Activity-Guided Purification Identifies Lupeol, a Pentacyclic Triterpene, As a Therapeutic Agent Multiple Pathogenic Factors of Acne  Hyuck Hoon Kwon, Ji Young Yoon, Seon Yong Park, Seonguk Min, Yong-il Kim, Ji Yong Park, Yun-Sang Lee, Diane M. Thiboutot, Dae Hun Suh  Journal of Investigative Dermatology  Volume 135, Issue 6, Pages 1491-1500 (June 2015) DOI: 10.1038/jid.2015.29 Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Acidic hexane fraction of Solanum melongena L. (SM) demonstrated antiacne characteristics. (a) Extracts of five candidate medicinal plants were separated on the basis of polarity and acidity. The acidic hexane fraction of SM demonstrated antiacne characteristics by displaying (b) antiproliferative effects in SEB-1 sebocytes with no general cytotoxicity on the HaCaT keratinocytes and 3T3-L1 adipocytes measured by MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, (c) antilipogenic effects for SEB-1 sebocytes based on relative percentile quantification of Nile red assay after normalization by cell numbers, and (d) anti-inflammatory effects in SEB-1 sebocytes stimulated by heat-inactivated P. acnes after 3 and 6hours based on reverse transcription PCR (RT-PCR) test for tumor necrosis factor-α (TNF-α), IL-6, and IL-8. All experiments were repeated a minimum of three times (mean±SEM). *P<0.05 between control and each concentration in acidic hexane fraction of the SM-treated group (Student’s t-test). Journal of Investigative Dermatology 2015 135, 1491-1500DOI: (10.1038/jid.2015.29) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Lupeol was isolated and identified on the basis of activity-guided purification procedures. (a) Activity-guided purification procedures were performed based on repetitive chromatographic separation steps with open column chromatography and preparatory HPLC methods for acidic hexane Solanum melongena L. (SM) extract. Instrumental analyses including (b) gas chromatography–mass spectrometry (GC–MS), (c) Fourier transform infrared spectroscopy (FT-IR), and (d) 1H nuclear magnetic resonance (NMR) were performed for molecular characterization of the final isolated product that had the most desired biologic activities (Fr 5-5-1) in terms of toxicity, antilipogenesis, anti-inflammation, and antimicrobial activities. (e) Lup-20 (29)-en-3β (lupeol), a pentacyclic triterpene, was finally identified. Journal of Investigative Dermatology 2015 135, 1491-1500DOI: (10.1038/jid.2015.29) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Lupeol decreased lipogenesis in SEB-1 sebocytes by suppressing the IGF-1R/phosphatidylinositide 3 kinase (PI3K)/Akt/sterol response element–binding protein (SREBP) signaling pathway. (a) Nile red assay was performed for SEB-1 sebocytes treated with lupeol for 24hours (bar=10μm). (b) Relative percentile quantification after cell count normalization. (c) Fatty acid methyl ester–gas chromatography (FAME-GC) analysis was performed to detect quantitative changes in fatty acid profiles. (d) Changes of 14C acetate incorporation levels in specific lipid components of SEB-1 cells after lupeol treatments were analyzed using thin-layer chromatography (C, cholesterol; FA, fatty acid; SQ, squalene) (left). Representative radio-TLC data for fatty acid (red line) of control and 20μM lupeol samples (right). After 24hours of lupeol treatment, (e) western blotting of phospho-IGF-1R, phospho IRS-1, phospho-PI3K, phospho-Akt, precursor, and mature SREBP-1 was performed, and (f) RNA was subjected to quantitative real-time PCR to determine the abundance of SREBP-1a, SREBP-1c, SREBP-2, fatty acid synthase (FAS), acetyl CoA carboxylase (ACC), HMG-CoA reductase (HMGCR), and HMG-CoA synthase (HMGCS). After pretreatment with LY294002 at 30minutes before lupeol treatment, (g) western blotting of phospho-Akt, precursor, and mature SREBP-1 was performed, and (h) quantification of Nile red assay was done. All experiments were repeated a minimum of three times (mean±SEM). *P<0.05 between each lupeol-treated group and control (Student’s t-test). Journal of Investigative Dermatology 2015 135, 1491-1500DOI: (10.1038/jid.2015.29) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Lupeol decreased inflammation induced by heat-inactivated P. acnes through the inhibition of the NF-κB pathway in both SEB-1 sebocytes and HaCaT keratinocytes. ELISA was performed for (a) IL-8 and (b) IL-6 in the supernatant of SEB-1 sebocytes stimulated with heat-inactivated P. acnes before and after treatments with lupeol for 24hours. (c) Western blotting of NF-κB p65 and phospho-IκBα under the same condition. (d) Immunocytofluorescence staining was done for IL-8 (green) with cell nuclei counterstained with 4,6-diamidino-2-phenylindole (blue) (bar=50μm). Parallel experiments were performed with HaCaT keratinocytes. ELISA for (e) tumor necrosis factor-α (TNF-α), (f) IL-6, (g) western blotting of NF-κB p65, and phospho-IκBα, and (h) quantitative real-time PCR for IL-6, IL-8, and TNF-α in HaCaT keratinocytes. All experiments were repeated a minimum of three times (mean±SEM). *P<0.05 between control and each P. acnes–stimulated group, †P<0.05 between P. acnes–stimulated, lupeol nontreated group, and each lupeol-treated group (Student’s t-test). Journal of Investigative Dermatology 2015 135, 1491-1500DOI: (10.1038/jid.2015.29) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Lupeol exhibited only a marginal effect on cell viability, possibly modulated dyskeratosis of epidermal keratinocytes, and inhibited the growth of P. acnes. Cell viability of both SEB-1 sebocytes and HaCaT keratinocytes was measured by both (a) MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and (b) CCK-8 (cell counting kit-8) assays after treatment with lupeol for 24 and 48hours. For HaCaT keratinocytes stimulated with heat-inactivated P. acnes before and after treatments with lupeol for 24hours, (c) western blotting of IL-1α, Toll-like receptor-2 (TLR-2), and keratin 16, and (d) quantitative real-time PCR were performed. (e) Immunocytofluorescence staining was done for keratin 16 (green) with cell nuclei counterstained with 4,6-diamidino-2-phenylindole (blue) (bar=50μm). (f) P. acnes was incubated with increasing concentrations of lupeol and vehicle control. (g) Their antibacterial effects were measured and minimal inhibition concentration of lupeol was determined. (h) Possible therapeutic mechanisms based on overall experimental results. *P<0.05 between control and each P. acnes–stimulated group, †P<0.05 between P. acnes–stimulated, lupeol nontreated group, and each lupeol-treated group (Student’s t-test). Journal of Investigative Dermatology 2015 135, 1491-1500DOI: (10.1038/jid.2015.29) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Histopathological changes from human acne tissues after applying lupeol for 4 weeks confirmed proposed therapeutic mechanisms in vivo. To detect changing patterns of both acne inflammation levels around comedones or sebaceous glands and expression of several target proteins associated with acne pathogenesis in vivo, typical acne lesions were acquired from patients before and after applying 2% lupeol twice daily for 4 weeks. (a) Histopathological inflammation severity assessment in hematoxylin and eosin staining (H&E), and immunohistochemical staining intensities of target proteins from patient skin tissues for (b) IGF-1R, (c) sterol response element–binding protein-1 (SREBP-1), (d) NF-κB p65, (e) IL-8, (f) IL-1α, (g) Toll-like receptor-2 (TLR-2), and (h) keratin 16 were analyzed at baseline and final visit (bar=100μM, arrows: inflammation around comedones or sebaceous glands). *P<0.05 compared with baseline (Student’s t-test). BL, baseline. Journal of Investigative Dermatology 2015 135, 1491-1500DOI: (10.1038/jid.2015.29) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions