybin does not cause irritation or photosensitivity, making it an appealing “retinol-like” alternative for improving skin aging safely. Recent clinical evidence supports silybin’s efficacy: a formulation with Silybum marianum extract was shown to increase collagen III and hyaluronic acid in human skin, producing anti-wrinkle results comparable to retinol but without the side effects. Despite these benefits, silybin has been relatively underutilized in ECM-focused skincare studies, and its potential synergistic interactions with pro-collagen peptides are largely unexamined.
Building on this background, we developed a comprehensive anti-aging formula that merges the multi-mechanistic peptide approach with silybin’s protective and collagen-boosting properties. To our knowledge, this study is the first to evaluate a cosmetic formulation containing such a broad combination of peptides together with silybin. We conducted a series of investigations to assess its impact from the molecular level up to clinical outcomes: (1) rapid gene activation of collagen subtypes and LOX in cultured human fibroblasts and keratinocytes, (2) restoration of UV-damaged ECM proteins in human skin explants, (3) rapid in vitro activation of anti-aging proteins by the cream containing multi-peptides and silybin, and (4) a 56-day clinical trial to measure improvements in wrinkles, elasticity, and dermal structure in human volunteers. By examining both mechanistic biomarkers and visible skin changes, we aim to determine whether the seven-peptide–silybin complex delivers synergistic anti-aging effects that are superior to peptides alone, and to elucidate a potential new paradigm for skin anti-aging therapy.
The composition of multi-peptides includes cyclopeptide-161, Linum usitatissimum (linseed) seed extract, tetradecyl aminobutyroylvalylaminobutyric urea trifluoroacetate, palmitoyl tripeptide-5, acetyl tetrapeptide-11, acetyl tetrapeptide-9, and hexapeptide-9. Peptide test articles were used in their as-supplied cosmetic ingredient forms, i.e., peptide solutions or dispersions in inert carriers (e.g., water, glycerin, caprylyl glycol, or triglyceride solvents). Reported concentrations for the ‘multi-peptides’ refer to the as-supplied mixtures unless otherwise noted. Active-equivalent concentrations, calculated from supplier certificates of analysis, are provided. Silybin was used as a phospholipid complex (lecithin and 33% silybin (w/w)), and concentrations are expressed as silybin equivalents.
For the model of damage caused by UV irradiation to human skin explants, multi-peptides and silybin were added to a minimal formula without similar efficacy ingredients. The ratio of multi-peptides and silybin was the same as that in the cream. The vehicle (minimal formulation with the least interference to the results, composed only of essential excipients, including emulsifiers, preservatives, thickeners, and penetration enhancers) was prepared. The cream (the ingredients are listed) for clinical study containing polypeptides and silybin was provided by Proya Cosmetics Co., Ltd. (Hangzhou, China). Multi-peptides, silybin, and the cream were diluted to different concentration for cell experiments.
Human skin fibroblasts (HSFs, Biocell, batch No. Fb220309, Guangzhou, China) were cultured with Dulbecco’s modified Eagle’s medium (DMEM, GIBCO BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin/streptomycin (GIBCO, USA). Normal human epidermal keratinocytes (NHEKs, Biocell, batch No. Ep23013101, Guangzhou, China) were cultured with KcGrowth (Biocell, Guangzhou, China) culture solution. Human skin explants were immersed in 75% alcohol for 30 s and washed with sterile phosphate buffer saline (PBS) three times. Consequently, the explants were cut into 24 ± 2 mm 2 blocks, with the epidermis facing up and the dermis facing down, and put into the culture mold. Then the explants were transferred into the 6-well plates, containing 3.7 mL culture solution (Biocell, Guangzhou, China). The solution was replaced every day. All cells and tissues were maintained at 37 °C in a humidified atmosphere containing 5% CO 2.
HSFs and NHEKs were incubated for 24 h and then pretreated with polypeptides, silybin, or the cream. Then the cells were collected after incubation for several hours.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich, USA. Antibodies of collagen I and collagen III were purchased from Proteintech, Rosemont, IL, USA. Antibodies of collagen IV, collagen XVII, and lysyl oxidase (LOX) were purchased from Abcam, Cambridge, UK. RNAiso Plus, reverse transcription (RT) reagent kit, and fluorochrome were purchased from Accurate Biology, Changsha, China. PBS (Solarbio, Beijing, China) and paraformaldehyde (PFA, Biosharp, Hefei, China) were used for cell culture and fixation. Vitamin C and E (Sigma-Aldrich, St. Louis, MO, USA) and TGF-β1 (PeproTech, Rocky Hill, NJ, USA) were used as a positive control.
A CO 2 incubator (Thermo Fisher Scientific, Waltham, MA, USA) and an ultra-clean workbench (Airtech, Suzhou, China) were used for cell culture. Microplate spectrophotometer (BioTek, Winooski, VT, USA), polymerase chain reaction (PCR, BioRad, Hercules, CA, USA), quantitative real-time PCR (qRT-PCR, BioRad, USA), LED optical microscopes (Leica, Wetzlar, Germany), BioImage Lab device (Bio-Rad, USA), and upright light microscopes (Olympus, Tokyo, Japan) were used for analysis. UVA and UVB lamps (Philips, Amsterdam, The Netherlands) were used for irradiation.
In the clinical study, the parameters related to skin wrinkles and pores were measured with Primos CR (Canfield, Parsippany, NJ, USA), Visia 7 (Canfield, USA), and Antera 3D (Miravex, Dublin, Ireland). Dermal density was measured with a DUB® SkinScanner (tpm taberna pro medicum GmbH, Lüneburg, Germany). Skin elasticity and firmness were measured with Cutometer dual MPA580 (Courage + Khazaka electronic GmbH, Köln, Germany).
After 2 days of culture, human skin explants were treated with drugs and then irradiated with ultraviolet A (UVA, 30 J/cm 2) and ultraviolet B (UVB, 50 mJ/cm 2). Treatment and irradiation were performed at an interval of 8 h every day for four days. Only treatment of drugs performed for the next three days. The model group (negative control, NC) was only irritated by UV without treatment. Then, 100 μg/mL VC + 7 μg/mL VE were added to the medium as the positive control. The human skin explants were treated with multi-peptides alone or in combination with 0.033% silybin. After treatment, the human skin explants were fixed with 4% PFA and then sliced for analysis.
Compared with the multi-peptides group, the synergistic enhancement rate of multi-peptides combined with silybin was calculated by the following formula: Rate of synergistic enhancement = 100% × (expressi
