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A 1,1′-biuracil from Epidermidibacterium keratini EPI-7 shows anti-aging effects on human dermal fibroblasts

A 1,1′-biuracil from Epidermidibacterium keratini EPI-7 shows anti-aging effects on human dermal... Our previous study we isolated novel bacterial stain, Epidermidibacterium keratini, called EPI‑7 from skin samples. Repeated column separation yielded one new pyrimidine compound, 1,1′‑biuracil, from EPI‑7 culture solutions grown in R2A medium. Its chemical structure was determined based on spectroscopic data, IR, FAB/MS, and NMR. And 1,1′‑biuracil and EPI‑7 culture solutions showed regulating effects of anti‑aging associated mRNA expressions in UV‑irradiated fibroblasts without toxicity in Hs68 cells. These results demonstrates the cosmetic potential of 1,1′‑biura‑ cil and EPI‑7 as anti‑aging agents. Keywords: Epidermidibacterium keratini EPI‑7, Keratinocyte, Skin bacteria, Human skin, 1,1′‑Biuracil Introduction was aerobic and heterotrophic, and consisted of non- Human skin provides a habitat for various microorgan- motile, non-spore-forming, rod-shaped cells (Fig.  1). isms that stably maintain communities through commen- Good growth was obtained on R2A agar but not on NA, sal relationships. The symbiotic relationships between ISP 2, or TSA. the skin and the microbiome produce complex protec- Skin microorganisms produce various metabolites and tive barriers against external environmental factors. influence skin cells directly or indirectly [3]. Among these Diverse metabolites produced by skin microbiome pro- microorganisms, the most common are of the genus vide favorable efficacy to human skin [1]. In this study, we Staphylococcus, which has been reported to be involved collected skin-microbiome samples from two different in Toll-like receptor (TLR) signaling in inflammation age groups of females. One group was in their 20  s, and and wound regeneration [4, 5]. Indeed, several species the other was in their 40  s. Analysis of 16S rRNA gene in the genus Staphylococcus have been linked to various sequences showed a newly found bacterium that shares inflammatory diseases of the skin; however, no study has 93.4% homology with the genus Sporichthya, indicating linked these organisms to aging. In addition, no report the discovery of a novel genus. We isolated a novel bac- has described the relationships between aging and skin terial stain, Epidermidibacterium keratini, called EPI-7 microorganisms. Therefore, in the current study, we ana - [2], from skin samples. Additionally, the younger skin lyzed the distribution of EPI-7 by age and investigated appeared to have high proportion of EPI-7 , while the mechanisms related to aging. older skin had no EPI-7 but rather other types of bac- teria. Skin probiotic strain EPI-7 stained gram-positive, Experimental Materials Epidermidibacterium keratini EPI-7 in R2A medium was *Correspondence: shyunk@cosmax.com; nibaek@khu.ac.kr provided by COSMAX R&I Center, Seongnam, Repub- Yeong‑ Geun Lee and Dong‑ Geol Lee made equal contributions to this work (co‑first author) lic of Korea, in May 2018. A voucher specimen (KHU- Department of Oriental Medicine Biotechnology, Graduate School NPCL-201805) has been deposited at the Laboratory of of Biotechnology, Kyung Hee University, Yongin 17104, Republic of Korea Natural Products Chemistry, Kyung Hee University. R&I Center, COSMAX BTI, Seongnam, Republic of Korea © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Lee et al. Appl Biol Chem (2019) 62:14 Page 2 of 6 1 13 Table 1 H- and  C-NMR data of 1,1′-biuracil (1) and uracil (2) (600 MHz, DMSO-d , δ ; 150 MHz, DMSO-d , δ ) 6 H 6 C 1,1′‑Biuracil (1) Uracil (2) δ δ * δ δ * C H C H 1, 1′ – – 1 – 10.82, br. s 151.5 – 2 152.0 – 2, 2′ 3, 3′ – 10.98, br. s 3 – 11.02, br. s 164.3 – 4 164.8 – 4, 4′ 5, 5′ 100.2 5.44, br. d, 7.2 5 100.7 5.45, ddd, 6.6, 1.8, 1.2 6, 6′ 142.2 7.38, d, 7.2 6 142.7 7.40, dd, 6.6, 4.8 * Coupling pattern, J in Hz Fig. 1 Morphology of strain EPI‑7 viewed by SEM modified Eagle’s medium supplemented with 1% Antibi - otic Antimycotic Solution (DMEM; HyClone Laborato- General experimental procedures ries, Inc., Logan, UT, USA) and 10% fetal bovine serum Instruments and chemicals used in this study were pre- at 37 °C in an atmosphere of 5% CO . For UV irradiation pared based on the previously described methods [6]. and treatment, Hs68 cells were seeded at 80% conflu - ence into 6-well plates and incubated in an atmosphere of Extraction and isolation 5% CO at 37 °C. After 24 h, the cells were washed once EPI-7 culture solutions grown in R2A medium (50  L) with phosphate-buffered saline (PBS) and placed in fresh were centrifuged, filtrated, and evaporated under reduced PBS. Next, 12 mJ/cm of UVB (wavelength 290–320 nm, pressure. The concentrates were extracted in 80% aque - maximum peak 311 nm) was applied in the presence of ous MeOH (500  mL × 3). The combined concentrates crosslinker (UVP; Upland, CA, USA), and then EPI-7 (144  g) were poured into H O (2.2  L) and successively (0.1– 1%) or 1,1′-biuracil (0.1–10 ppm) was administered extracted with EtOAc (2.2 L × 3) and n-BuOH (2.0 L × 3). into the cells through serum-free medium for 24 h. Each layer was concentrated under reduced pressure to obtain EtOAc (EPE, 10.7  g), n-BuOH (EPB, 42.8  g), and Cell viability test H O (EPH, 90.5  g) fractions. Fraction EPE (10.7  g) was Hs68 cells were seeded in 48-well plates, incubated for applied to silica gel (SiO ) column chromatography (c.c.) 24 h, and then treated with the indicated concentrations (Φ 5.5 × 35  cm) and eluted with EtOAc-n-BuOH–H O of EPI-7 or 1,1′-biuracil for another 24  h. After remov- (20:3:1 → 7:3:1, 2 L of both) with monitoring by TLC to ing the culture medium, 3-(4, 5-dimethylthiazol-2-yl)-2, provide 12 fractions (EPE-1 to EPE-12). Fraction EPE-3 5-diphenyltetrazolium bromide (MTT) solution was [168.0  mg, elution volume/total volume (Ve/Vt) 0.200– added to each well and incubated for 4  h. After termi- 0.250] was subjected to octadecyl SiO (ODS) c.c. [Φ nation of the reaction, the medium was discarded, and 2 × 10  cm, MeOH–H O (2:1), 400 mL] to yield four frac- dimethylsulfoxide (DMSO) was added to dissolve the tions (EPE-3-1 to EPE-3-4) and a purified compound formazan crystals. Optical density (OD) values were 1 [EPE-3-1, 20.5  mg, Ve/Vt 0.000–0.225, TLC (SiO) R 2 f measured at 570  nm using a microplate reader and nor- 0.70, EtOAc-n-BuOH–H O (15:3:1), TLC (ODS) R 0.88, 2 f malized to that of the control. MeOH–H O (2:1)]. 1,1′-Biuracil (1) White amorphous powder; IR (KBr) −1 RNA isolation and real‑time PCR ν 1715, 1674, 1418 cm ; low resolution positive ESI/ max + + Total RNA was isolated from cells using TRIzol reagent MS m/z 113 [M/2 + 1] , 223 [M + 1] , 267 [M + 2Na- according to the manufacturer’s instruction (TaKaRa, H] ; High resolution positive ESI/MS m/z 223.0459 + 1 13 Shiga, Japan). cDNA was synthesized from 1  µg of total [M + 1] (Calcd. for C H N O 223.0467); H- and C- 8 7 4 4 RNA using Reverse Transcription Premix (Elpis-biotech, NMR (600 and 150  MHz, DMSO-d , δ and δ ), see 6 H C Daejeon, Korea) under the following reaction conditions: Table 1. 45  °C for 45  min and 95  °C for 5  min. Gene expression signals were quantified with real-time PCR, and the data Cell culture and treatment were analyzed using StepOne PlusTM system software The human fibroblast cell line (Hs68) was purchased (Applied Biosystems, Foster City, CA, USA). Real-time from the American Type Culture Collection (Manas- PCR amplification reactions were performed using SYBR sas, VA, USA). The cells were cultured in Dulbecco’s Lee et al. Appl Biol Chem (2019) 62:14 Page 3 of 6 Fig. 2 H‑NMR spectrum of 1,1′‑biuracil (1) and uracil (2) (600 MHz, DMSO ‑d ) Green PCR Master Mix with premixed ROX (Applied ACC ACC CT-3′ and R: 5′-CAG CTG GAT GGC CAC ATC Biosystems, Foster City, CA, USA). The following primer GG-3′), fibrillin (F: 5′-AAT GTC AGA CGA AGC CAG pairs (Bioneer, Daejeon, Korea) were used in the reac- GG-3′ and R: 5′-GAT TTG GTG ACG GGG TTC CT-3′), tions performed in an ABI 7300 following the manufac- MMP-1 (F: 5′-CGA ATT TGC CGA CAG AGA TGA- turer’s protocol: β-actin (F: 5′-GGC CAT CTC TTG CTC 3′ and R: 5′-GTC CCT GAA CAG CCC AGT ACTT-3′), GAA GT-3′ and R: 5′-GAC ACC TTC AAC ACC CCA MMP-3 (R: 5′-ATT CCA TGG AGC CAG GCT TTC-3′ and GC-3′), type I procollagen (F: 5′-CTC GAG GTG GAC R: 5′-CAT TTG GGT CAA ACT CCA ACT GTG -3′). The Lee et al. Appl Biol Chem (2019) 62:14 Page 4 of 6 Fig. 3 Cell viability of EPI‑7 culture solutions and 1,1′‑biuracil in Hs68 human fibroblasts reaction conditions were as follows: initiation at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 10  s and 60  °C for 1  min. The expression of β-actin was used as an internal control. Results and discussion Concentrated R2A medium including E. keratini EPI-7 were successively partitioned into EtOAc, n-BuOH, and aqueous fractions. Repeated SiO and ODS c.c. of the n-BuOH fraction yielded one new uracil derivative. Compound 1, white amorphous powder, showed an −1 IR absorbance band of carboxyl (1715, 1674 cm ), aro- −1 −1 matic (1674  cm ), and amide group (1418  cm ). IR spectrum of it was very similar to uracil with the excep- tion for the absence of an amide moiety [7]. Its molec- ular weight and molecular formula were respectively 222  Da and C H N O from the molecular ion peak 8 6 4 4 m/z 223.0459 [M + 1] (Calcd. for C H N O 223.0467) 8 7 4 4 in positive HR ESI/MS. The four carbon signals at δ 164.3 (s, C-4,4′), 151.5 (s, C-2,2′), 142.2 (d, C-6,6′), and 100.2 (d, C-5,5′) in the C-NMR spectrum suggested that compound 1 was very similar to uracil (2), one of Fig. 4 The effect of EPI‑7 culture solutions on the regulation of the pyrimidine bases of nucleic acids [8, 9]. The car - anti‑aging associated mRNA expressions in UV ‑irradiated fibroblasts bon signals of two amide carbonyls [δ 164.3 (C-4,4′) and δ 151.5 (C-2,2′)], a nitrogenated olefine methine [δ 142.2], and an olefine methine [δ 100.2] were C C compound 1 had only one chemical shift, indicating observed. The above mentioned C-NMR and ESI/MS the uracil dimer was linked through an N–N linkage. J data suggested compound 1 to be a dimer of uracil (2, values (7.2 Hz) of the nitrogenated aromatic proton sig- molecular weight 112  Da) with a symmetrical struc- nals (H-6,6′) confirmed the proton signals to show only ture. The H-NMR spectrum (600 MHz, DMSO-d , δ ) 6 H one J coupling in the heterocyclic structure, which also showed amine (δ 10.96, 2H, br. s, H-NH-3,3′) and aro- indicated an N–N linkage between NH-1 and NH-1′. matic (δ 7.38, 2H, d, J = 7.2 Hz , H-6,6′; δ 5.40, 2H, br. H H As shown in Fig.  2, the nitrogenated aromatic proton d, J = 7.2 Hz , H-5,5′) proton signals. As shown in Fig. 2, signals (H-6,6′) in uracil (2) were split as dd through the uracil (2) showed amine proton signals at two dif- two J couplings with H-5 and NH-1. Also, the gHMBC ferent chemical shifts in the H-NMR spectrum, while Lee et al. Appl Biol Chem (2019) 62:14 Page 5 of 6 to 10  ppm for 24  h. The mRNA expression levels were measured using RT-qPCR. As shown in Fig.  4, EPI- 7 culture solutions increased type I procollagen and fibrillin mRNA expression, which were suppressed by UV irradiation. In addition, the mRNA expression level of MMP-1, the major collagen-degrading proteinase, was significantly reduced by EPI-7 culture solutions. These results indicated that EPI-7 culture solutions exerted skin anti-aging effects. Furthermore, we measured the mRNA expression levels of type I procollagen, fibrillin, and MMP-1 after treatment with 1,1′-biuracil in UV-irradiated Hs68 fibroblasts to clarify the anti-aging effects of 1,1′ -biura- cil derived from EPI-7 culture solutions. Application of 1,1′-biuracil did not regulate type I procollagen or MMP-1 mRNA expression (data not shown), whereas it significantly increased fibrillin mRNA expression and reduced that of MMP-3, the fibrillin-degrading protein - ase [13], in the UV-irradiation condition (Fig. 5). Taken together, these results suggest that 1,1′-biuracil is a key molecule in EPI-7 culture solutions, exerting protec- tive effects against UV-irradiated skin aging. Additional file Additional file 1. Spectroscopic data of 1,1’‑biuracil are available as sup ‑ Fig. 5 Anti‑aging functions of 1,1′‑biuracil in UV ‑irradiated fibroblasts plementary material. Authors’ contributions spectrum (Additional file  1: Fig. S5) showed correla- Y‑ GL, D‑ GL, SHK, and N‑IB planned study and made paper. Y ‑ GL, JEG, H‑ GK, tions between the nitrogenated aromatic proton signal J‑HK, and N‑IB isolated biuracil. Y ‑ GL and N‑IB identified compound. D ‑ GL, (H-6,6′) and two amide carbonyl carbons [(C-4,4′) and MSK, MJK, HJY, and SHK performed anti‑aging experiments. All authors read and approved the final manuscript. (C-2,2′)] as well the olefine methine carbon (C-5,5′ ) and between the aromatic proton (H-5,5′) and the amide carbonyl carbon (C-4,4′) as well the nitrogenated ole- Acknowledgements MAFRA (317071‑03‑2‑SB020) funded this study. fine methine carbon (C-6,6′ ). Based on these findings, compound 1 was identified as 1,1′ -biuracil, which was Competing interests revealed to be a new compound. A similar pyrimidine The authors declare that they have no competing interests. compound, 5,5′-biuracil, has been reported as a syn- thetic compound [10]. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ To determine the effects of EPI-7 culture solutions lished maps and institutional affiliations. and 1,1′-biuracil, we first examined the effects of EPI- 7 culture solutions and 1,1′-biuracil on cell viability Received: 29 December 2018 Accepted: 25 February 2019 of Hs68 human fibroblasts using MTT assay (Fig.  3), which showed no cytotoxicity at concentrations less than 1% and 10 ppm, respectively. References Next, we evaluated the expression levels of MMP-1 1. Cogen AL, Nizet V, Gallo RL (2008) Skin microbiota: a source of disease or and the skin aging-associated factors type I procolla- defence? Br J Dermatol 158:442–455 gen, fibrillin, in UV-irradiated fibroblasts [11, 12]. Hs68 2. Lee DG, Trujillo ME, Kang S, Nam JJ, Kim YJ (2018) Epidermidibacterium keratini gen. nov., sp. nov., a member of the family Sporichthyaceae, human fibroblasts were irradiated with UVB (12  mJ/ 2 T isolated from keratin epidermis. Int J Syst Evol Microbiol 68:745–750 cm ) and then treated with EPI-7 culture solutions 3. Cogen AL, Yamasaki K, Sanchez KM, Dorschner RA, Lai YD, MacLeod ranging from 0.1 to 1% or 1,1′-biuracil ranging from 0.1 TJ, Torpey W, Otto M, Nizet V, Kim JE, Gallo RL (2010) Selective antimi‑ crobial action is provided by phenol‑soluble modulins derived from Lee et al. Appl Biol Chem (2019) 62:14 Page 6 of 6 Staphylococcus epidermidis, a normal resident of the skin. J Invest Derma‑ 8. Ding ZG, Zhao JY, Yang PW, Li MG, Huang R, Cui XL, Wen ML (2009) 1H tol 130:192–200 and 13C NMR assignments of eight nitrogen containing compounds 4. Wanke I, Steffen H, Christ C, Krismer B, Gotz F, Peschel A, Schaller M, Schit ‑ from Nocardia alba sp. nov (YIM 30243T ). Magn Reson Chem 47:366–370 tek B (2011) Skin commensals amplify the innate immune response to 9. Blicharska B, Kupka T (2002) Theoretical DFT and experimental NMR stud‑ pathogens by activation of distinct signaling pathways. J Invest Dermatol ies on uracil and 5‑fluorouracil. J Mol Struct 613:153–166 131:382–390 10. Ishihara H, Wang SY (1966) Photochemistry of 5‑bromouracils: isolation of 5. Iwase T, Uehara Y, Shinji H, Tajima A, Seo H, Takada K, Agata T, Mizunoe 5, 5′‑ diuracils. Nature 210:1222 Y (2010) Staphylococcus epidermidis Esp. inhibits Staphylococcus aureus 11. Fligiel SE, Varani J, Datta SC, Kang S, Fisher GJ, Voorhees JJ (2003) Collagen biofilm formation and nasal colonization. Nature 465:346 degradation in aged/photodamaged skin in vivo and after exposure to 6. Nguyen NT, Song HS, Oh EJ, Lee YG, Ko JH, Kwon JE, Kang SC, Lee DY, matrix metalloproteinase‑1 in vitro. J Invest Dermatol 120:842–848 Jung IH, Baek NI (2017) Phenylpropanoids from Lilium asiatic hybrid flow‑ 12. Watson RE, Griffiths CE, Craven NM, Shuttleworth CA, Kielty CM (1999) ers and their anti‑inflammatory activities. Appl Biol Chem 60:527–533 Fibrillin‑rich microfibrils are reduced in photoaged skin. Distribution at 7. Kumar N, Thomas S, Tokas RB, Kshirsagar RJ (2014) SEIRA studies of uracil the dermal—epidermal junction. J Invest Dermatol 112:782–787 adsorbed on wet‑ chemically prepared gold nanoparticles film on glass 13. Ashworth JL, Murphy G, Rock MJ, Sherratt MJ, Shapiro SD, Shuttleworth substrate—effect of morphology of film. Spectrochim Acta A Mol Biomol CA, Kielty CM (1999) Fibrillin degradation by matrix metalloproteinases: Spectrosc 129:359–364 implications for connective tissue remodelling. Biochem J 340:171–181 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Applied Biological Chemistry Springer Journals

A 1,1′-biuracil from Epidermidibacterium keratini EPI-7 shows anti-aging effects on human dermal fibroblasts

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Abstract

Our previous study we isolated novel bacterial stain, Epidermidibacterium keratini, called EPI‑7 from skin samples. Repeated column separation yielded one new pyrimidine compound, 1,1′‑biuracil, from EPI‑7 culture solutions grown in R2A medium. Its chemical structure was determined based on spectroscopic data, IR, FAB/MS, and NMR. And 1,1′‑biuracil and EPI‑7 culture solutions showed regulating effects of anti‑aging associated mRNA expressions in UV‑irradiated fibroblasts without toxicity in Hs68 cells. These results demonstrates the cosmetic potential of 1,1′‑biura‑ cil and EPI‑7 as anti‑aging agents. Keywords: Epidermidibacterium keratini EPI‑7, Keratinocyte, Skin bacteria, Human skin, 1,1′‑Biuracil Introduction was aerobic and heterotrophic, and consisted of non- Human skin provides a habitat for various microorgan- motile, non-spore-forming, rod-shaped cells (Fig.  1). isms that stably maintain communities through commen- Good growth was obtained on R2A agar but not on NA, sal relationships. The symbiotic relationships between ISP 2, or TSA. the skin and the microbiome produce complex protec- Skin microorganisms produce various metabolites and tive barriers against external environmental factors. influence skin cells directly or indirectly [3]. Among these Diverse metabolites produced by skin microbiome pro- microorganisms, the most common are of the genus vide favorable efficacy to human skin [1]. In this study, we Staphylococcus, which has been reported to be involved collected skin-microbiome samples from two different in Toll-like receptor (TLR) signaling in inflammation age groups of females. One group was in their 20  s, and and wound regeneration [4, 5]. Indeed, several species the other was in their 40  s. Analysis of 16S rRNA gene in the genus Staphylococcus have been linked to various sequences showed a newly found bacterium that shares inflammatory diseases of the skin; however, no study has 93.4% homology with the genus Sporichthya, indicating linked these organisms to aging. In addition, no report the discovery of a novel genus. We isolated a novel bac- has described the relationships between aging and skin terial stain, Epidermidibacterium keratini, called EPI-7 microorganisms. Therefore, in the current study, we ana - [2], from skin samples. Additionally, the younger skin lyzed the distribution of EPI-7 by age and investigated appeared to have high proportion of EPI-7 , while the mechanisms related to aging. older skin had no EPI-7 but rather other types of bac- teria. Skin probiotic strain EPI-7 stained gram-positive, Experimental Materials Epidermidibacterium keratini EPI-7 in R2A medium was *Correspondence: shyunk@cosmax.com; nibaek@khu.ac.kr provided by COSMAX R&I Center, Seongnam, Repub- Yeong‑ Geun Lee and Dong‑ Geol Lee made equal contributions to this work (co‑first author) lic of Korea, in May 2018. A voucher specimen (KHU- Department of Oriental Medicine Biotechnology, Graduate School NPCL-201805) has been deposited at the Laboratory of of Biotechnology, Kyung Hee University, Yongin 17104, Republic of Korea Natural Products Chemistry, Kyung Hee University. R&I Center, COSMAX BTI, Seongnam, Republic of Korea © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Lee et al. Appl Biol Chem (2019) 62:14 Page 2 of 6 1 13 Table 1 H- and  C-NMR data of 1,1′-biuracil (1) and uracil (2) (600 MHz, DMSO-d , δ ; 150 MHz, DMSO-d , δ ) 6 H 6 C 1,1′‑Biuracil (1) Uracil (2) δ δ * δ δ * C H C H 1, 1′ – – 1 – 10.82, br. s 151.5 – 2 152.0 – 2, 2′ 3, 3′ – 10.98, br. s 3 – 11.02, br. s 164.3 – 4 164.8 – 4, 4′ 5, 5′ 100.2 5.44, br. d, 7.2 5 100.7 5.45, ddd, 6.6, 1.8, 1.2 6, 6′ 142.2 7.38, d, 7.2 6 142.7 7.40, dd, 6.6, 4.8 * Coupling pattern, J in Hz Fig. 1 Morphology of strain EPI‑7 viewed by SEM modified Eagle’s medium supplemented with 1% Antibi - otic Antimycotic Solution (DMEM; HyClone Laborato- General experimental procedures ries, Inc., Logan, UT, USA) and 10% fetal bovine serum Instruments and chemicals used in this study were pre- at 37 °C in an atmosphere of 5% CO . For UV irradiation pared based on the previously described methods [6]. and treatment, Hs68 cells were seeded at 80% conflu - ence into 6-well plates and incubated in an atmosphere of Extraction and isolation 5% CO at 37 °C. After 24 h, the cells were washed once EPI-7 culture solutions grown in R2A medium (50  L) with phosphate-buffered saline (PBS) and placed in fresh were centrifuged, filtrated, and evaporated under reduced PBS. Next, 12 mJ/cm of UVB (wavelength 290–320 nm, pressure. The concentrates were extracted in 80% aque - maximum peak 311 nm) was applied in the presence of ous MeOH (500  mL × 3). The combined concentrates crosslinker (UVP; Upland, CA, USA), and then EPI-7 (144  g) were poured into H O (2.2  L) and successively (0.1– 1%) or 1,1′-biuracil (0.1–10 ppm) was administered extracted with EtOAc (2.2 L × 3) and n-BuOH (2.0 L × 3). into the cells through serum-free medium for 24 h. Each layer was concentrated under reduced pressure to obtain EtOAc (EPE, 10.7  g), n-BuOH (EPB, 42.8  g), and Cell viability test H O (EPH, 90.5  g) fractions. Fraction EPE (10.7  g) was Hs68 cells were seeded in 48-well plates, incubated for applied to silica gel (SiO ) column chromatography (c.c.) 24 h, and then treated with the indicated concentrations (Φ 5.5 × 35  cm) and eluted with EtOAc-n-BuOH–H O of EPI-7 or 1,1′-biuracil for another 24  h. After remov- (20:3:1 → 7:3:1, 2 L of both) with monitoring by TLC to ing the culture medium, 3-(4, 5-dimethylthiazol-2-yl)-2, provide 12 fractions (EPE-1 to EPE-12). Fraction EPE-3 5-diphenyltetrazolium bromide (MTT) solution was [168.0  mg, elution volume/total volume (Ve/Vt) 0.200– added to each well and incubated for 4  h. After termi- 0.250] was subjected to octadecyl SiO (ODS) c.c. [Φ nation of the reaction, the medium was discarded, and 2 × 10  cm, MeOH–H O (2:1), 400 mL] to yield four frac- dimethylsulfoxide (DMSO) was added to dissolve the tions (EPE-3-1 to EPE-3-4) and a purified compound formazan crystals. Optical density (OD) values were 1 [EPE-3-1, 20.5  mg, Ve/Vt 0.000–0.225, TLC (SiO) R 2 f measured at 570  nm using a microplate reader and nor- 0.70, EtOAc-n-BuOH–H O (15:3:1), TLC (ODS) R 0.88, 2 f malized to that of the control. MeOH–H O (2:1)]. 1,1′-Biuracil (1) White amorphous powder; IR (KBr) −1 RNA isolation and real‑time PCR ν 1715, 1674, 1418 cm ; low resolution positive ESI/ max + + Total RNA was isolated from cells using TRIzol reagent MS m/z 113 [M/2 + 1] , 223 [M + 1] , 267 [M + 2Na- according to the manufacturer’s instruction (TaKaRa, H] ; High resolution positive ESI/MS m/z 223.0459 + 1 13 Shiga, Japan). cDNA was synthesized from 1  µg of total [M + 1] (Calcd. for C H N O 223.0467); H- and C- 8 7 4 4 RNA using Reverse Transcription Premix (Elpis-biotech, NMR (600 and 150  MHz, DMSO-d , δ and δ ), see 6 H C Daejeon, Korea) under the following reaction conditions: Table 1. 45  °C for 45  min and 95  °C for 5  min. Gene expression signals were quantified with real-time PCR, and the data Cell culture and treatment were analyzed using StepOne PlusTM system software The human fibroblast cell line (Hs68) was purchased (Applied Biosystems, Foster City, CA, USA). Real-time from the American Type Culture Collection (Manas- PCR amplification reactions were performed using SYBR sas, VA, USA). The cells were cultured in Dulbecco’s Lee et al. Appl Biol Chem (2019) 62:14 Page 3 of 6 Fig. 2 H‑NMR spectrum of 1,1′‑biuracil (1) and uracil (2) (600 MHz, DMSO ‑d ) Green PCR Master Mix with premixed ROX (Applied ACC ACC CT-3′ and R: 5′-CAG CTG GAT GGC CAC ATC Biosystems, Foster City, CA, USA). The following primer GG-3′), fibrillin (F: 5′-AAT GTC AGA CGA AGC CAG pairs (Bioneer, Daejeon, Korea) were used in the reac- GG-3′ and R: 5′-GAT TTG GTG ACG GGG TTC CT-3′), tions performed in an ABI 7300 following the manufac- MMP-1 (F: 5′-CGA ATT TGC CGA CAG AGA TGA- turer’s protocol: β-actin (F: 5′-GGC CAT CTC TTG CTC 3′ and R: 5′-GTC CCT GAA CAG CCC AGT ACTT-3′), GAA GT-3′ and R: 5′-GAC ACC TTC AAC ACC CCA MMP-3 (R: 5′-ATT CCA TGG AGC CAG GCT TTC-3′ and GC-3′), type I procollagen (F: 5′-CTC GAG GTG GAC R: 5′-CAT TTG GGT CAA ACT CCA ACT GTG -3′). The Lee et al. Appl Biol Chem (2019) 62:14 Page 4 of 6 Fig. 3 Cell viability of EPI‑7 culture solutions and 1,1′‑biuracil in Hs68 human fibroblasts reaction conditions were as follows: initiation at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 10  s and 60  °C for 1  min. The expression of β-actin was used as an internal control. Results and discussion Concentrated R2A medium including E. keratini EPI-7 were successively partitioned into EtOAc, n-BuOH, and aqueous fractions. Repeated SiO and ODS c.c. of the n-BuOH fraction yielded one new uracil derivative. Compound 1, white amorphous powder, showed an −1 IR absorbance band of carboxyl (1715, 1674 cm ), aro- −1 −1 matic (1674  cm ), and amide group (1418  cm ). IR spectrum of it was very similar to uracil with the excep- tion for the absence of an amide moiety [7]. Its molec- ular weight and molecular formula were respectively 222  Da and C H N O from the molecular ion peak 8 6 4 4 m/z 223.0459 [M + 1] (Calcd. for C H N O 223.0467) 8 7 4 4 in positive HR ESI/MS. The four carbon signals at δ 164.3 (s, C-4,4′), 151.5 (s, C-2,2′), 142.2 (d, C-6,6′), and 100.2 (d, C-5,5′) in the C-NMR spectrum suggested that compound 1 was very similar to uracil (2), one of Fig. 4 The effect of EPI‑7 culture solutions on the regulation of the pyrimidine bases of nucleic acids [8, 9]. The car - anti‑aging associated mRNA expressions in UV ‑irradiated fibroblasts bon signals of two amide carbonyls [δ 164.3 (C-4,4′) and δ 151.5 (C-2,2′)], a nitrogenated olefine methine [δ 142.2], and an olefine methine [δ 100.2] were C C compound 1 had only one chemical shift, indicating observed. The above mentioned C-NMR and ESI/MS the uracil dimer was linked through an N–N linkage. J data suggested compound 1 to be a dimer of uracil (2, values (7.2 Hz) of the nitrogenated aromatic proton sig- molecular weight 112  Da) with a symmetrical struc- nals (H-6,6′) confirmed the proton signals to show only ture. The H-NMR spectrum (600 MHz, DMSO-d , δ ) 6 H one J coupling in the heterocyclic structure, which also showed amine (δ 10.96, 2H, br. s, H-NH-3,3′) and aro- indicated an N–N linkage between NH-1 and NH-1′. matic (δ 7.38, 2H, d, J = 7.2 Hz , H-6,6′; δ 5.40, 2H, br. H H As shown in Fig.  2, the nitrogenated aromatic proton d, J = 7.2 Hz , H-5,5′) proton signals. As shown in Fig. 2, signals (H-6,6′) in uracil (2) were split as dd through the uracil (2) showed amine proton signals at two dif- two J couplings with H-5 and NH-1. Also, the gHMBC ferent chemical shifts in the H-NMR spectrum, while Lee et al. Appl Biol Chem (2019) 62:14 Page 5 of 6 to 10  ppm for 24  h. The mRNA expression levels were measured using RT-qPCR. As shown in Fig.  4, EPI- 7 culture solutions increased type I procollagen and fibrillin mRNA expression, which were suppressed by UV irradiation. In addition, the mRNA expression level of MMP-1, the major collagen-degrading proteinase, was significantly reduced by EPI-7 culture solutions. These results indicated that EPI-7 culture solutions exerted skin anti-aging effects. Furthermore, we measured the mRNA expression levels of type I procollagen, fibrillin, and MMP-1 after treatment with 1,1′-biuracil in UV-irradiated Hs68 fibroblasts to clarify the anti-aging effects of 1,1′ -biura- cil derived from EPI-7 culture solutions. Application of 1,1′-biuracil did not regulate type I procollagen or MMP-1 mRNA expression (data not shown), whereas it significantly increased fibrillin mRNA expression and reduced that of MMP-3, the fibrillin-degrading protein - ase [13], in the UV-irradiation condition (Fig. 5). Taken together, these results suggest that 1,1′-biuracil is a key molecule in EPI-7 culture solutions, exerting protec- tive effects against UV-irradiated skin aging. Additional file Additional file 1. Spectroscopic data of 1,1’‑biuracil are available as sup ‑ Fig. 5 Anti‑aging functions of 1,1′‑biuracil in UV ‑irradiated fibroblasts plementary material. Authors’ contributions spectrum (Additional file  1: Fig. S5) showed correla- Y‑ GL, D‑ GL, SHK, and N‑IB planned study and made paper. Y ‑ GL, JEG, H‑ GK, tions between the nitrogenated aromatic proton signal J‑HK, and N‑IB isolated biuracil. Y ‑ GL and N‑IB identified compound. D ‑ GL, (H-6,6′) and two amide carbonyl carbons [(C-4,4′) and MSK, MJK, HJY, and SHK performed anti‑aging experiments. All authors read and approved the final manuscript. (C-2,2′)] as well the olefine methine carbon (C-5,5′ ) and between the aromatic proton (H-5,5′) and the amide carbonyl carbon (C-4,4′) as well the nitrogenated ole- Acknowledgements MAFRA (317071‑03‑2‑SB020) funded this study. fine methine carbon (C-6,6′ ). Based on these findings, compound 1 was identified as 1,1′ -biuracil, which was Competing interests revealed to be a new compound. A similar pyrimidine The authors declare that they have no competing interests. compound, 5,5′-biuracil, has been reported as a syn- thetic compound [10]. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ To determine the effects of EPI-7 culture solutions lished maps and institutional affiliations. and 1,1′-biuracil, we first examined the effects of EPI- 7 culture solutions and 1,1′-biuracil on cell viability Received: 29 December 2018 Accepted: 25 February 2019 of Hs68 human fibroblasts using MTT assay (Fig.  3), which showed no cytotoxicity at concentrations less than 1% and 10 ppm, respectively. References Next, we evaluated the expression levels of MMP-1 1. Cogen AL, Nizet V, Gallo RL (2008) Skin microbiota: a source of disease or and the skin aging-associated factors type I procolla- defence? Br J Dermatol 158:442–455 gen, fibrillin, in UV-irradiated fibroblasts [11, 12]. Hs68 2. Lee DG, Trujillo ME, Kang S, Nam JJ, Kim YJ (2018) Epidermidibacterium keratini gen. nov., sp. nov., a member of the family Sporichthyaceae, human fibroblasts were irradiated with UVB (12  mJ/ 2 T isolated from keratin epidermis. Int J Syst Evol Microbiol 68:745–750 cm ) and then treated with EPI-7 culture solutions 3. Cogen AL, Yamasaki K, Sanchez KM, Dorschner RA, Lai YD, MacLeod ranging from 0.1 to 1% or 1,1′-biuracil ranging from 0.1 TJ, Torpey W, Otto M, Nizet V, Kim JE, Gallo RL (2010) Selective antimi‑ crobial action is provided by phenol‑soluble modulins derived from Lee et al. Appl Biol Chem (2019) 62:14 Page 6 of 6 Staphylococcus epidermidis, a normal resident of the skin. J Invest Derma‑ 8. Ding ZG, Zhao JY, Yang PW, Li MG, Huang R, Cui XL, Wen ML (2009) 1H tol 130:192–200 and 13C NMR assignments of eight nitrogen containing compounds 4. Wanke I, Steffen H, Christ C, Krismer B, Gotz F, Peschel A, Schaller M, Schit ‑ from Nocardia alba sp. nov (YIM 30243T ). Magn Reson Chem 47:366–370 tek B (2011) Skin commensals amplify the innate immune response to 9. Blicharska B, Kupka T (2002) Theoretical DFT and experimental NMR stud‑ pathogens by activation of distinct signaling pathways. J Invest Dermatol ies on uracil and 5‑fluorouracil. J Mol Struct 613:153–166 131:382–390 10. Ishihara H, Wang SY (1966) Photochemistry of 5‑bromouracils: isolation of 5. Iwase T, Uehara Y, Shinji H, Tajima A, Seo H, Takada K, Agata T, Mizunoe 5, 5′‑ diuracils. Nature 210:1222 Y (2010) Staphylococcus epidermidis Esp. inhibits Staphylococcus aureus 11. Fligiel SE, Varani J, Datta SC, Kang S, Fisher GJ, Voorhees JJ (2003) Collagen biofilm formation and nasal colonization. Nature 465:346 degradation in aged/photodamaged skin in vivo and after exposure to 6. Nguyen NT, Song HS, Oh EJ, Lee YG, Ko JH, Kwon JE, Kang SC, Lee DY, matrix metalloproteinase‑1 in vitro. J Invest Dermatol 120:842–848 Jung IH, Baek NI (2017) Phenylpropanoids from Lilium asiatic hybrid flow‑ 12. Watson RE, Griffiths CE, Craven NM, Shuttleworth CA, Kielty CM (1999) ers and their anti‑inflammatory activities. Appl Biol Chem 60:527–533 Fibrillin‑rich microfibrils are reduced in photoaged skin. Distribution at 7. Kumar N, Thomas S, Tokas RB, Kshirsagar RJ (2014) SEIRA studies of uracil the dermal—epidermal junction. J Invest Dermatol 112:782–787 adsorbed on wet‑ chemically prepared gold nanoparticles film on glass 13. Ashworth JL, Murphy G, Rock MJ, Sherratt MJ, Shapiro SD, Shuttleworth substrate—effect of morphology of film. Spectrochim Acta A Mol Biomol CA, Kielty CM (1999) Fibrillin degradation by matrix metalloproteinases: Spectrosc 129:359–364 implications for connective tissue remodelling. Biochem J 340:171–181

Journal

Applied Biological Chemistry – Springer Journals

Published: Dec 1, 2019

Keywords: applied microbiology; bioorganic chemistry; biological techniques

References

  • Skin microbiota: a source of disease or defence?
    AL Cogen, V Nizet, RL Gallo
  • Epidermidibacterium keratini gen. nov., sp. nov., a member of the family Sporichthyaceae, isolated from keratin epidermis
    DG Lee, ME Trujillo, S Kang, JJ Nam, YJ Kim
  • Selective antimicrobial action is provided by phenol-soluble modulins derived from Staphylococcus epidermidis, a normal resident of the skin
    AL Cogen, K Yamasaki, KM Sanchez, RA Dorschner, YD Lai, TJ MacLeod, W Torpey, M Otto, V Nizet, JE Kim, RL Gallo
  • Skin commensals amplify the innate immune response to pathogens by activation of distinct signaling pathways
    I Wanke, H Steffen, C Christ, B Krismer, F Gotz, A Peschel, M Schaller, B Schittek
  • Staphylococcus epidermidis Esp. inhibits Staphylococcus aureus biofilm formation and nasal colonization
    T Iwase, Y Uehara, H Shinji, A Tajima, H Seo, K Takada, T Agata, Y Mizunoe
  • Phenylpropanoids from Lilium asiatic hybrid flowers and their anti-inflammatory activities
    NT Nguyen, HS Song, EJ Oh, YG Lee, JH Ko, JE Kwon, SC Kang, DY Lee, IH Jung, NI Baek
  • SEIRA studies of uracil adsorbed on wet-chemically prepared gold nanoparticles film on glass substrate—effect of morphology of film
    N Kumar, S Thomas, RB Tokas, RJ Kshirsagar
  • 1H and 13C NMR assignments of eight nitrogen containing compounds from Nocardia alba sp. nov (YIM 30243T)
    ZG Ding, JY Zhao, PW Yang, MG Li, R Huang, XL Cui, ML Wen
  • Theoretical DFT and experimental NMR studies on uracil and 5-fluorouracil
    B Blicharska, T Kupka
  • Photochemistry of 5-bromouracils: isolation of 5, 5′-diuracils
    H Ishihara, SY Wang
  • Collagen degradation in aged/photodamaged skin in vivo and after exposure to matrix metalloproteinase-1 in vitro
    SE Fligiel, J Varani, SC Datta, S Kang, GJ Fisher, JJ Voorhees
  • Fibrillin-rich microfibrils are reduced in photoaged skin. Distribution at the dermal—epidermal junction
    RE Watson, CE Griffiths, NM Craven, CA Shuttleworth, CM Kielty
  • Fibrillin degradation by matrix metalloproteinases: implications for connective tissue remodelling
    JL Ashworth, G Murphy, MJ Rock, MJ Sherratt, SD Shapiro, CA Shuttleworth, CM Kielty