Natalenamides A-C, Cyclic Tripeptides Los ntawm Termite-Associated Actinomadura Sp. RB 99

Abstract:

Nyob rau hauv xyoo tas los no, kev tshawb fawb txog biochemistry ntawm cov kab mob ntsig txog kab mob tau nce ntxiv. Thaum ua ke nrog cov txheej txheem analytical dereplication, cov kev tshawb fawb no muab lub tswv yim muaj zog los txheeb xyuas cov txheej txheem thiab / lossis cov khoom siv roj ntsha tshiab. Non-ribosomally synthesized cyclic peptides muaj ib tug dav bioactivity spectrum nrog siab tshuaj muaj peev xwm.

Ntawm no, peb tshaj tawm qhov kev tshawb pom ntawm peb qhov tshiab cyclic tripeptides: natalenamides A–C (cov tshuaj 1–3). Cov tebchaw no tau txheeb xyuas los ntawm kab lis kev cai broth ntawm fungus-loj hlob termite-associated Actinomadura sp. RB99 siv cov kua chromatography (LC)/ultraviolet (UV)/mass spectrometry (MS)-raws li dereplication method. Cov qauv tshuaj ntawm cov khoom sib txuas tshiab (1–3) tau tsim los ntawm kev tshuaj xyuas ntawm cov txheej txheem spectroscopic, suav nrog ib sab (1H thiab 13C) thiab ob sab (1H-1H-COSY, HSQC, HMBC) nuclear magnetic Resonance spectroscopy (NMR), ua ke nrog cov ntaub ntawv siab tshaj plaws electrospray ionization mass spectrometry (HR-ESIMS). Qhov tseeb configurations ntawm lub tebchaw tshiab tau elucidated siv Marfey txoj kev tsom xam. Los ntawm ntau qhov kev sim bioactivity rau tripeptides, peb pom tias compound 3 tau nthuav tawm cov teebmeem tseem ceeb ntawm 3-isobutyl-1-methylxanthine (IBMX)-induced melanin ntau lawm. Cov nyhuv ntawm compound 3 yog zoo ib yam li kojic acid, ib tug compound feem ntau siv los ua cov khoom siv tshuaj pleev ib ce nrog rau cov nyhuv ntawm daim tawv nqaij dawb.

Cov tawv nqaij ncaj ncees, du, thiab muag heev yeej ib txwm yog lub hom phiaj nrhiav los ntawm cov poj niam oriental. Kev tshawb fawb thiab kev tsim cov tshuaj ntxuav hniav dawb rau cov khoom tu tawv nqaij feem ntau yog ua raws li inhibition ntawm tyrosinase kev ua haujlwm.

Cov tshuaj muaj cov benzene rings lossis phenolic hydroxyl cov qauv muaj peev xwm ua kom dawb huv, xws li tam sim no siv cov tshuaj ntxuav hniav dawb arbutin, uas tuaj yeem ua kom cov nyhuv whitening los ntawm inhibiting cov haujlwm ntawm tyrosinase.

Thiab peb pom tias Cistanche deserticola muaj cov nyhuv whitening. Lub ntsiab active ingredient ntawm Cistanche deserticola, phenylethanoid glycosides, yog ib hom ntawm ntuj glycoside compound. Cov kev tshawb fawb tau pom tias phenylethanoid glycosides tuaj yeem cuam tshuam kev ua haujlwm ntawm tyrosinase thiab tsim cov melanin hauv tib neeg epidermal melanocytes, thiab muaj cov nyhuv whitening. kev ua tau zoo ntawm tus neeg sawv cev.

Nyem cistanche tubulosa extract hmoov

Ntsiab lus:

fungus loj hlob termite; Actinomadura sp.; tripeptides; natalenamides A-C; cov teebmeem ntawm daim tawv nqaij dawb.

1. Taw qhia

Kev tshuaj xyuas tshuaj ntawm kev tiv thaiv kab mob sib kis ntawm cov kab ua liaj ua teb tau nyiam cov neeg siv khoom ntuj tsim nyob rau xyoo tas los no [1-5]. Siv lub xeev-of-the-art nuclear magnetic resonance (NMR) / mass spectrometry (MS)-raws li dereplication txheej txheem thiab ecologically cuam tshuam bioassays, ib tug impressive npaum li cas ntawm structurally intriguing tej yam ntuj tso khoom, nrog rau ob peb non-ribosomally synthesized cyclic peptides, tau uncovered. los ntawm microbial symbionts [6] Cov piv txwv tseem ceeb tshaj plaws suav nrog cov tshuaj tua kab mob dentigerumycin, cyclic depsipeptide cais tawm ntawm cov kab mob fungus-loj hlob-ant-associated Pseudonocardia sp. [7], thiab cov gerumycins A-C, piperazic acid-bearing cyclic depsipeptides txheeb xyuas los ntawm Pseudonocardia sp. [8].

Cyclic peptides tau pom tias muaj ntau yam kev ua haujlwm lom neeg, txhawb nqa ntau txoj hauv kev rau cov qauv tshuaj muaj txiaj ntsig zoo [9–12]. Ntau tshaj 40 cov tshuaj cyclic peptide tam sim no tau ua lag luam thiab siv dav hauv thaj chaw kho mob, thiab kwv yees li ib qho tshuaj tshiab cyclic peptide nkag mus rau hauv khw txhua xyoo, qhov nruab nrab [13].

Raws li ib feem ntawm peb qhov kev mob siab txuas ntxiv los txheeb xyuas cov txheej txheem tshiab thiab / lossis bioactive theem nrab metabolites los ntawm cov kab mob ntsig txog kab mob [14–17], peb tau tsom mus rau cov kab mob ntsig txog Actinomadura sp. RB99, cais tawm ntawm cov kab mob fungus-loj hlob, Macrotermes natalensis. Peb cov kua chromatography (LC) / ultraviolet (UV) / mass spectrometry (MS)-raws li dereplication lub tswv yim yielded muaj peev xwm tshiab bioactive natural khoom. Nyob rau hauv txoj kev tshawb no, peb tshaj tawm txog kev cais thiab tshuaj txheeb xyuas ntawm peb tus tshiab cyclic tripeptides, natalenamide A-C (cov tshuaj 1–3, daim duab 1), siv LC / UV / MS-based dereplication txoj kev nrog rau lawv cov kev ntsuam xyuas lom rau cytotoxic. , anti-inflammatory, thiab daim tawv nqaij-dawb kev ua ub no.

2. Cov txiaj ntsig thiab kev sib tham

2.1. Liquid chromatography (LC)/Ultraviolet (UV)/Mass Spectrometry (MS)-Based Isolation of Compounds 1–3

Actinomadura sp. RB99 raug cais tawm ntawm qhov chaw ntawm tus neeg ua haujlwm termite (M. natalensis). Phylogenetic tsom xam ntawm ze li ntawm ua tiav 16S ribosomal ribonucleic acid (rRNA) ib ntus qhia tias hom RB99 belongs rau genus Actinomadura, nrog rau cov neeg nyob ze Actinomadura nitritigenes NBRC 15918T. Tom qab LC / UV / MS-raws li kev tshuaj xyuas ntawm cov kab lis kev cai zoo tshaj plaws tau nthuav tawm cov yam ntxwv ntawm UV nqus nrog cov cim molecular ion peaks uas muaj nitrogen atoms.

Txhawm rau txheeb xyuas cov metabolites thiab tshawb xyuas lawv cov dej num, qhov loj-loj fermentation siv cov kev loj hlob zoo (100 agar daim hlau los ntawm 2 L ISP2 agar, pH 6, 10 hnub ntawm 30 ◦C) thiab cov txheej txheem tsim ua haujlwm thiab ua ntej purification yog siv tau [17]. Tom qab LC-UV / MS-guided fractionation thiab repetitive semi-preparative high-performance liquid chromatography (HPLC) ua rau kev sib cais ntawm peb metabolites nrog ib tug tshwj xeeb NMR / MS qauv. Peb tau kawm txog kev loj hlob thiab kev tshaj tawm xov xwm uas siv cov ntsev sib txawv (NaCl, KBr 1–3 feem pua) thiab cov khoom siv hauv xov xwm (ISP1-6 media) kom ua raws li ib puag ncig ntuj thiab txhawb cov metabolite ntau lawm, uas kuj ua rau muaj peb qhov tshiab. metabolites (saib daim duab ntxiv S19).

2.2. Structure Elucidation of the Compounds

Natalenamide A (1) tau txais los ua cov hmoov amorphous. Cov mis mos molecular ntawm 1 tau txiav txim siab yog C19H25N3O5 los ntawm sodium-adducted molecular ion ntawm m/z 398.1691 [M ntxiv Na] ntxiv (xws li rau C19H25N3O5Na, 398.1692) siv qhov zoo-ion hom high-resolution electrospray ionization ESIMS) cov ntaub ntawv. Lub infrared (IR) spectrum pom muaj zog nqus band ntawm 1670 cm−1, qhia tias muaj cov pab pawg amide hauv cov molecule. Cov ncauj lus kom ntxaws tsom xam ntawm 1H NMR cov ntaub ntawv ntawm 1 (Table 1) qhia cov cim qhia ntawm lub cev pob txha peptide, qhia ob lub cim methyl (δH 0.91 (3H, d, J=7). }} Hz, H-3) thiab 0.92 (3H, d, J=7.0 Hz, H-4)), peb lub teeb liab (δH 1.95 (1H, m, H-7a), 2.27 (1H, m, H{42}}a), 2.36 (1H, m, H-8b), 2.48 (1H , m, H{{50}}b), 2.97 (1H, dd, J=14, 8.0 Hz, H{{58} }a), thiab 3.20 (1H, dd, J=14). , m, H-2), 4.16 (1H, d, J=7.5 Hz, H-1), 4.20 (1H, dd, J=8.5, 4.5 Hz, H-6), thiab 4.63 (1H, dd, J=8.0, 5.0 Hz, H-11), thiab tsib overlapped aromatic protons (δH 7.18 (1H, m, H-16), 7.23 (2H, m, H{100}}/18), thiab 7.24 (2H, m, H-15/17)).

13C NMR spectrum (Table 1), nrog kev pab los ntawm HSQC thiab HMBC spectra, pom tag nrho 19 carbon resonances lub luag hauj lwm rau ob lub methyl signals (δC 18.9 thiab 19.9), peb methylene signals (δC 27.0, 30.6, thiab 38.8), cuaj lub cim methine (δC 32.1, 55.6, 58.0, 60.4, 127.8, 129.5 (×2), thiab 130.6 (×2)), suav nrog tsib cov pa roj carbon monoxide, ib qho olefinic carbon teeb liab (δC 138.8), thiab plaub carbonyl cov paib (δC 173.2, 173.3, 174.8, thiab 181.3). Ua ke nrog cov ntaub ntawv spectroscopic saum toj no, cov ncauj lus kom ntxaws soj ntsuam ntawm ob-dimensional (2D) NMR (1H-1H COSY, HSQC, thiab HMBC kev sim) qhia pom muaj peb cov amino acids hauv 1: glutamate (Glu), phenylalanine ( Phe), thiab valine (Val) (Daim duab 2). Kev sib txuas ntawm cov amino acids no tau txiav txim siab los ntawm HMBC kev sib raug zoo ntawm H-1/C-5 thiab C-19, H-11/C-10 thiab C{ {50}}, thiab H-6/C-5 thiab C-10 (Daim duab 2).

Txhawm rau txheeb xyuas qhov kev teeb tsa ntawm 1, Marfey txoj kev tau siv los txiav txim siab stereochemistry ntawm -H multiples (C-1, C-6, thiab C-11). Cov kua qaub hydrolysates ntawm 1 thiab tus qauv amino acids (L/D-Glu, Phe, thiab Val) tau muab derivatized nrog 1-fluoro-2, 4-dinitrophenyl-5-L-alanine amide (L-FDA). Ua tiav, qhov sib xyaw derivatized ntawm 1 thiab cov qauv amino acids tau txheeb xyuas los ntawm LC / MS los tshuaj xyuas lawv lub sijhawm khaws cia. Qhov tseeb configurations ntawm Glu, Phe, thiab Val moieties tau txiav txim siab los ua L-forms, raws li lub sijhawm khaws cia ntawm L-FDAA derivatives ntawm 1 piv nrog cov qauv amino acids. Yog li, tus qauv ntawm 1 tau elucidated raws li cyclic tripeptide qhia hauv daim duab 1.

Natalenamide B (2) raug cais raws li cov hmoov amorphous. Nws cov mis molecular (C20H27N3O5) tau txiav tawm los ntawm hydrogen thiab sodium-adducted molecular ion peaks ntawm 390.2032 [M ntxiv rau H] ntxiv (xws li rau C20H28N3O5, 390.2029) thiab 412.1849 [M ntxiv rau 18.4 calculated rau Na. Piv nrog rau cov mis molecular thiab NMR spectral cov ntaub ntawv ntawm 1, compound 2 tshwm sim kom muaj ib tug ntxiv CH2 pab pawg neeg nyob rau hauv lub peptide skeleton. Kev tshuaj xyuas tag nrho ntawm ib-dimensional (1D) (1H thiab 13C NMR) thiab 2D NMR (1H– 1H COSY, TOCSY, HSQC, thiab HMBC) spectra ntawm 2 qhia txog qhov muaj peb cov amino acid residues: Glu, Phe, thiab Leu (leucine). Cov kab ke ntawm cov amino acids no tau tsim los ntawm HMBC kev sib raug zoo ntawm H-1/C-6 thiab C-20, H-12/C-11 thiab C -20, thiab H-7/C-6 thiab C-11 (Daim duab 2). Txhawm rau txiav txim siab qhov tseeb ntawm 2, derivatization ntawm Glu, Phe, thiab Leu residues nrog L-FDAA tau ua thiab tom qab ntawd txheeb xyuas los ntawm LC / MS. Hauv LC/MS cov ntaub ntawv, L-Glu, L-Phe, thiab L-Leu tau kuaj pom los ntawm kev sib piv lub sijhawm khaws cia rau L-FDAA derivatives ntawm 2 nrog rau cov qauv amino acids. Yog li, cov qauv tshuaj ntawm 2 tau tsim los ua cyclic tripeptide qhia hauv daim duab 1.

Cov ntaub ntawv zoo-hom HR-ESIMS ntawm natalenamide C (3) tso tawm hydrogen thiab sodium-adducted molecular ions ntawm 424.1870 [M ntxiv rau H] ntxiv (xws li C23H26N3O5, 424.1872) thiab 446.1694 [M ntxiv rau Na] C ntxiv 424.1692). Qhov no qhia tias nws cov mis molecular yog C23H25N3O5. Kev soj ntsuam ntxaws ntxaws ntawm 1D thiab 2D NMR spectra ntawm 3 tau qhia tias nws cov qauv tshuaj zoo ib yam li ntawm 2, tshwj tsis yog Leu tau hloov nrog Phe hauv 3. Cov kev sib txuas ntawm peb cov amino acids uas tau txheeb xyuas hauv 3 tau txheeb xyuas siv HMBC kev sib raug zoo ntawm H-1/C{31}} thiab C-23, H-15/C-14 thiab C-23, thiab H-10/ C-9 thiab C-14 (Daim duab 2). Los ntawm kev siv Marfey txoj kev mus rau compound 3, qhov tseeb configurations ntawm -H multiples (C-1, C-10, thiab C-15) tau elucidated los ua ib L-Glu thiab ob L. -Poj niam. Raws li, cov qauv ua tiav ntawm 3 tau txiav txim siab raws li qhia hauv daim duab 1.

Natalenamides A-C yog tricyclic peptides, txawm tias tsis yog-ribosomal keeb kwm. Tam sim no, ob peb bioactive cyclic tripeptides tau pom tias yog cov khoom ntuj tsim: cytotoxic 17-cov tswv cuab cyclic tripeptides (piv txwv li, OF4949 I-IV) cais los ntawm Penicillium rugulose [18]; antifungal sclerotomies A-K, txheeb xyuas los ntawm fermentation broth ntawm cov kab mob halotolerant [10]; thiab antiproliferative psychrophilic E, cais los ntawm ib tug sib xyaw kab lis kev cai ntawm ob marines alga-derived fungal hom ntawm genus Aspergillus [19].

2.3. Kev Ua Si lom zem ntawm Cov Cheeb Tsam 1-3

Txij li thaum cyclic peptides tau tshaj tawm los nthuav tawm ntau yam kev ua ub no lom neeg lom zem ntawm cov kev sib cais cyclic tripeptides ({{0}}}) raug soj ntsuam siv peb bioactivities. Lub cytotoxicity ntawm cov tebchaw 1-3 tau tshawb xyuas tawm tsam plaub kab mob qog noj ntshav (MCE7 lub mis mob qog noj ntshav, HeLa hlwb qog noj ntshav, A549 tib neeg lub ntsws qog noj ntshav, thiab HepG2 lub siab mob qog noj ntshav) ntawm qhov sib txawv (0, 6.25 , 12.5, 25, 50, thiab 100 uM). Compound 1 tsis cuam tshuam rau kev ua haujlwm ntawm tes ntawm MCF-7, HeLa, lossis A549 hlwb. Txawm li cas los xij, kev kho mob ntawm HepG2 hlwb nrog 100 M ntawm compound 1 txo qis cell viability mus rau 78.5 ntxiv rau 3.2 feem pua ​​(Daim duab 3). Compound 2 txo cov cell viabilities ntawm HeLa thiab A549 hlwb rau 82.9  2.1 feem pua ​​​​thiab 73.5=3.0 feem pua, raws li, tab sis tsis cuam tshuam rau kev muaj peev xwm ntawm MCF-7 lossis HepG2 hlwb (Daim duab 3). Compound 3 tsis cuam tshuam rau kev muaj peev xwm ntawm ib qho ntawm cov kab ntawm tes (Daim duab 3).

Tom ntej no, RAW264.7 macrophages tau siv los tshawb xyuas cov tshuaj tiv thaiv kab mob ntawm cov tshuaj sib cais. Txhawm rau tshem tawm qhov yuam kev hauv kev tsim cov NO tshwm sim los ntawm kev hloov pauv cell ciaj sia taus, cov tshuaj tsis muaj tshuaj lom ntawm txhua qhov sib xyaw tau raug tshuaj xyuas. Raws li pom nyob rau hauv daim duab 4, cov tebchaw 1–3 muaj ob peb cytotoxic teebmeem nyob rau hauv RAW264.7 hlwb (Daim duab 4A–C) thiab exerted tsis muaj inhibitory los ntawm TSIS MUAJ ntau lawm nyob rau hauv lipopolysaccharide (LPS)-stimulated RAW264.7 hlwb (Daim duab 4D–F) .

Cov teebmeem inhibitory ntawm cov tebchaw 1-3 ntawm cov ntsiab lus melanin hauv B16F10 hlwb raug tshuaj xyuas raws li cov pov thawj ntawm lawv cov kev ua kom tawv nqaij dawb (Daim duab 5). Txij li thaum hloov pauv ntawm cell viability ua yuam kev hauv kev tsim thiab ntsuas melanin, peb thawj zaug tshuaj xyuas cov teebmeem ntawm cov tshuaj 1-3 ntawm B16F10 cell ciaj sia taus. Cov tshuaj 1-3 tsis muaj kev cuam tshuam cytotoxic hauv B16F10 hlwb ntawm ib qho ntawm cov concentrations siv (Daim duab 5A-C). Tom qab ntawd peb tau soj ntsuam cov teebmeem inhibitory ntawm cov tebchaw ntawm 3-isobutyl-1-methylxanthine (IBMX)-induced melanin ntau lawm hauv B16F10 hlwb (Daim duab 5D-F). IBMX, ib tug paub zoo stimulator ntawm melanogenesis, induces ib tug tseem ceeb nce nyob rau hauv melanin ntau lawm tom qab ib tug kho mob nyob rau hauv melanoma hlwb. Ntawm cov tshuaj ntsuam xyuas, compound 3 (ntawm 5-100 µM) tau nthuav tawm cov teebmeem tseem ceeb ntawm IBMX-mediated melanin synthesis nyob rau hauv koob tshuaj. Kojic acid, peb txoj kev tswj hwm zoo, tau siv dav los ua cov khoom siv kho kom zoo nkauj nrog cov teebmeem ntawm daim tawv nqaij dawb [20]. Cov nyhuv inhibitory ntawm compound 3 ntawm IBMX-induced melanin ntau lawm zoo ib yam li kojic acid (Daim duab 5F), qhia tias cov compound 3 ua haujlwm raws li lub zog inhibitor ntawm IBMX-induced melanin ntau lawm hauv B16F10 melanoma hlwb.

Tsis tas li ntawd, qhov sib xyaw 3 tau paug nrog qee qhov tsis huv, uas tau txiav txim siab tias yog fatty acid analogs los ntawm kev txhais cov ntaub ntawv NMR spectroscopic thiab LC/MS tsom xam (Cov Khoom Siv Ntxiv, Figures S11–S15 thiab S23). Txhawm rau txheeb xyuas cov haujlwm ntawm cov impurities, kev sim ntxiv nrog cov fatty acid analogs tau ua rau lawv cov teebmeem inhibitory ntawm IBMX-induced melanin ntau lawm hauv B16F10 hlwb, uas tau qhia tias cov tshuaj ntsuam xyuas tsis muaj cov nyhuv whitening (Cov ntaub ntawv ntxiv, daim duab S24). Yog li, txawm hais tias peb tsis tuaj yeem cais tawm tias cov impurities hauv compound 3 yog lub luag haujlwm rau cov nyhuv inhibitory ntawm IBMX-induced melanin ntau lawm, qhov no zoo li tsis zoo li.

3. Cov ntaub ntawv thiab cov txheej txheem

3.1. Cov txheej txheem kev sim dav dav

Infrared spectra tau txais los ntawm Bruker IFS-66/S FT-IR (Bruker, Billerica, MA, USA) spectrometer. Cov electrospray ionization thiab HR-ESIMS spectra tau ntsuas ntawm SI-2/LCQ DecaXP kua chromatography (LC)-mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). NMR spectra, suav nrog 1H–1H COSY, HSQC, thiab HMBC kev sim, tau ua los ntawm Varian UNITY INOVA 800 NMR spectrometer (Varian, Palo Alto, CA, USA) ua haujlwm ntawm 800 MHz (1H) thiab 200 MHz (13C), nrog Cov tshuaj hloov pauv hauv ppm (δ). Kev Npaj HPLC tau siv Waters 1525 Binary HPLC twj tso kua mis nrog Waters 996 Photodiode Array Detector (Waters Corporation, Milford, CT, USA). Silica gel 60 (230–400 mesh, Merck, Kenilworth, NJ, USA) thiab RP-C18 silica gel (230–400 mesh, Merck, tau siv rau kab chromatography. Semi-preparative HPLC siv Shimadzu Prominence HPLC System nrog SPD{ {22}}A/20AV Series Prominence HPLC ultraviolet-pom (UV-Vis) Detectors (Shimadzu, Tokyo, Nyiv). Teb Chaws Asmeskas) nruab nrog lub diode array detector thiab 6130 Series ESI huab hwm coj spectrometer nrog kev ntsuas Kinetex (4.6 × 100 mm, 3.5 µm). Merck pre-coated silica gel F254 daim hlau thiab RP-18 F254s daim hlau tau siv rau nyias -txheej chromatography (TLC) Cov chaw raug kuaj pom ntawm TLC hauv qab UV teeb lossis los ntawm cua sov tom qab txau nrog cov tshuaj anisaldehyde-sulfuric acid.

3.2. Cov khoom siv microbial

Actinomadura sp. RB99 raug cais tawm ntawm qhov chaw ntawm tus neeg ua haujlwm termite ntawm genus, M. natalensis, (colony Mn103, GPS S25 43 45.9 E28 14 08.9) thaum Lub Ib Hlis 2{ {13}}10. Biomaterial tau muab tso rau hauv cov hnab yas huv thiab ua tiav hauv ib hnub ntawm kev sau. Cov kab mob tau ntxuav hauv cov dej tsis huv deionized, thiab cov kab mob raug cais los ntawm kev muab cov tshuaj tshem tawm ntawm cov khoom noj uas tsis muaj chitin (ib litre: 4 g chitin, 0.7 g K2HPO4, 0.3 g KH2PO4, 0.5 g MgSO4·5H2O, 0.01 g FeSO4·7H2O, 0.001 g ZnSO4, 0.001 g MnCl2, thiab 20 g agar) [21]. Kev rho tawm nrog Actinobacteria zoo li morphology raug xa mus rau ISP2 agar (ib liter: 10 g malt extract, 4 g poov xab extract, 4 g qabzib, thiab 20 g agar), thiab sub-cultured kom txog thaum ntshiab cais tau.

3.3. DNA Extraction thiab Polymerase Chain Reaction Amplification

Actinomadura sp. RB99 tau loj hlob hauv cov kua dej uas muaj cov khoom noj muaj txiaj ntsig ISP2 rau tsib mus rau xya hnub ntawm 30 ◦C. Cells raug sau, thiab genomic DNA tau muab rho tawm siv GenJet genomic DNA purification kit (#K0721, Thermo Scientific, Waltham, MA, USA) ua raws li cov chaw tsim khoom cov lus qhia nrog cov kev hloov hauv qab no: (a) lysozyme kev kho mob tau txuas ntxiv mus rau 40 min, thiab (b) kev kho proteinase K tau txuas ntxiv mus rau 40 feeb. DNA tau txheeb xyuas cov duab thaij duab siv Nanodrop Lite spectrometer (Thermo Scientific).

Rau kev tshawb fawb phylogenetic, 16S rRNA noob tau nthuav dav siv cov txheej txheem primer, 1492R / 27F. Txhua qhov tshuaj tiv thaiv amplification tau npaj rau hauv 25 µL qhov kawg cov tshuaj tiv thaiv ntim uas muaj 7.25 µL ntawm cov dej distilled, 5 µL ntawm HF tsis, 5 µL ntawm txhua primer (2.5 µM), {{10}}.5 µL ntawm dNTPs (10 µM), 0.25 µL ntawm Phusion High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA), thiab 2 µL ntawm rho tawm DNA (template). Polymerase chain reaction (PCR) tau ua raws li cov hauv qab no: 98 ◦C rau 38 s; 32 lub voj voog ntawm 98 ◦ rau 30 s, 52 ◦C rau 45 s, 72 ◦C rau 1 min 20 s; thiab qhov kawg txuas ntxiv ntawm 72 ◦C rau 8 min. Cov khoom PCR tau pom los ntawm agarose gel electrophoresis, thiab PCR cov tshuaj tiv thaiv tau raug ntxuav los ntawm PCR purification kit (Thermo Scientific). DNA fragments tau sequenced ntawm GATC (Konstanz, Lub teb chaws Yelemees).

3.4. Sequencing thiab Species Identification

Cov kev sib tw tau raug soj ntsuam rau kev coj dawb huv thiab tsis sib haum xeeb siv BioEdit [22]. Cov tom ntej thiab thim rov qab tau txais rau txhua hom kab mob tau sib sau ua ke nrog BioEdit thiab sim rau chimeras siv DECIPHER (http://decipher.cee.wisc.edu/FindChimerasOutputs.html). Cov txiaj ntsig tshwm sim tau muab tso rau hauv GenBank (tus lej nkag: KY558684). Kev ntsuam xyuas tawg nrog yuav luag tiav 16S rRNA sequences (1368 bp) tau ua los ntawm National Center for Biotechnology Information (NCBI) database (reference RNA sequences). Cov txiaj ntsig tau qhia tias hom RB99 yog ib tus tswv cuab ntawm genus, Actinomadura. Cov kab ke ntawm thawj 10 hits tau rub tawm los ntawm NCBI database thiab ua raws li 16S rRNA ntu ntawm Actinomadura sp. RB99 siv Sina sequences alignment service [23]. Ob qhov sib txawv phylogenetic ntoo tau rov tsim dua nrog cov neeg zej zog koom nrog lossis qhov siab tshaj plaws-zoo li algorithms siv MEGA software version 7.0.26 [24–26]. Tus qauv evolutionary nrug ntawm Tamura thiab Nei tau siv los tsim cov evolutionary nrug matrices rau qhov siab tshaj plaws-zoo li thiab cov neeg zej zog koom nrog algorithms, nrog rau kev tshem tawm tag nrho cov khoob thiab cov ntaub ntawv ploj lawm [27]. Rau qhov siab tshaj plaws-zoo li algorithm, discrete gamma faib tau siv (ntxiv rau G), thiab tus qauv hloov pauv tau tso cai rau qee qhov chaw yuav hloov pauv hloov pauv tsis tau (ntxiv rau kuv). Rau cov neeg nyob sib ze-sib koom algorithm, tus nqi sib txawv ntawm cov chaw tau ua qauv nrog gamma faib (ntxiv rau G). Kev ntseeg siab qhov tseem ceeb ntawm cov nodes tau soj ntsuam los ntawm kev tshuaj ntsuam bootstrap raws li 1000 resampling cov kauj ruam [28].

3.5. Extraction thiab rho tawm

Actinomadura sp. RB99 tau loj hlob hauv 50 mL ISP2 kua zaub rau xya hnub ntawm 30 ◦C (ua ntej kab lis kev cai) thiab siv los ua 100 ISP2 agar daim hlau. Cov phaj tau incubated rau 10 hnub ntawm 30 ◦C, txiav mus rau hauv me me pieces, sib sau ua ke, thiab raus rau ib hmos nyob rau hauv MeOH. MeOH theem tau lim thiab evaporated nyob rau hauv lub siab txo. MeOH extract (20 g) yog yaj nyob rau hauv dej distilled (700 mL) thiab ces hnyav-partitioned nrog EtOAc (700 mL) peb zaug, affording 1.1 g ntawm residue. EtOAc-soluble feem (1.1 g) los ntawm MeOH extract tau thauj mus rau silica gel (230-400 mesh) kem rau chromatography thiab eluted nrog gradient hnyav system ntawm CH2Cl2-MeOH (100: 1 txog 1: 1, v/v). Qhov no muab rau 6 feem (A–F), uas tau raug rau LC-UV / MS-raws li kev tsom xam. Kev tshuaj xyuas tsis zoo siv lub tsev qiv ntawv UV spectral hauv tsev tau qhia txog qhov muaj me me peptide analogs hauv feem E. Cov no tso tawm cov qauv UV yooj yim nyob ib puag ncig 220 nm thiab cov cim molecular mis ion peaks uas muaj nitrogen atoms. Qhov polar feem E (233 mg) yog fractionated los ntawm preparative reversed-phase HPLC (Phenomenex Luna C18, 250 × 21.2 mm id, 5 µm, Torrance, CA, USA) siv CH3CN/H2O (1:9 txog 9:1, v /v, gradient system, ntws tus nqi: 5 mL / min) kom tawm tau tsib ntu ntu (E1-E5). Sub-fraction E2 (27 mg) tau purified los ntawm semi-preparative reversed-phase HPLC (Phenomenex Luna C18, 250 × 10.0 mm id, 5 µm) nrog 33 feem pua ​​MeOH/H2O (isocratic system, flow rate: 2 mL/min ) kom tawm los compound 1 (5.8 mg, tR=57.0 min). Cov tshuaj 2 (1.6 mg, tR=42.0 min) thiab 3 (1.5 mg, tR=55.0 min) raug cais tawm los ntawm cov seem E3 (15 mg) los ntawm ib nrab npaj rov qab-theem HPLC eluting 43 feem pua ​​MeOH/H2O (isocratic system, flow rate: 2 mL/min).

3.5.1. Natalamide A (1)

3.5.2. Natalamide B (2)

3.5.3. Natalamide C (3)

3.6. Acid Hydrolysis ntawm Compounds 1-3

Ib qho nyiaj tag nrho 0.4 mg ntawm txhua qhov sib xyaw (1–3) yog hydrolyzed nrog 6 N HCl (500 µL) rau 1 h ntawm 110 ◦C. Tom qab txias rau chav tsev kub, cov hydrolysates ntawm 1-3 tau evaporated kom tshem tawm cov kab ntawm HCl. Cov dej distilled (500 µL) tau ntxiv rau cov hydrolyzate sib tov thiab ces evaporated kom tshem tawm cov kab ntawm HCl; txheej txheem no tau ua peb zaug.

3.7. Kev txiav txim siab ntawm Kev Txhim Kho Kom Zoo ntawm Amino Acids hauv 1–3

Cov tshuaj sib xyaw hydrolyzate (1–3), nrog rau cov qauv amino acids (L/D-Leu, Glu, Phe, thiab Val), tau yaj hauv 1 N NaHCO3 (100 µL) thiab tom qab ntawd kho. nrog 50 µL ntawm L-FDAA (10 mg / mL hauv acetone). Ua tiav, txhua qhov hydrolyzate tau rhaub rau 10 feeb ntawm 80 ◦C. Txhua qhov sib tov yog quenched nrog 2 N HCl (50 µL) thiab concentrated hauv vacuo. Cov residue tau yaj hauv 300 µL ntawm MeOH. Txhua aliquot (5 µL) tau txais los ntawm hydrolyzate sib tov tau ncaj qha mus rau LC / MS (Phenomenex Luna C18, 4.6 × 100 mm, 3.5 µm, ntws tus nqi ntawm 0.3 mL / min), thiab tag nrho scan hauv qhov zoo thiab tsis zoo. ion hom (scan range ntawm m / z 100 txog 1000) tau siv los txheeb xyuas lub sijhawm khaws cia ntawm L-FDAA-derivatized amino acids.

Cov theem ntawm lub xov tooj ntawm tes, suav nrog formic acid hauv cov dej distilled ({{0}}}.1 feem pua ​​v/v) (A) thiab acetonitrile (B), tau nqa nrog cov kuab tshuaj gradient raws li hauv qab no: 20–40 feem pua ​​(B) rau 10 min, 100 feem pua ​​(B) isocratic rau 5 min, thiab ces 20 feem pua ​​(B) isocratic rau 5 min, mus ua ib tug tom qab-khiav cov txheej txheem ntxuav rau kab. Lub sijhawm khaws cia ntawm L-FDAA derivatized amino acids siv raws li cov qauv yog 16.7 min (L-Glu, m / z 400 [M ntxiv rau H] ntxiv), 18.2 min (D-Glu, m / z 400 [M ntxiv H] ntxiv ), 22.1 min (L-Val, m/z 370 [M ntxiv rau H] ntxiv), 25.1 min (D-Val, m/z 370 [M ntxiv rau H] ntxiv), 25.6 min (L-Phe, m/ z 418 [M plus H] plus ), 27.0 min (D-Phe, m/z 418 [M plus H] plus ), 25.5 min (L-Leu, m/z 384 [M plus H] plus ), thiab 28.2 min (D-Leu, m/z 384 [M plus H] plus ). Lub sijhawm khaws cia ntawm derivatized hydrolyzates ntawm 1-3 yog L-Glu (16.9 min), L-Phe (25.7 min), thiab L-Val (22.5 min) los ntawm 1; L-Glu (16.7 min), L-Phe (25.7 min), thiab L-Leu (25.6 min) los ntawm 2; thiab L-Glu (16.9 min) thiab L-Phe (25.7 min) los ntawm 3.

3.8. Cytotoxic Assays siv cov qog nqaij hlav cancer

MCF7, HeLa, thiab A549 hlwb tau yuav los ntawm American Type Culture Collection (Rockville, MD, USA). HepG2 hlwb tau yuav los ntawm Kauslim Cell Line Bank (Seoul, Kauslim). MCF7 hlwb tau coj los ua kab ke hauv Roswell Park Memorial Institute (RPMI) 1640 nruab nrab ntxiv nrog 10 feem pua ​​​​fetal bovine serum. HeLa thiab A549 hlwb tau coj mus rau hauv Dulbecco qhov nruab nrab qhov tseem ceeb tsawg (DMEM, Cellgro, Manassas, VA, USA) ntxiv nrog 10 feem pua ​​​​fetal bovine serum. Cov kab mob HepG2 tau coj mus rau hauv Yam Tsawg Tseem Ceeb Nruab Nrab ntxiv nrog 10 feem pua ​​​​fetal bovine serum. Cov kab lis kev cai tau khaws cia ntawm 37 ◦C hauv qhov chaw muaj av noo uas muaj 5 feem pua ​​​​CO2. Cytotoxicity tau sim ntawm plaub kab ntawm tib neeg (MCF7, HeLa, A549, thiab HepG2) siv EZ-CyTox cell viability assay kit (Dojindo Laboratories, Kumamoto, Nyiv). Luv luv, 5 × 103 cell /well tau noob nyob rau hauv 96-zoo daim hlau. Tom qab 24 teev, cov hlwb raug kho nrog cov tshuaj sib txuas ntawm qhov qhia tau hais tseg. Tom qab 72 teev ntawm incubation, EZ-CyTox cell viability assay kit tau siv. Tom qab 2 teev ntawm incubation, qhov nqus tau ntsuas ntawm 450 nm nrog kev siv ntawm 620 nm. Cell viability raug xam raws li feem pua ​​ntawm kev tswj.

3.9. Anti-Inflammatory Activity

Tus nas macrophage RAW264.7 xov tooj ntawm tes tau yuav los ntawm American Type Culture Collection. Cells tau coj mus rau hauv DMEM ntxiv nrog 10 feem pua ​​​​fetal bovine serum (FBS), 100 units / mL penicillin, thiab 100 µg / mL streptomycin, thiab incubated ntawm 37 ◦C nyob rau hauv ib tug humidified cua nrog 5 feem pua ​​CO2. Cell viability raug ntsuas siv Ez-Cytox cell viability detection kit. Cells tau incubated nyob rau hauv 96-cov phiaj zoo ntawm ib tug concentration ntawm 2500 hlwb ib qhov dej rau 24 teev thiab kho nrog cov qhia concentrations ntawm 1-3 rau 24 teev. Tom ntej no, Ez-Cytox reagents tau ntxiv rau txhua qhov dej. Qhov ceev ntawm qhov muag ntawm 450 nm tau ntsuas tom qab 1 teev siv lub microplate nyeem ntawv (PowerWave XS; Bio-Tek Instruments, Winooski, VT, USA) los kwv yees kev muaj peev xwm ntawm tes. Hauv ib qho kev sim cais, RAW264.7 hlwb tau tsim nyob rau hauv 96-cov phiaj zoo ntawm qhov concentration ntawm 30,{23}} hlwb rau ib qhov dej rau 24 teev. Tom qab cov ntshav qabzib tshaib plab rau 12 teev, cov hlwb raug pretreated nrog cov tebchaw 1-3 rau 1 h thiab tom qab ntawd txhawb nqa nrog 1 µg / mL ntawm LPS rau 24 teev. Kev nqus ntawm kab lis kev cai xov xwm hauv Griess cov tshuaj tiv thaiv tau ntsuas ntawm 540 nm siv PowerWave XS microplate nyeem ntawv los kwv yees qhov tsis muaj qib.

3.10. Kev ntsuas ntawm Melanin Cov ntsiab lus

Tus nas melanoma cell kab, B16F10, tau yuav los ntawm American Hom Kab lis kev cai sau. Cells tau coj mus rau hauv DMEM ntxiv nrog 10 feem pua ​​​​FBS, 100 units / mL penicillin, thiab 100 µg / mL streptomycin, thiab incubated ntawm 37 ◦C nyob rau hauv ib tug humidified cua nrog 5 feem pua ​​CO2. B16F10 hlwb raug incubated nyob rau hauv 6 cm kab lis kev cai tais diav ntawm 250, 000 hlwb ib qhov dej nyob rau hauv DMEM ntxiv nrog 10 feem pua ​​FBS, 100 µg/mL streptomycin, thiab 100 U/mL penicillin rau 24 teev. Cov hlwb raug ntxuav ob zaug nrog phosphate-buffered saline (PBS) thiab tom qab ntawd txhawb nqa los ntawm IBMX thiab incubated nrog ntau qhov sib txawv ntawm cov tshuaj 1-3 lossis kojic acid (zoo tswj) hauv phenol liab-dawb RPMI nruab nrab ntxiv nrog 10 feem pua ​​FBS, 100 units. / mL penicillin, thiab 100 µg / mL streptomycin rau 24 teev thiab 48 teev. Qhov nqus ntawm kab lis kev cai nruab nrab yog ntsuas ntawm 405 nm siv PowerWave XS microplate nyeem ntawv los kwv yees cov ntsiab lus melanin. Cov txiaj ntsig tau nthuav tawm raws li kev hloov pauv piv rau kev tswj hwm (cov hlwb tsis txhawb nqa los ntawm IBMX).

3.11. Kev txheeb cais

Tag nrho cov ntaub ntawv suav nrog cell viability, NO, thiab melanin productions tau nthuav tawm raws li tus nqi nruab nrab thiab tus qauv sib txawv (SD). Tag nrho cov kev ntsuam xyuas tau ua tiav hauv triplicate thiab rov ua dua tsawg kawg peb zaug. Nyob rau hauv txoj kev tshawb no, tsuas yog ob peb tus lej ntawm kev rov ua dua ntawm txhua qhov kev sim ntawm tes tau suav nrog, yog li txoj kev ntsuas tsis yog parametric tau raug coj los siv rau kev txheeb xyuas. Kev xeem Kruskall-Wallis tau siv rau kev txheeb xyuas kev txheeb xyuas ntawm txhua qhov sib txawv. Cov pob ntawv SPSS tau siv rau txhua qhov kev txheeb xyuas (International Business Machines Corporation (IBM) SPSS cov txheeb cais version 21, Boston, MA, USA). Kev txheeb xyuas qhov tseem ceeb tau txiav txim siab ntawm p-tus nqi qis dua 0.05.

4. Cov lus xaus

Peb daim ntawv tshaj tawm muab cov tshuaj ntsuam xyuas ntawm microbial cais tawm ntawm cov kab mob fungus-loj hlob termite. Peb qhov tshiab cyclic tripeptides, natalenamides A-C (1-3), raug cais tawm thiab cov qauv tsim los ntawm kab lis kev cai broth ntawm fungus-loj hlob -termite-associated Actinomadura sp. RB99 siv LC-UV / MS-based dereplication txoj kev. Tag nrho cov kev sib cais sib cais tau raug soj ntsuam rau cov dej num lom neeg siv cov kev ntsuam xyuas ntawm tes. Cov tshuaj 1 thiab 2 pom muaj zog cytotoxicity tawm tsam HepG2 thiab HeLa/A549 hlwb, raws li. Tsis muaj ib qho ntawm cov tshuaj sib cais tau pom muaj qhov cuam tshuam loj heev ntawm TSIS MUAJ ntau lawm hauv LPS-stimulated RAW264.7 hlwb. Compound 3 tau nthuav tawm cov teebmeem tseem ceeb ntawm IBMX-mediated melanin synthesis nyob rau hauv koob tshuaj. Cov nyhuv zoo ib yam li kojic acid, uas yog siv los ua cov khoom siv tshuaj pleev ib ce nrog cov teebmeem ntawm cov tawv nqaij dawb.

Kev lees paub:

Peb ua tsaug ntau rau Michael Thomas-Poulsen (Department of Biology, University of Copenhagen, Denmark) rau kev txhawb nqa peb txoj haujlwm.

Kev tsis sib haum xeeb ntawm kev txaus siab:

Cov kws sau ntawv tshaj tawm tsis muaj kev sib cav txog kev txaus siab.

Cov ntaub ntawv

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Qauv Muaj:

Cov qauv ntawm cov tebchaw tsis muaj los ntawm cov kws sau ntawv.

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