Effects of a complex mixture prepared from agrimonia, houttuynia, licorice, peony, and phellodendron on human skin cells

Bioprospecting screens bioactive molecules from natural sources such as fungi, algae, and bacteria, and many cosmetic actives are plant-derived. Examples include astaxanthin used as an antioxidant and hyaluronic acid for anti-aging. Motivated by the widespread use of natural products, the study evaluated a mixture of five herbal extracts—Agrimonia pilosa, Houttuynia cordata, Glycyrrhiza uralensis (licorice), Paeonia lactiflora, and Phellodendron amurense—chosen for reported benefits relevant to skin barrier function, anti-inflammation, and anti-aging. Prior work suggests A. pilosa accelerates barrier restoration via TRPV3 and has antioxidant/anti-inflammatory properties; H. cordata promotes hair growth, upregulates filaggrin, and ameliorates atopic dermatitis; licorice exerts immune-modulating and anti-photoaging effects; P. lactiflora confers anti-inflammatory and immunoregulatory benefits and protects against ROS-induced skin damage; P. amurense shows anti-pigmentation, anti-inflammatory, and anti-acne activities. Hydrogels, widely used for skin due to biocompatibility and capacity to carry actives, were considered as delivery matrices, with ion-crosslinking favored for mild processing and minimal impact on actives. The study asked whether the combined AHLPP mixture modulates cytokine secretion and gene expression in human keratinocytes and fibroblasts; whether γ-irradiation could sterilize the extract while maintaining bioactivity to enable preservative-free use; and whether hydrogel-incorporated, γ-irradiated AHLPP affects nearby skin cells and benefits acne-prone skin.

The paper cites extensive prior evidence for each plant: A. pilosa extracts accelerate skin barrier restoration via TRPV3 and possess antioxidant/anti-inflammatory flavonoids (e.g., tiliroside, quercetin, luteolin, apigenin). H. cordata extracts promote hair growth, upregulate filaggrin in an aryl hydrocarbon receptor-dependent manner, and ameliorate atopic dermatitis; bioactive constituents include flavonoids and polyphenols (quercetin, rutin, hyperin, chlorogenic acid). Licorice (G. uralensis) provides immuno-enhancing polysaccharides and anti-inflammatory constituents (e.g., licoricidin) with anti-photoaging effects. P. lactiflora (paeony) total glucosides/paeoniflorin have anti-inflammatory and immunoregulatory effects and protect keratinocytes from UVB-induced apoptosis. P. amurense (Amur cork tree) contains limonoids, alkaloids, and phenolics with melanogenesis-inhibitory and anti-inflammatory effects and has been used for acne. The authors also review hydrogel technologies (ion-crosslinking, chemical, radical) highlighting ion-crosslinking for gentle processing. They note radiosterilization (γ-irradiation) advantages over heat/chemical methods and its known capacity to alter physicochemical properties of natural products, potentially modulating bioactivity. Prior examples of synergistic plant extract combinations in dermatology/pharmacology are referenced.

Preparation of AHLPP extracts: Decoctions were prepared by boiling 500 g each of H. cordata, A. pilosa, P. amurense bark, P. lactiflora root, and G. uralensis root with 10× water for 3 h. After three filtrations (0.45 μm), extracts were concentrated and freeze-dried. The AHLPP complex was made by incubating P. amurense extract (65 g), P. albiflora extract (26 g), A. pilosa extract (14 g), G. uralensis extract (5 g), and H. cordata extract (3.5 g) in 1 L water for 48 h, followed by lyophilization. Stock solutions were prepared in water. Gamma irradiation: AHLPP was exposed to 3 kGy γ-irradiation using an MB10-30 accelerator (10 MeV, 30 kW; Mevex, Canada). Hydrogel preparation: A hydrogel polymer was fabricated with carrageenan 1%, gellan gum 1%, carob gum 1%, glycerin 15%, EDTA 0.03%, polyglyceryl laurate 0.1%, lavender oil 0.01%, and AHLPP 1% (w/w), balance distilled water. Mixing was done at 2500 rpm, 75 °C, per stated sequence. Final composition is in Table 2 of the paper. Cell culture: Human dermal fibroblasts (Hs68) and human epidermal keratinocytes (HaCaT) were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin at 37 °C, 5% CO2. Viability was measured by CCK-8. Cytokine analysis: HaCaT cells were treated with control or γ-irradiated AHLPP for 48 h. Supernatants were clarified (15,000×g, 30 min) and analyzed using a cytokine array (AAH-CYT-3, RayBiotech). Densitometry was done in ImageJ; statistics by one-way ANOVA. Gene expression: Total RNA was extracted (TRIzol), 100 ng reverse-transcribed (iScript), and qPCR performed (TaqMan Universal PCR Master Mix, QuantStudio 3). Probes were from Roche UPL; G6PD was the loading control. Target genes included FLG, TGM1, DSP, SPTLC1/2/3, COL1A1, TERT (exon 2/3 total, exon 7/8 catalytically active forms), CDKN1A, CDKN2A/CDKN1B. Procollagen: Procollagen type I C-peptide in Hs68 supernatants was quantified by ELISA (Takara PIP EIA Kit). Telomerase activity: RQ-TRAP qPCR assay using TS and ACX primers; lysates prepared in NP-40 buffer; heat-inactivated controls included. Sterility testing: Hydrogel polymer filled with control or γ-irradiated AHLPP was cultured on selective media for Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC 8739), Candida albicans (ATCC 10231), Acinetobacter baumannii (ATCC 16404), and Staphylococcus aureus; monitored at 7 and 14 days. Hydrogel co-culture and clinical assessment: HaCaT cells were co-cultured 48 h with hydrogel containing 1% γ-irradiated AHLPP; FLG, CDKN1A, CDKN1B mRNA measured. A small clinical test (Korea Institute of Dermatological Sciences approval KIDS-AIB030-PTP) recruited 10 adults (20–35 years) with acne; Global Acne Grading System (GAGS) and sebum levels (Sebumeter) were measured at 2 and 4 weeks of using the hydrogel with AHLPP.

- Cytotoxicity: AHLPP exhibited low cytotoxicity in HaCaT and Hs68 cells; minimal cell death up to approximately 3 μg/mL and no significant cytotoxicity under 5 μg/mL. γ-irradiation did not alter viability effects. - Cytokines (HaCaT, 0.5 μg/mL, 48 h): Proinflammatory mediators G-CSF, IL-1β, IL-7, MIG/CXCL9, SDF-1/CXCL12, and PDGF-BB were significantly reduced by both control and γ-irradiated AHLPP (one-way ANOVA, p<0.05 to p<0.005). Control AHLPP significantly increased IL-6, MCP-1, and angiogenin, whereas γ-irradiated AHLPP did not show the same increases, indicating altered bioactivity post-irradiation. - Barrier gene expression (HaCaT, 0.5 μg/mL, 48 h): FLG mRNA increased vs vehicle (RU: vehicle 1.0; control 2.0; γ-irradiated 2.5). TGM1 mRNA increased (vehicle 1.0; control 1.5; γ-irradiated 1.5). DSP mRNA markedly increased with control (vehicle 1.0; control 4.0) but only modestly with γ-irradiated (1.5). Statistical significance indicated by one-way ANOVA (*p<0.05 to ***p<0.005). - Ceramide synthesis genes (HaCaT): SPTLC2 mRNA increased with both treatments (vehicle 1.0; control 2.0; γ-irradiated 2.0). SPTLC1 increased notably with γ-irradiated (vehicle 1.0; control 1.5; γ-irradiated 2.0). SPTLC3 unchanged. Indicates enhanced de novo sphingolipid/ceramide pathway potential. - Collagen synthesis (Hs68): Procollagen type I C-terminal propeptide in supernatant increased upon AHLPP treatment (colorimetric RU elevated; one-way ANOVA significance *p<0.05 to ****p<0.0001). COL1A1 mRNA significantly upregulated only with non-irradiated AHLPP at 0.5 μg/mL; suggests enhanced procollagen processing and collagen production. - Anti-aging markers (HaCaT): Telomerase activity significantly increased with both control and γ-irradiated AHLPP at 0.5 and 2 μg/mL; heat-inactivated controls abrogated activity. CDKN1B (p16) mRNA significantly downregulated; CDKN1A (p21) unchanged. TERT total (exon 2/3) and catalytically active (exon 7/8) transcripts significantly upregulated with both treatments (ANOVA *p<0.05 to ****p<0.0001). - Hydrogel incorporation: Hydrogel containing control or γ-irradiated AHLPP showed no microbial growth for S. aureus, P. aeruginosa, E. coli, C. albicans, or A. baumannii at 7 and 14 days. Co-culture of 1% γ-irradiated AHLPP hydrogel with HaCaT for 48 h increased FLG mRNA (t-test **p<0.01), did not change CDKN1A, and decreased CDKN1B (**p<0.01); no notable cytotoxicity. - Clinical pilot (n=10 acne subjects): GAGS scores improved after 2 and 4 weeks of hydrogel with AHLPP use (ANOVA significance indicated; improvement evident after >2 weeks). Sebum levels decreased nearly 40% after 4 weeks with statistical significance p<0.001.

The combined AHLPP extract demonstrated multi-faceted benefits aligned with the study aims: anti-inflammatory effects via downregulation of key chemokines/cytokines; enhancement of epidermal barrier-related genes (FLG, TGM1, DSP) and ceramide biosynthesis enzymes (SPTLC1/2); promotion of dermal collagen synthesis; and anti-aging effects through increased telomerase activity, TERT upregulation, and CDKN1B downregulation. These cellular changes support the hypothesis that a synergistic plant extract mixture can beneficially modulate skin physiology. γ-Irradiation for sterilization preserved major activities and, in some cases (e.g., SPTLC1 induction and lack of IL-6/MCP-1/angiogenin increases), altered the bioactivity profile, consistent with known irradiation-induced physicochemical changes in phytochemicals. Incorporation into a hydrogel enabled sterile, preservative-free delivery that still modulated adjacent keratinocyte gene expression. Preliminary clinical observations in acne-prone skin suggest potential for improving inflammatory lesions (GAGS) and reducing sebum. Collectively, the findings indicate feasibility of AHLPP as a cosmeceutical active for skin protection, barrier support, anti-inflammatory action, collagen support, and anti-aging, with γ-irradiation a viable processing step that may fine-tune activity while ensuring sterility.

This study introduces AHLPP, a γ-irradiation-compatible mixture of five herbal extracts, as a candidate cosmeceutical active that: (1) reduces proinflammatory cytokines; (2) upregulates skin barrier and ceramide synthesis genes; (3) enhances collagen production in dermal fibroblasts; and (4) exerts anti-aging effects via telomerase activation and modulation of senescence-associated genes. The mixture retained activity post-γ-irradiation and was effective when delivered via a hydrogel, which remained sterile and modulated keratinocyte gene expression; a small clinical pilot indicated improved acne grading and reduced sebum. Future research should identify and quantify the specific active constituents, elucidate molecular changes induced by γ-irradiation and their structure–activity relationships, expand in vivo and clinical evaluations with larger, controlled trials, and optimize formulation/dosing strategies for different skin indications.

The study did not identify specific active components responsible for the observed effects within the AHLPP mixture. Mechanistic reasons for differential cytokine responses between control and γ-irradiated extracts were not elucidated. Many findings derive from in vitro cell line models, which may not fully recapitulate in vivo skin physiology. The clinical component was a small, uncontrolled pilot with 10 participants, limiting generalizability and causal inference. Additional analytical characterization of irradiation-induced chemical modifications was not performed.