Aqueous two-phase system as a greener extraction method and the synergistic effects of the herbal mixture for acne-prone skin cosmetic applicationsOA

Background:Acne-prone skin requires long-term,user-friendly cosmetic approaches that can address oxidative stress,microbial imbalance,and biofilm formation without solely relying on antibiotics.Objective:This study evaluated whether an ethanol/(NH_(4))_(2)SO_(4)aqueous two-phase system(ATPS)could serve as a greener,polarity-directed extraction method for a four-herb mixture(Houttuynia cordata,Scutellaria baicalensis,Chamaecyparis obtusa,and Artemisia capillaris)and examined whether the resulting fractions were suitable for downstream cosmetic development.Materials and Methods:ATPS fractions prepared with 35%ethanol and 10%,12%,or 14%ammonium sulfate(N10,N12,and N14)were compared with the crude extract using TLC profiling,total phenolic content(TPC),total flavonoid content(TFC),total triterpenoid content(TTC),and DPPH radical-scavenging assays.Biological relevance was further assessed using zebrafish toxicity and H_(2)O_(2)-induced oxidative-stress models with qPCR analysis of antioxidant-related genes.Because the crude extract showed a more favorable in vivo safety profile than the ATPS fractions,prototype cleanser and spot gel serum formulations were developed using the crude extract rather than the ATPS fractions and were evaluated for short-term physicochemical stability,antibacterial activity,and antibiofilm formation against Cutibacterium acnes and Staphylococcus epidermidis.Results:ATPS selectively enriched phenolic and flavonoid constituents while reducing triterpenoid content,with N12 showing the highest TPC(690.8 mg GAE/g)and TFC(42.6 mg QE/g)and strong DPPH scavenging(IC_(50)10.67μg/mL),markedly improved over the crude extract(IC_(50)50.18μg/mL).However,zebrafish assays favored the crude extract,which showed better tolerance at higher exposure and clearer protection under oxidative stress,with modest upregulation of prdx1(∼1.44-fold)at 50μg/mL.Both crude extract-based prototypes remained physically stable over 4 weeks,while the cleanser showed clear antibacterial activity and near-complete suppression of mono-and mixed-species biofilms.Conclusion:Although ATPS improved chemical antioxidant enrichment,the crude extract was safer and more suitable for prototype development,and the crude extract-based cleanser emerged as the most promising formulation for repeated,user-friendly application to acne-prone skin.