Tuliposides H-J Thiab Bioactive Cheebtsam Los Ntawm Lub Teeb Ntawm Amana Edulis
Mar 20, 2023
Abstract:
Peb qhov tshiab tulip sab H-J (1-3) thiab 11 paub cov tebchaw tau txais los ntawm cov tshuaj methanolic ntawm cov qij ntawm Amana edulis thawj zaug. Lawv cov qauv tau piav qhia los ntawm NMR, MS, thiab IR spectroscopic cov ntaub ntawv, kev sib hloov kho qhov muag, thiab Mosher txoj kev. Cov khoom melanogenesis ntawm tag nrho cov kev sib cais tau raug soj ntsuam hauv B16 melanoma hlwb. Yog li ntawd, tributyl citrate (9) muaj cov tshuaj tiv thaiv melanogenesis tab sis yog cytotoxic rau B16. (ntxiv )-Pyroglutamic acid (4), (ntxiv )-butyl 5-oxo pyrrolidine-2-carboxylate (6), (–)-3-hydroxy-2-methyl butyrolactone (10 ), thiab 5- (hydroxymethyl) furfural (12) tau nce melanin ntau lawm thiab tyrosinase kev ua ub no. Cov khoom xyaw nquag tuaj yeem kawm ntxiv raws li cov neeg sib tw tiv thaiv melanoma thiab vitiligo rau cov kab mob ntawm daim tawv nqaij los yog whitening/hypopigmentation rau cov plaub hau.
Hauv kev tshawb fawb whitening, peb pom tias Cistanche kuj muaj cov nyhuv whitening
Ua ntej tshaj plaws, Cistanche yog nplua nuj nyob rau hauv polysaccharides thiab flavonoids. Cov tebchaw no ua raws li cov tshuaj antioxidants thiab tuaj yeem pab lub cev tshem tawm cov dawb radicals, ua kom cov tawv nqaij qeeb qeeb thiab yog li txhim kho cov tawv nqaij.
Thib ob, Cistanche kuj muaj ntau yam amino acids, minerals, thiab vitamins, uas muaj txiaj ntsig zoo rau kev kho tawv nqaij thiab rov tsim dua tshiab. Cistanche tuaj yeem txhawb cov metabolism hauv cov hlwb ntawm daim tawv nqaij, txhim kho tus kheej lub peev xwm ntawm daim tawv nqaij, thiab kho cov tawv nqaij kom muaj xim zoo.
Thaum kawg, Cistanche kuj muaj qee cov organic acids thiab alkaline tshuaj, thiab cov khoom xyaw no kuj muaj qee yam cuam tshuam rau kev txias thiab metabolism ntawm daim tawv nqaij cuticles. Cov tshuaj no tuaj yeem sib npaug pH tus nqi ntawm daim tawv nqaij, txhim kho lub thickness ntawm stratum corneum, thiab ua rau daim tawv nqaij ntau translucent thiab muag heev.
1. Taw qhia
Amana edulis (A. edulis; Miq.) Honda, syn. Tulipa edulis (Miq.) Baker belongs rau tsev neeg Liliaceae thiab yog ib tug pej xeem tshuaj nroj tsuag siv los kho cov kab mob cancer [1,2]. Txawm li cas los xij, muaj qee qhov kev tshawb fawb phytochemical thiab lom ntawm cov tsiaj no tau tshaj tawm txog niaj hnub no.
Piv txwv li, 95 feem pua thiab 50 feem pua ntawm EtOH rho tawm tuaj yeem ua rau apoptosis hauv tib neeg lub plab (SGC7901) [1] thiab hepatoma (BEL7404, HepG2, thiab Huh7) [2] carcinoma cells, raws li. Cov polysaccharides npaj los ntawm A. edulis muaj cov khoom tiv thaiv oxidant, suav nrog DPPH, OH-, thiab ABTS ntxiv rau kev ua ub no [3–5]. Hauv kev tshawb fawb ntawm cov neeg ua haujlwm tshiab los ntawm cov khoom ntuj tsim rau cov tawv nqaij tsis zoo, peb pom tias MeOH extracts ntawm qhov muag teev ntawm A. edulis qhia txog cov kev cai melanogenesis uas tseem tsis tau tshawb fawb. Thaum raug hluav taws xob ultraviolet ntawm lub hnub ci los yog cov tshuaj tsis zoo thiab cov kab mob pathogenic, cov melanogenesis nruab nrab tuaj yeem tiv thaiv peb cov tawv nqaij los ntawm reactive oxygen hom tsim nyob rau hauv keratinocytes thiab melanocytes [6]. Tyrosinase yog ib qho enzyme tseem ceeb hauv melanogenesis los tsim ntau melanin rau peb los tiv thaiv cov xwm txheej uas tau hais los saum no.
Yog li ntawd, cov khoom xyaw nquag ntawm A. edulis tsim nyog yuav raug tshem tawm rau kev siv tshuaj kho mob ntxiv. Hauv qhov kev tshawb nrhiav no, peb qhov tshiab tulip sab H–J (1–3) thiab 11 paub cov tebchaw (4–14) (Daim duab 1) raug cais tawm ntawm A. edulis thiab cov txheej txheem txheeb xyuas los ntawm NMR, MS, thiab IR spectroscopic cov ntaub ntawv, kev sib hloov kho qhov muag. , los yog Mosher ester cov tshuaj tiv thaiv. Compound 9 tuaj yeem txo cov ntsiab lus melanin tab sis yog cytotoxic rau B16 melanoma hlwb. Cov tshuaj 4, 6, 10, thiab 12 tau nce melanin ntau lawm thiab tyrosinase kev ua ub no. Zuag qhia tag nrho, txoj haujlwm no tau ua pov thawj tias A. edulis thiab nws cov khoom nquag tuaj yeem yog cov neeg ua haujlwm tshiab rau cov kab mob melanin, xws li melanoma thiab hypopigmentation (dawb tawv vitiligo lossis cov plaub hau ntxuav).
2. Cov txiaj ntsig thiab kev sib tham
2.1. Structure Elucidations of Isolates 1–3
Lub MeOH extract (TEM) ntawm qhov muag teev ntawm A. edulis tau muab faib ua EtOAc-soluble(TEE), n-BuOH-soluble (TEB), thiab HO-soluble (TEW) feem cais. Qhov feem. qhov kev txiav txim ntawm TEE thiab TEB rho tawm tau them nyiaj tshiab isoprenoid glycosides 1-3 thiab paub cov tebchaw 4-14 (Daim duab 1). Tsis tas li ntawd, cov khoom tseem ceeb ntawm TEW extract yog carbohydrates.
Lub HRESIMS (high-resolution electrospray ionization mass spectrometry) ntawm 1 qhia ib (M - H- ion ntawm m/z 351.1652, qhia ib tug molecular mis ntawm CsH2gOg (calcd forC; 5HoOg; 351.1655) thiab ob degrees ntawm unsaturation. pom kev nqus rau hydroxyl (3376 cm-l) thiab carbonyl (1725 cm-l) functionalities. Kaum tsib cov paib carbon, suav nrog ob methyls, tsib methylene, xya methines, thiab ib qho quaternary carbon, tau pom nyob rau hauv ib-dimensional (1D) NMR. Spectra ntawm 1 (Table 1). Cov quaternary carbon tau txheeb xyuas tias yog carbonyl carbon raws li kev hloov tshuaj lom neeg 6c 176.6. Lub 1D thiab HSOC (heteronuclear ib leeg quantum coherence) NMR spectra tso tawm ib qho anomeric ({22}}/8c: 4.27 (d, J=8.0 Hz)/1{{30}}4.9), tsib oxymetholone (0h/8c: 3.21(t, J { {34}}. } Hz)/77.9, thiab 3.89 (td, I=7.0, 2.5 Hz)/73.6), thiab peb oxymethylene (6,/6c: 3.57 (dd, J {{64}) }.5, 7.0 Hz)4.01 (dd, I {{70}}.5, 2.5 Hz)/73.1, 3.66 (dd, I {78}}.0, 5.0 Hz), 3.85 (brd, I=12.0 Hz)/62.7and 4.10 (2H, t, J=7.5 Hz)/65.5) cov cim. HMBC (heteronuclear ntau daim ntawv cog lus sib raug zoo) kev sib raug zoo (Daim duab 2) ntawm compound 1 ntawm methine protons H-2 (8 2.67, quint nrog C-1 (6 176. 6)/C-3 ({104}}.6)/C{106}} ({107}}.1)/C{109}} (6 13.9) , H-3 ({113}}.89, td) nrog C-1/C-2({117}}.6)/C-4/C -5, H-1' (6 4.27, d) nrog C-4, thiab cov methyl protons H-5 (8 1.14 , d) nrog C-1/C{129}}/C{130}} qhia tias cov methyl thiab hydroxyl moieties tau muab tso rau ntawm C-2 thiab C{132}}, ntsig txog Ntxiv mus, cov methylene protons ntawm H,-1" (8 4.10, t) thiab H-4 (6 3.57dd; 4.01, dd) nthuav tawm 3 kev cuam tshuam nrog C{{ 142}} thiab C-1' (8 104.9), feem, nyob rau hauv HMBCspectrum (Daim duab 2) uas txhawb nqa cov haujlwm ntawm pawg n-butyl ntawm C-1 thiab ib qho beta -glucose (H{150}}', 8 4.27, d, J=8.0 Hz) ntawm C-4 8].
Daim duab 2 qhia qhov tseem ceeb COZY thiab HMBC kev sib raug zoo rau 1. Cov txheeb ze configurations ntawm C-2 thiab C-3 (3-hydroxy-2- methyl moiety) ntawm 1 tuaj yeem txiav txim siab tiv thaiv -form los ntawm 3 JH2-H3 tus nqi ntawm 7.0 Hz (anti-form: 7.0 Hz; syn-form: 4.0 Hz) [ 9]. Txhawm rau txiav txim siab qhov kev teeb tsa tsis raug, cov tshuaj sib xyaw 1 raug kho nrog (R)- thiab (S)- -methoxy- -(trifluoromethyl)-phenyl acetyl chloride [(R)- thiab (S)-MTPA- Cl] nyob rau hauv lub xub ntiag ntawm pyridine-d5 kom tau cov (S)- thiab (R)-MTPA esters (1a thiab 1b), feem. MTPA esters tau tsim ua tiav ntawm C-3 thiab C-20/C-30/C-40, raws li tau piav qhia los ntawm 1H NMR thiab 1H-1H COZY spectra (1a, H-3, δ 5.82, m; H-20 , δ 5.70, t, J=9.5 Hz; H-30 , δ 4.39, t, J=9.5 Hz; H-40 , δ 5.76, t, J=9.5 Hz; 1b, H-3, δ 5.81, m; H{{64} }} , δ 5.56, t, J=9.5 Hz; H-30 , δ 4.12, t, J=9.5 Hz; H-40 , δ 5.66, t, J=9.5 Hz). Qhov sib txawv ntawm 1H NMR tshuaj hloov pauv rau 1a thiab 1b (∆ qhov tseem ceeb hauv daim duab 3) tau ua rau cov haujlwm ntawm S- thiab R-configurations ntawm C-3 thiab C-2, feem. Yog li ntawd, compound 1 tau elucidated li butyl 4- -D-glucopyranose-(S)-3-hydroxy-(R)-2-methyl butanoate thiab hu ua tulip sab H.
Compound 2, tulip side I, muab cov mis molecular, C15H28O8, tsim los ntawm HRESIMS (m/z 359.1686 [M ntxiv Na] ntxiv, calcd rau 359.1682). Lub 1D NMR spectra ntawm 2 kuj zoo ib yam li cov 1, tshwj tsis yog qhov tseeb tias cov tshuaj hloov pauv ntawm methylene teeb liab δH 1.65 (sext, J=7.0 Hz) thiab 1.96 (sext, J=7. Raws li cov ntaub ntawv COZY thiab HMBC (Daim duab 2), cov khoom sib xyaw 2 tau muab tso ua ke los ntawm cov isoprenoid, ib qho qabzib, thiab pawg n-butyl ib yam nkaus. HMBC 3 J kev sib raug zoo ntawm H2-1" (δ 4.04, 2H, t, J=7.5 Hz/δC 65.4) nrog C-1 (δ 178.5) thiab H-10 (δ 4.19, d, J=8.0 Hz/δC 104.4) nrog C-4 (δ 68.4) qhia tias pawg n-butyl thiab ib-glucose moiety [8] tau txuas ntawm C-1 thiab C-4, raws li (Daim duab 2). Cov kev sib raug zoo ntawm HMBC thiab COZY yog qhia hauv daim duab 2. R-configuration ntawm H3-5 ntawm C-2 ntawm 2 tau txiav txim siab los ntawm acid hydrolysis ntawm 2 los muab cov sib xyaw tulipalin compound, 3-methyldihydrofuran-2(3H)-ib qho (Daim duab S28) [8] nrog kev sib hloov kho qhov muag zoo tus nqi ([ ] 23 D plus 18.7, CH2Cl2) (R: [ ] 25 D plus 14.7, CHCl3) [10]. 81}}methyl butanoate.
Cov mis mos molecular ntawm 3 raug txiav tawm raws li C11H20O8 vim qhov pom ntawm [M ntxiv Na] ntxiv ion ntawm m/z 303.1049 (calcd rau 303.1050) hauv HRESIMS. Kaum ib cov paib carbon, suav nrog ib qho methyl (δ 17.6), peb methylene (δ 34.7, 62.8, thiab 68.6), rau methines (δ 37.6, 71.6, 75.1, 77.9, 78.0, thiab 104.4), thiab ib qho quaternary. , tau pom nyob rau hauv 1D NMR spectra ntawm 3 (Table 1). Cov pa roj carbon quaternary tau pom tias yog carbonyl carbon raws li cov pa roj carbon monoxide hloov ntawm δ 180.6. Tsis tas li ntawd, compound 3 tau pom cov ntaub ntawv zoo sib xws rau ntawm 2, tshwj tsis yog cov cim n-butyl. Hauv HMBC spectrum (Daim duab S15), cov anomeric proton ntawm δ 4.23 (d, J=8.0 Hz) nthuav tawm 3 J cuam tshuam nrog C-4 (δ 68.6) ua rau -glucose nyob ntawm C-4. Cov kev sib txuas tseem ceeb HMBC thiab COZY tau pom nyob rau hauv daim duab 2. Compound 3 tsis raug cais nyob rau hauv kom muaj nuj nqis txaus los txiav txim siab qhov tseeb ntawm C-2. Thaum kawg, tulip sab J (3), 4- -D-glucopyranose-(R)-2-methyl butanoic acid, tau txheeb xyuas vim yog cov qauv kev qhia saum toj no ntawm 1 thiab 2 hauv txoj kev tshawb no.
Cov tshuaj 1–3 yog tulip sab, ib yam khoom tshwj xeeb muaj xws li 4-hydroxy2-methylene butanoic acid (HMBA) thiab/los yog (3S)-3,4-dihydroxy -2-methylene butanoic acid (DHMBA) pawg acylated ntawm C-1 thiab/los yog C-6 txoj haujlwm ntawm -D-glucose (Glc) feem ntau pom nyob rau hauv genus Tulipa [8,11] . Txog tam sim no, tulip sab analogs, suav nrog 1-tulip sab A, 6-tulip sab A, 1-tulip sab B, 6-tulip sab B, thiab tulip sab D-G , tau raug cais tawm ntawm Tulipa [8,11]. Txawm li cas los xij, cov qauv ntawm tulip sab C tsis tau tshaj tawm thiab tuaj yeem ploj mus hauv cov ntaub ntawv dhau los [11]. Tulipalins, xws li tulipalin A thiab (–)-tulipalin B, yog cov aglycone feem ntawm tulip sab, uas spontaneously lactones tom qab tso HMBA thiab/los yog DHMBA pawg los ntawm tulip sab-hloov enzymes [8,11]. Hauv txoj kev tshawb no, cov tebchaw 1–3 lub npe hu ua tulip sab H-J yog nyob rau sab tulip, tab sis lawv ob daim ntawv cog lus ntawm C-2 raug txo thiab -D-glucose pawg tau txuas rau C-4 (oxymethylene ), tsis yog C-1 (O-acyl functionality) hauv HMBA lossis DHMBA (Daim duab 1). Tsis tas li ntawd, compound 10 (2S,3S) yog tulipalin tab sis tsis tuaj yeem metabolized los ntawm 1 (2R,3S) vim muaj qhov sib txawv ntawm C-2. Qhov ua tau biosyntheses ntawm tulip sab 1-3 muaj nyob rau hauv daim duab S29 [8,11].
Tsis tas li ntawd, 11 lub tebchaw paub suav nrog peb cov pyroglutamic acid analogs (4–6), peb citric acid derivatives (7–9), ob furanone (10–11), ib furan (12), thiab ob steroids (13–14) yog cais tawm ntawm A. edulis thawj zaug. Cov tshuaj 1–9 thiab 10–14 tau los ntawm TEB thiab TEE crude feem, feem. Lawv tau txheeb xyuas raws li (ntxiv rau )-pyroglutamic acid (4) [12], (ntxiv )-methyl 5-oxo pyrrolidine-2-carboxylate (5) [12], (ntxiv)- butyl {{23} }}oxo pyrrolidine-2-carboxylate (6) [12], 1-butyl citrate (7) [13], 1,10 -dibutyl 5-methyl citrate (8) [ 13], tributyl citrate (9) [13], (–)-3-hydroxy-2-methyl butyrolactone (10) [14–16], (–)- methyl 3-hydroxy{{ 44}}oxo tetrahydrofuran-3-carboxylate (11) [17], 5-(hydroxymethyl) furfural (12) [18], sitosterol (13) [19], thiab sitosterol-3- -D -glucose (14) [20] los ntawm cov ntaub ntawv spectroscopic thiab piv nrog cov ntaub ntawv.
2.2. Chemical Components Elucidation of TEW
1H thiab 13C NMR spectra ntawm TEW, ib qho dej-soluble crude feem ntawm TEM extract, pom zoo heev rau cov carbohydrates (Figures S16 thiab S17). Polysaccharides npaj los ntawm hom no tau tshaj tawm tias muaj rhamnose, xylose, arabinose, galactose, mannose, qabzib, thiab fructose tom qab kev tshuaj xyuas monosaccharide [3,8]. Cov ntaub ntawv NMR spectroscopic thiab TLC (thin-txheej chromatography) khaws cia qhov tseem ceeb ntawm TEW piv nrog cov qauv suab thaj (Figures S18-S23) qhia D-glucose thiab D-fructose yog cov khoom tseem ceeb hauv TEW extracts.
2.3. Cov teebmeem ntawm Extracts thiab cais tawm ntawm Melanogenesis
Lub MeOH extract (TEM) ntawm cov tsiaj no tau muab cais ua TEE, TEB, thiab TEW crude feem, thiab cov lus hais plaub tau raug soj ntsuam rau cytotoxicity thiab melanogenesis cuam tshuam hauv B16 melanoma hlwb ntawm qhov ntau ntawm 12.5 txog 200 µg / mL (Daim duab S24) . TEE tau pom tseeb cytotoxicity rau B16 ntawm 200 µg/mL thiab TEB nrog rau TEW muaj cov haujlwm cytotoxic ntawm qhov siab ntawm 12.5 txog 200 µg/mL (Daim duab S24A-1, B-1, C{13} }, D-1 ib. Hauv bioassay, -melanocyte-stimulating hormone (-MSH) tau siv los ua kom B16 hlwb los ua ke ntau melanin, thiab TEM tuaj yeem txhawb nqa melanogenesis (Daim duab S24A-2).
Feem ntau ntawm tag nrho cov kev cais tawm (Daim duab 1), tsis suav nrog 13 thiab 14, tau soj ntsuam ntxiv rau kev tswj hwm ntawm cov teebmeem ntawm melanogenesis ntawm qhov siab ntawm 10 mus rau 40 µM, nrog arbutin siv los tswj qhov zoo (Daim duab 4). Raws li pom nyob rau hauv daim duab 4, compound 9 pom tseeb thiab koob tshuaj-raws li inhibited melanin ntau lawm uas tshwm sim los ntawm cytotoxicity mus rau B16 hlwb nrog ib tug IC50 (ib nrab maximal inhibitory concentration) tus nqi ntawm 81.9 µM (Daim duab S25). Kev cais tawm 4, 6, 10, thiab 12 nce melanin cov ntsiab lus koob tshuaj-nyob ntawm qhov siab tshaj plaws ntawm 11.9 feem pua, 29.8 feem pua, 27.2 feem pua, thiab 17.6 feem pua, raws li, ntawm 40 µM uas tsis muaj cellular toxicity. Tyrosinase plays lub luag haujlwm tseem ceeb hauv thawj ob kauj ruam ntawm melanogenesis [6]; Yog li ntawd, cov teebmeem ntawm cov tebchaw 4, 6, 10, thiab 12 ntawm intracellular tyrosinase tau soj ntsuam ntxiv. -MSH nce cov tyrosinase kev ua si mus txog 254.3-305.5 feem pua thaum piv nrog pawg tswj hwm (100 feem pua) (Daim duab 5 thiab Table S1).
Yog li ntawd, 4 (14.1–21.9 feem pua), 6 (15.8–21.9 feem pua), 10 (8.5–13.1 feem pua), thiab 12 (7.5–11.8 feem pua) nce kev ua haujlwm ntawm cov enzyme, tyrosinase, nyob rau hauv koob tshuaj. ntawm qhov siab ntawm 10 mus rau 40 µM, thaum piv nrog -MSH pawg (Daim duab 5 thiab Table S1). Ib daim duab sib raug zoo ntawm cov cellular melanin cov ntsiab lus thiab cov teebmeem ntawm tyrosinase ntawm cov tebchaw 4, 6, 10, thiab 12 yog qhia hauv daim duab S26. Tsis tas li ntawd, txhawm rau paub meej txog cov teebmeem melanogenesis ntawm 4, 6, 10, thiab 12, txhua qhov sib xyaw tau ntxiv rau hauv B16 hlwb yam tsis muaj kev sib koom ua ke -MSH uas tau siv los ua kev tswj xyuas zoo hauv qhov kev ntsuam xyuas no (Daim duab S27). Yog li ntawd, cov tib neeg 4, 6, 10, thiab 12 tsis tau nce melanin ntau lawm ib leeg (Daim duab S27) zoo ib yam li cov uas tau pom hauv daim duab 4. Nws tau pom tias plaub lub tebchaw uas tau hais los saum toj no tuaj yeem txhim kho cov teebmeem ntawm melanogenesis ntawm -MSH. Yog li, ntxiv cov teeb liab txoj hauv kev hauv melanogenesis, xws li tyrosinase-related protein-1 (TRP-1), TRP-2, microphthalmia-associated transcription factor, melanocortin 1 receptor, cyclic adenosine monophosphate, protein. kinase A, cAMPresponse element-binding protein, c-Jun N-terminal kinases, extracellular signal-regulated kinase, p38, thiab phosphoinositide 3-kinase/protein kinase B [7,21], yuav tsum tau kuaj ntxiv thiab lees paub rau cov active Cheebtsam.
3. Cov ntaub ntawv thiab cov txheej txheem
3.1. Cov txheej txheem kev sim dav dav
NMR spectra tau ntsuas ntawm Bruker Avance Ill 500 MHz ntsuas (BrukerBillerica, America) rau 1H thiab 125 MHz rau 13C-NMR. Chemical hloov (0) qhov tseem ceeb nyob rau hauv ppm, thiab coupling tas li () nyob rau hauv Hz nrog CD; OD, CDCl, thiab / lossis D, O tau siv los ua cov kuab tshuaj. Kev sib hloov kho qhov muag tau txais ntawm JASCO-P-2000 polarimeter (JASCO, TokyoJapan) (cell length 10 mm). IR spectra raug kaw rau ntawm PerkinElmer Spectrum TwoFT-IR spectrometer (PerkinElmer, Waltham, MA, USA). Tsawg- thiab high-resolution ESISelectrospray ionization huab hwm coj spectrometry) tau ntsuas ntawm Bruker Daltonics EsquireHCT ultra siab muaj peev xwm ntxiab huab hwm coj spectrometer thiab Orbitrap huab hwm coj spectrometer (LIOOrbitrap XL, Thermo Fisher Scientific), feem. TLC tau ua tiav ntawm Kieselgel60 F254 (0.25 mm; Merck) thiab / lossis RP-18 F254S (0.25 mm; Merck), coated daim hlau thiab tom qab ntawd stained los ntawm txau nrog 5 feem pua (o/u) sulfuric acid nyob rau hauv ib tug MeOH tov thiab cua sov rau ntawm lub phaj kub. Silica gel (Silicycle: 70 230 thiab 230 400 mesh), RP-18 (LiChroprepe 4063 um: Merck. Sephadexn C20 CE episode 0 pe qhib w Bre thov foSupelcoTM, Bellefonte), thiab MCI CHP20P (SupelcoTcolumn chromatography. A Shimadzu LC-20AT twj tso kua mis thiab Shimadzu RID-10Ib lub ntsuas ntsuas qhov ntsuas tsis pom tseeb (Shimadzu Inc, Kyoto, Nyiv), nrog rau Cosmosil 5C18-MS-II ( 250 x10 mm id, 5 um) kem ntawm tus nqi ntws ntawm 2.0 mL / min, tau siv rau HPLC. Cov qab zib reagents suav nrog D-glucose (MP Biomedicals, LLC, Illkirch, Fabkis) thiab D-fructose (TCI, Tokyoapan) yog siv rau cov dej-soluble crude feem (TEW) tsom xam.
3.2. Khoom siv cog
Cov qij qhuav ntawm A. edulis (yav tas los T. edulis) tau yuav los ntawm ib lub tsev muag tshuaj suav tshuaj hauv Taichung, Taiwan, thaum lub Cuaj Hlis 2018 thiab txheeb xyuas los ntawm tus kws sau ntawv Prof. Chang. Daim ntawv voucher (TE201809) tau muab tso rau ntawm Tuam Tshoj Tshuaj Tshawb Fawb thiab Txhim Kho Chaw, CMUH Taiwan.
3.3. Extraction thiab rho tawm
Lub qhov muag teev ntawm A. edulis (5.0 kg) tau muab rho tawm yim zaug nrog MeOH (8.0 L txhua) nyob rau hauv chav tsev kub kom tau txais cov khoom nyoos extract. MeOH extract (TEM, 525.0 g) tau muab faib peb zaug ntawm ethyl acetate (EtOAc) thiab H2O (15{11}}0: 1500, v/v) los muab EtOAc -soluble feem (TEE, 45.0 g) thiab ib theem aqueous, uas tau ntxiv partitioned nrog n-BuOH/H2O (1500:1500, v/v × 3), thiab ces muab cais ua n-BuOH-soluble (TEB, 53.6 g. ) thiab H2O-soluble (TEW, 410.0 g) feem.
TEE tau raug qhib kab chromatography ntawm silica gel ({{0}}}063–{11}}.200 mm, kab: 7 × 26 cm, txoj kab uas hla × ntev), siv gradients ntawm hexane–EtOAc–MeOH (3{{20}}}:1:0; 1:1:0; { {67}}:0:1, v/v/v) thiab muab 14 subfractions (TEE1–TEE14). Precipitate 14 (249.0 mg; cais tawm tawm: 0.{{100}}0498 feem pua ) tau los ntawm subfraction TEE12 (2.2 g) tau lim thiab ntxuav nrog MeOH. TEE9 (3.6 g) tau muab faib ua xya subfractions (TEE9-1 rau 9-7) los ntawm silica gel (kem: 5 × 24 cm; CH2Cl2–MeOH, 7:1; 1:1; 0:1 , v/v), nrog subfraction TEE9-4 (1.1 g), thiab tom qab ntawd raug rau Sephadex LH-20 kem chromatography (kem: 5 × 54 cm; CH2Cl2–MeOH, 1: 1 txog 0: 1, v/v), muab xya subfractions (TEE9-4-1 rau 9-4-7). TEE9- 4-5 thiab TEE9-4-6 tau ua ke (tag nrho 551.7 mg) thiab rov ua kom huv los ntawm RP-HPLC (MeOH–H2O, 45:55; 20:80, v/v) kom muab 12 (21.0 mg. ; tR=13 min; cais tawm tawm: 0.00042 feem pua ) thiab niam cawv (131.2 mg), uas tau ntxiv rau RP-HPLC (MeOH–H2O; 10:90, v/v) kom muab cov tshuaj 10 ( 35.5 mg; tR=13 min; rho tawm tawm los: 0.00071 feem pua ) thiab 11 (1.2 mg; tR=15 min; cais tawm tawm: 0.000024 feem pua). Fraction TEE6 (2.7 g) raug cais los ntawm Sephadex LH-20 (kem: 2.5 × 45 cm; CH2Cl2–MeOH, 1: 1; 0: 1, v / v) kom tau 13 (6.8 mg; cais tawm tawm: 0.000136 feem pua).
TEB tau chromatographed hla Diaion HP{{0}} kem (6 × 60 cm; H2O–MeOH– acetone, 100:{{15} }:0; 25:75:{18}}; 50:5{{2{26}}}}:0; 75:25:{{41} }}; 0:10{{70}}:{{9{96}}}}; {{1{1{111}}5} }0}}:0:100, v/v/v) muab 6 subfractions (TEB1–TEB6). Subfraction TEB5 (502. } min; cais tawm: 0.000186 feem pua) thiab 9 (144.2 mg; tR {{4{{2{2{2{218}} }9}}3}}}} min; cais tawm: 0.002884 feem pua ). Fraction TEB3 (5.5 g) raug rau RP-18 chromatography (kem: 7 × 25 cm; MeOH–H2O, 1:1; 1:0, v/v), thiab subfraction TEB3-6 ( 1.0 g) tau cais ntxiv los ntawm Sephadex LH-20 (kem: 5 × 54 cm; MeOH) kom tau txais xya qhov feem cuam tshuam (TEB3-6-1 rau 3-6-7). TEB3-6-4 (377.1 mg) tau cais los ntawm MCI gel chromatography (kem: 2.5 × 23 cm; H2O–MeOH, 100:0; 80:20; 60:40; 40:60; 20:80; 0: 100, v/v) muab xya subfractions (TEB3-6-4-1 rau 3-6-4-7). TEB3-6-4-4 (38.1 mg) tau purified los ntawm RP-HPLC (MeOH–H2O, 45:55 hauv 0.1 feem pua formic acid, v/v) kom them tau cov ntshiab 1 (12.2 mg; tR=17 min; Kev rho tawm tawm los: 0.000244 feem pua). Tsis tas li ntawd, TEB3-6-4-6 (31.0 mg) tau ua nrog RP-HPLC (MeOH–H2O, 40:60 hauv 0.1 feem pua formic acid, v/v) kom tawm los 6 (7.1 mg; tR=28 min ; cais tawm tawm: 0.000142 feem pua ). TEB3-6-5 (410.7 mg) raug cais los ntawm MCI gel chromatography (kem: 2.5 × 23 cm; H2O–MeOH, 100:0; 80:20; 60:40; 50:50; 40:60; 30: 70; 20:80; 0:100, v/v) kom muab yim feem (TEB3-6-5-1 rau 3-6-5-8). TEB3-6-5-3 (75.1 mg) yog purified los ntawm MCI gel chromatography (kem: 1 × 20 cm; H2O–MeOH, 80:20; 70:30; 0:100, v/v) kom them tau cov tebchaw 4 (59.3 mg; rho tawm tawm los: 0.001186 feem pua ) thiab 5 (10.2 mg; cais tawm: 0.000204 feem pua). Subfraction TEB3-6-5-4 (97.0 mg) yog purified los ntawm RP-HPLC (MeOH–H2O, 40:60 hauv 0.1 feem pua formic acid, v/v) kom tau 7 (46.4 mg; tR=18 min; Kev rho tawm tawm: 0.000928 feem pua). TEB3-10 (925.4 mg) tau chromatographed hla MCI gel chromatography (kem: 2.5 × 28 cm; H2O–MeOH, 100:0; 80:20; 60:40; 50:50; 40:60; 30 :70; 10:90; 0:100, v/v) muab yim subfractions (TEB3-10-1 rau 3-10-8) thiab ntshiab 2 (393.4 mg; cais tawm tawm: 0.007868 feem pua). Compound 3 (6.3 mg; tR=8 min; kev rho tawm tawm: 0.000126 feem pua) tau los ntawm TEB3-10-2 (20.9 mg) los ntawm RP-HPLC purification (MeOH–H2O, 30:70 hauv 0.1 feem pua formic acid, v/v).
3.3.1. Tuliposide H (1)
[ ] 23 D −23.1 (c 0.12, MeOH); IR (zoo) νmax 3376 (OH), 2960, 2934, 2875 (CH), 1725 (C=O), 1640, 1589, 1459, 1382, 1259, 1167, 10415 (C{10}}O), C), 926, 900, 632, 521 cm-1; 1H thiab 13C NMR spectroscopic cov ntaub ntawv, saib Table 1; HRESIMS m/z 351.1652 [M − H]− (calcd for C15H27O9, 351.1655).
3.3.2. Tuliposide II (2)
[ ] 23 D −25.8 (c 0.2, MeOH); IR (zoo) νmax 3404 (OH), 2961, 2934, 2876 (CH), 1729 (C=O), 1638, 1461, 1378, 1277, 1185, 1165, 1077 (C) C), 897, 736, 625, 517 cm-1; 1H thiab 13C NMR spectroscopic cov ntaub ntawv, saib Table 1; HRESIMS m/z 359.1686 [M plus Na] plus (calcd for C15H28O8Na, 359.1682).
3.3.3. Tuliposide J (3)
[ ] 23 D plus 9.0 (c 0.1, MeOH); IR (neat) νmax 3424 (OH), 2953, 2924, 2853 (CH), 1740 (C=O), 1632, 1441, 1401 (C–O–C), 1284, 1205, 1182, 1 , 1078, 1039, 893, 768, 721 cm-1; 1H thiab 13C NMR spectroscopic cov ntaub ntawv, saib Table 1; HRESIMS m/z 303.1049 [M plus Na] plus (calcd for C11H20O8Na, 303.1050).
3.4. (R)- thiab (S)-MTPA Derivatives ntawm 1
Kev npaj ntawm (S)-MTPA ester derivative ntawm 1 tau ua los ntawm txoj kev yooj yim Mosher ester [22,23]. 1 (3.4 mg, 0). Tsis tas li ntawd, C5D5N ({{20}}.5 mL) thiab (R)-(−)- -methoxy- -(trifluoromethyl)-phenyl-acetyl chloride (MTPA chloride, 12.2 mg, 0.05 mmole) tau ntxiv tam sim ntawd rau hauv lub kaw NMR raj uas txuas ntxiv co ua tib zoo sib xyaw cov qauv thiab MTPA chloride. Lub 1H NMR thiab 1H-1H COZY spectra ntawm qhov sib tov tom qab reacting rau 24 teev ntawm chav tsev kub tau sau tseg. Tus (R)-MTPA ester ntawm 1 kuj tau npaj zoo sib xws los ntawm cov txheej txheem hais saum toj no. Compound 1: 1H NMR (C5D5N, 500 MHz) δ 0.78 (H{35}}, t), 1.26 (H-300, sext), 1.28 (H3-5, d), 1.52 ( H-200, quint), 3.08 (H-2, quint), 3.95 (H-50, m), 4.03 (H-4a, dd), 4.05 (H -20, sib tshooj), 4.15 (H-100, t), 4.23 (H-30, sib tshooj), 4.23 (H-40, sib tshooj), 4.36 (H{{ 68}}a, dd), 4.39 (H-3, sib tshooj), 4.43 (H-4b, dd), 4.53 (H-60b, d), thiab 4.95 (H -10, d).
3.4.1. (S)-MTPA Ester ntawm 1 (1a)
1H NMR (C5D5N, 500 MHz) δ 0.77 (H-400, t), 1.17 (H-300, sext), 1.21 (H3-5, d) , 1.37 (H-200, quint), 3.18 (H-2, quint), 3.92 (H-100, t), 3.97 (H{24}}a, dd), 4.33 (H-50 , m), 4.39 (H-30 , t), 4.49 (H-4b, brd), 4.64 (H-60a, dd), 4.96 (H{39}} , d), 5.05 (H-60b, d), 5.70 (H-20 , t), 5.76 (H-40 , t), thiab 5.82 (H-3, m).
3.4.2. (R)-MTPA Ester ntawm 1 (1b)
1H NMR (C5D5N, 500 MHz) δ 0.79 (H-400, t), 1.26 (H-300, sext), 1.30 (H3-5, d) , 1.50 (H-200, quint), 3.33 (H-2, quint), 3.60 (H-50 , m), 3.94 (H{24}}a, dd), 4.11 (H-100, t), 4.12 (H-30 , t), 4.13 (H-60a, dd), 4.50 (H-4b, brd), 4.70 (H-10 , d), 4.85 (H-60b, d), 5.56 (H-20 , t), 5.66 (H-40 , t), thiab 5.81 (H-3, m).
3.5. Acid Hydrolysis ntawm Tuliposide I (2)
Isolate 2 (78.6 mg) tau hydrolyzed hauv 1 M HCl (1.5 mL) ntawm 100 ◦C rau 2 h [8]. Tom qab txias, cov tshuaj tiv thaiv sib tov tau muab rho tawm nrog CH2Cl2 (2.0 mL × 3) los muab cov tshuaj tulipalin analog, 3-methyldihydrofuran-2(3H)-one (11.1 mg). [ ] 23 D ntxiv rau 18.7 (c 1.1, CH2Cl2); 1H NMR (CDCl3, 400 MHz) δ 1.30 (3H, d, J=7.0 Hz), 1.94 (m), 2.45 (m), 2.61 (m), 4.20 (ddd, J {{43}) }.8, 6.8, 6.4 Hz), 4.33 (ddd, J=8.8, 8.8, 2.8 Hz). 13C NMR (CDCl3, 100 MHz) δ 15.2, 30.7, 34.1, 66.2, 180.1 (Daim duab S28); ESIMS m/z 101.06 [M ntxiv rau H] ntxiv .
3.6. Cell Culture
Lub murine B16 melanoma hlwb tau coj los ua kab ke hauv Dulbecco's Modified Eagle Medium (DMEM; GIBCO Invitrogen corporation, New York, NY, USA) ntxiv nrog 10 feem pua fetal bovine serum, 100 U / mL ntawm penicillin, thiab 100 µg / mL ntawm streptom. ◦C hauv 5 feem pua CO2 incubator.
3.7. Kev ntsuas ntawm B16 Cell Viability
Lub cytotoxicity ntawm B16 hlwb rau cov tshuaj ntsuam xyuas tau ntsuas los ntawm MTT nrog cov txheej txheem piav qhia yav dhau los nrog kev hloov kho me ntsis [6]. Cov noob tau cog rau hauv 96-cov phiaj zoo (1 × 103 lub hlwb / lub qhov dej) thiab coj mus kuaj rau 24 teev ua ntej raug kho nrog cov tshuaj sib xyaw rau lwm 72 teev. Tom qab ntawd, qhov nruab nrab raug tshem tawm, 100 µL ntawm MTT reagent (Thermo Scientific, Waltham, MA, USA) (ntawm qhov kawg concentration ntawm 0.5 mg / mL) ntxiv rau txhua qhov dej thiab tom qab ntawd incubated ntawm 37 ◦C rau 1 h. Lub formazan siv lead ua tau yaj nyob rau hauv 100 µL DMSO, thiab qhov muag pom qhov muag tau ntsuas los ntawm kev siv lub microplate nyeem ntawv (SPECTORstar® Nano, BMG LABTECH, Ortenberg, Lub teb chaws Yelemees) ntawm 570 nm.
3.8. Kev ntsuas melanin ntsiab lus hauv B16 Cells
Cov ntsiab lus melanin hauv B16 hlwb raug soj ntsuam raws li ib txoj kev piav qhia yav dhau los nrog kev hloov kho me ntsis [6]. Cov noob tau cog rau ntawm qhov ntom ntawm 5 × 103 lub hlwb / qhov dej hauv 24-cov phiaj zoo rau 24 teev. Qhov nruab nrab tau hloov los ntawm 500 µL tshiab kab lis kev cai nruab nrab uas muaj 0.5 µM -MSH nrog los yog tsis muaj tebchaw rau 72 h. Arbutin (1 mM) tau siv los ua kev tswj xyuas zoo. Tom qab ntawd, qhov nruab nrab raug tshem tawm thiab ntxuav nrog PBS (pH 6.8) ob zaug. Cov hlwb raug sau los ntawm NaOH (200 µL, 2 N) thiab tom qab ntawd rhaub ntawm 85 ◦C rau 30 min. Tom qab txias rau hauv chav tsev kub thiab raug centrifuged, qhov nqus tau ntsuas ntawm 405 nm siv tus nyeem ntawv microplate.
3.9. Intracellular Tyrosinase Kev Ua Haujlwm
Intracellular tyrosinase kev ua ub no tau ua los ntawm kev hloov kho me ntsis, uas tau tshaj tawm yav dhau los [6]. Cov noob tau cog rau hauv 24-cov phiaj zoo ntawm qhov ntom ntawm 5 × 103 lub hlwb / qhov dej rau 24 teev. Cov hlwb raug kho hauv DMEM uas muaj 0.5 µM -MSH nrog lossis tsis muaj tshuaj sib xyaw rau 72 teev. Tom qab tshem tawm qhov nruab nrab, cov hlwb raug ntxuav nrog txias PBS (pH 6.8) ob zaug. Cov hlwb raug lysed nrog 100 µL lysis buffer (1 feem pua triton X-100 hauv PBS) thiab khov ntawm −80 ◦C rau 15 min.
Tom qab ntawd, cov cell lysates tau centrifuged ntawm 13,200 × g ntawm 4 ◦C rau 30 feeb kom tau txais cov supernatant. Tag nrho cov ntsiab lus protein ntawm supernatant tau ntsuas los ntawm BCA protein assay. Tom ntej no, txhua qhov muaj pes tsawg leeg lysate (30 µg / 90 µL) tau sib xyaw nrog 10 µL ntawm L-DOPA (15 mM) hauv 96- cov phaj zoo thiab tom qab ntawd incubated ntawm 37 ◦C rau 1 h, thiab ntsuas qhov nqus tau. ntawm 405 nm siv lub microplate nyeem ntawv.
4. Cov lus xaus
Zuag qhia tag nrho, 14 cov tshuaj dawb huv, suav nrog peb lub isoprenoid glycosides tshiab, thiab tulip sab H–J (1–3), raug cais tawm ntawm A. edulis. Citric acid derivative 9, tributyl citrate, muaj kev ua haujlwm tiv thaiv melanogenesis tab sis pom cytotoxicity rau B16 melanoma hlwb uas tuaj yeem tshawb fawb ntxiv rau cov tshuaj tiv thaiv melanoma. Thaum pyroglutamic acid analogs 4 thiab 6, furanone 10, thiab furan 12 muaj koob tshuaj-raws li nce melanin cov ntsiab lus thiab activated tyrosinase uas tuaj yeem kawm ntxiv rau cov kab mob vitiligo ntawm daim tawv nqaij los yog cov tshuaj tiv thaiv dawb thiab tiv thaiv cov plaub hau. Txawm li cas los xij, cov txheej txheem hauv vitro thiab / lossis hauv vivo cov kev tshawb fawb yuav tsum tau tshawb xyuas kom ntxaws ntxiv txhawm rau txheeb xyuas qhov ua tau zoo ntawm cov khoom siv.
Cov khoom siv ntxiv:
Cov nram qab no muaj nyob online, Daim duab S1–S5: NMR spectral cov ntaub ntawv ntawm compound 1, Figures S6–S10: NMR spectral cov ntaub ntawv ntawm compound 2, Figures S11–S15: NMR spectral cov ntaub ntawv ntawm compound 3, Figures S16–S22: NMR spectral cov ntaub ntawv ntawm TEW, Daim duab S23: TLC tsom xam ntawm TEW, Daim duab S24: Cytotoxicity thiab cov kev cai ntawm melanogenesis teebmeem ntawm TEM, TEE, TEB, thiab TEW, Daim duab S25: Cytotoxicity cov ntaub ntawv ntawm cov tebchaw 1-12, Daim duab S26: Ib tug correlation graph ntawm lub cellular melanin cov ntsiab lus thiab cov teebmeem ntawm tyrosinase ntawm cov tebchaw 4, 6, 10, thiab 12, Daim duab S27: Melanogenesis teebmeem ntawm cov tebchaw 4, 6, 10, thiab 12 ib leeg yam tsis muaj -MSH, Daim duab S28: NMR spectral thiab optical rotation data of the Cov khoom tau los ntawm acid hydrolysis ntawm compound 2, Daim duab S29: Muaj peev xwm biosynthesis ntawm cov tebchaw 1-3, Table S1: Tyrosinase kev ua ub no los ntawm cov tebchaw 4, 6, 10, thiab 12.
Tus sau kev koom tes:
Conceptualization, C.-LL; cog cov ntaub ntawv txheeb xyuas, Y.-SC; kev coj tus kheej thiab kev huv huv, C.-LL thiab Z.-AG; kev coj ntawm bioassays, Y.-LJ; tag nrho spectral tsom xam thiab cov qauv txiav txim, C.-LL thiab C.-JC; kev sau ntawv ntawm thawj daim ntawv npaj, C.-LL Txhua tus kws sau ntawv tau nyeem thiab pom zoo rau cov ntawv luam tawm ntawm cov ntawv sau.
Nyiaj txiag:
Qhov kev tshawb fawb no tau txais nyiaj los ntawm Ministry of Science thiab Technology (Feem ntau 108-2320-B039-035-) thiab Tuam Tshoj Medical University (CMU108-MF-84), Taiwan. APC tau txais nyiaj los ntawm "Suav Tshuaj Tshawb Fawb Chaw, Tuam Tshoj Medical University" los ntawm Qhov Chaw Tshawb Fawb Qhov Chaw Tshawb Fawb nyob rau hauv lub moj khaum ntawm Kev Kawm Qib Siab Sprout Project los ntawm Ministry of Education (MOE) hauv Taiwan (CMRC-CHM{{5} }).
Institutional Review Board Statement:
Tsis siv tau.
Cov Lus Qhia Txog Kev Pom Zoo:
Tsis siv tau.
Cov ntaub ntawv muaj nyob:
Tsis siv tau.
Kev lees paub:
Txoj haujlwm no tau txais kev txhawb nqa nyiaj txiag los ntawm Ministry of Science thiab Technology (Feem ntau 108-2320-B-039-035-) thiab Tuam Tshoj Medical University (CMU108-MF-84), Taiwan, thiab lub "Suav Tshuaj Tshawb Fawb Chaw, Tuam Tshoj Medical University" los ntawm Qhov Chaw Tshawb Fawb Qhov Chaw Tshawb Fawb nyob rau hauv lub moj khaum ntawm Kev Kawm Qib Siab Sprout Project los ntawm Ministry of Education (MOE) hauv Taiwan (CMRC-CHM-1) tau muab rau C .-LL
Kev tsis sib haum xeeb ntawm kev txaus siab:
Cov kws sau ntawv tshaj tawm tsis muaj kev sib cav txog kev txaus siab.
Qauv Muaj:
Cov piv txwv ntawm cov tebchaw 1-14 muaj los ntawm cov kws sau ntawv.
Cov ntaub ntawv
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1 Department of Cosmeceutics, Tuam Tshoj Medical University, Taichung 406040, Taiwan.
2 Suav Tshuaj Tshawb Fawb thiab Kev Txhim Kho Center, Tuam Tshoj Medical University Tsev Kho Mob, Taichung 40402, Taiwan.
3 Suav Tshuaj Tshawb Fawb Chaw, Tuam Tshoj Medical University, Taichung 40402, Taiwan.
4 Department of Suav Pharmaceutical Sciences thiab Suav Tshuaj Kho Mob, Tuam Tshoj Medical University, Taichung 40402, Taiwan.
5 Graduate Institute of Integrated Medicine, Tuam Tshoj Medical University, Taichung 40402, Taiwan.
6 Proteomics Core Laboratory, Department of Medical Research, Tuam Tshoj Medical University Tsev Kho Mob, Taichung 40402, Taiwan.
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