The cosmetic industry relies heavily on natural sources for active ingredients. This study focuses on a mixture of five herbal extracts—agrimonia, houttuynia, licorice, peony, and phellodendron—known for their individual skin benefits. The research question is whether their combined effect in a mixture (AHLPP) would positively influence skin barrier function, inflammation, and aging in human skin cells. The study's purpose is to evaluate the efficacy of AHLPP in regulating skin cell physiology and to determine if a γ-irradiation sterilization method, eliminating the need for preservatives, maintains its biological activity. This research is important for the development of safe and effective natural cosmetic products. The use of natural extracts provides a potential avenue for creating sustainable and biocompatible skincare products, which is increasingly demanded by consumers who are seeking more natural and environmentally conscious options. Furthermore, investigating the combined effect of multiple herbs allows for the potential of synergistic effects and enhanced bioactivity, which may not be achieved using a single herbal extract. Finally, determining the stability of the extract after γ-irradiation is important for developing a scalable and efficient industrial process that maintains the integrity and effectiveness of the product.

Individual components of the AHLPP mixture have shown promising results in previous studies. *Agrimonia pilosa* has demonstrated skin barrier restoration and anti-inflammatory effects. *Houttuynia cordata* promotes hair growth and improves atopic dermatitis. Licorice exhibits immune-modulating activity and combats photoaging. *Paeonia lactiflora* has anti-inflammatory and anti-aging properties. Finally, *Phellodendron amurense* alleviates skin pigmentation and inflammatory responses. While each plant shows benefits, the combined effects on human skin cells were previously unknown. Hydrogels, chosen for their biocompatibility and ability to store active ingredients, are also widely used in the medical and cosmetic fields, and their potential as a delivery system for this plant extract mixture was investigated in this study.

AHLPP extracts were prepared by water-based extraction and lyophilization from the five dried plants (*Agrimonia pilosa*, *Houttuynia cordata*, *Glycyrrhiza uralensis*, *Paeonia lactiflora*, and *Phellodendron amurense*). A specific ratio of each extract was determined experimentally. The stock solution was then subjected to 3 kGy γ-irradiation for sterilization. Cell viability was assessed using the CCK-8 assay on HaCaT (human keratinocytes) and Hs68 (human fibroblasts) cells. Cytokine analysis using a cytokine array was conducted on supernatants of HaCaT cells treated with AHLPP extracts. Quantitative real-time PCR analyzed gene expression of FLG (filaggrin), TGM1 (transglutaminase 1), DSP (desmoplakin), SPTLC1, SPTLC2, SPTLC3, COL1A1, TERT, CDKN1A, and CDKN1B. Procollagen C-peptide levels were measured using an ELISA kit in Hs68 cells. Telomerase activity was determined using a quantitative real-time telomeric repeat amplification protocol (RQ-TRAP). A hydrogel polymer containing the γ-irradiated AHLPP extract was prepared (composition provided in Table 2). Microbial growth assessments on the hydrogel were conducted to assess sterility. HaCaT cells were co-cultured with the hydrogel-AHLPP combination to analyze gene expression. A clinical study evaluated the effects of the AHLPP hydrogel on acne-prone skin using the Global Acne Grading System (GAGS) and sebum measurements.

AHLPP showed minimal cytotoxicity up to 3 µg/mL in Hs68 and HaCaT cells. γ-irradiation did not significantly alter cytotoxicity. AHLPP significantly downregulated pro-inflammatory cytokines (IL-1β, IL-7, MIG, SDF-1, PDGF-BB) in HaCaT cells. Notably, some cytokines like IL-6 and MCP-1 were differently affected by the irradiated AHLPP complex. AHLPP significantly upregulated mRNA expression of filaggrin (FLG), indicating enhanced skin barrier function. The upregulation of TGM1 and DSP was observed only with non-irradiated AHLPP. Treatment with AHLPP significantly increased procollagen C-peptide synthesis and COL1A1 mRNA expression in Hs68 cells. AHLPP significantly increased telomerase activity in HaCaT cells, and downregulated CDKN1B (p16) mRNA expression. The γ-irradiated hydrogel-AHLPP formulation showed no microbial contamination and upregulated FLG mRNA expression in co-cultured HaCaT cells and downregulated CDKN2A. A clinical trial demonstrated significant improvement in acne and sebum reduction after 2-4 weeks of AHLPP hydrogel application.

The findings demonstrate that AHLPP possesses anti-inflammatory, skin barrier-enhancing, and anti-aging properties. The γ-irradiation sterilization method was effective and did not compromise the extract's activity. In some instances, γ-irradiation even seemed to enhance its activity. The hydrogel formulation served as an effective delivery system, maintaining the bioactivity of AHLPP. These findings support the potential of AHLPP as an active cosmeceutical ingredient. The observed differences in cytokine profiles between non-irradiated and irradiated AHLPP suggest that γ-irradiation may alter the chemical composition of the extract, potentially leading to differential modulation of inflammatory pathways. Further studies to elucidate the mechanisms underlying these effects and to fully characterize the active components of AHLPP are warranted.

This study provides compelling evidence supporting the use of AHLPP as a novel active cosmeceutical ingredient. AHLPP demonstrated significant improvements in skin barrier function, reduced inflammation, and anti-aging effects. The γ-irradiation sterilization method offers advantages for industrial application without compromising efficacy. Future research should focus on identifying the specific active components of AHLPP, investigating its long-term effects, and exploring potential applications beyond cosmetics.

The study primarily focused on in vitro analyses, which might not fully reflect in vivo effects. While a small clinical trial showed promising results, larger and longer-term studies are needed to validate these observations. The exact mechanisms underlying AHLPP's bioactivities require further investigation. Finally, although efforts were made to standardize the ratio of herbs used, further optimization might improve the formulation's efficacy.