Abstract:Kev tshawb nrhiav Phytochemical ntawm Egyptian mandarin txiv kab ntxwv (Citrus reticulata Blanco, F. Rutaceae) cov noob tau them kaum peb paub cov tebchaw, 1-13. Cov qauv ntawm cov khoom sib cais tau raug muab siv 1D thiab 2D NMR thiab HRESIMS analyses. Txhawm rau ua tus yam ntxwv ntawm cov tshuaj pharmacological ntawm cov tebchaw no, ntau qhov kev sib xyaw ua ke virtual kev tshuaj ntsuam-raws li thiab molecular dynamics simulation-based thwmsim tau siv. Raws li qhov tshwm sim, cov tebchaw 2, 3, thiab 5 tau raug txheeb xyuas tias yog hyaluronidase, xanthine oxidase, thiab tyrosinase inhibitors. Kev sim tom qab hauv vitro tau ua tiav los ua pov thawj cov kev sim hauv silico-raws li qhia txog lub peev xwm ntawm cov flavonoids raws li kev cog lus hyaluronidase, xanthine oxidase, thiab tyrosinase inhibitors nrog IC50 qhov tseem ceeb xws li 6.39 ± 0.36 µM 23. . Txoj kev tshawb fawb tam sim no tau pom lub peev xwm ntawm Egyptian mandarin txiv kab ntxwv cov khoom pov tseg (piv txwv li, nws cov noob) ua ib tus neeg saib xyuas kev noj qab haus huv ntawm daim tawv nqaij. Tsis tas li ntawd, nws tau nthuav tawm qhov kev siv tau ntawm kev sib koom ua ke rov qab docking-raws li kev tshuaj ntsuam xyuas virtual thiab MDS-raws li kev sim hauv kev kwv yees lub peev xwm lom neeg ntawm cov khoom ntuj tsim.

Glycoside ntawm cistanche tuaj yeem ua rau muaj kev ua haujlwm ntawm SOD hauv plawv thiab daim siab cov ntaub so ntswg, thiab txo cov ntsiab lus ntawm lipofuscin thiab MDA hauv txhua cov ntaub so ntswg, tshem tawm ntau yam reactive oxygen radicals (OH-, H₂O₂, thiab lwm yam) thiab tiv thaiv DNA puas. los ntawm OH-radicals. Cistanche phenylethanoid glycosides muaj peev xwm tshem tawm cov dawb radicals, muaj peev xwm txo tau ntau dua li cov vitamin C, txhim kho cov haujlwm ntawm SOD hauv cov phev ncua, txo cov ntsiab lus ntawm MDA, thiab muaj qee yam kev tiv thaiv ntawm cov phev ua haujlwm. Cistanche polysaccharides tuaj yeem txhim kho kev ua haujlwm ntawm SOD thiab GSH-Px hauv erythrocytes thiab ntsws cov ntaub so ntswg ntawm cov nas senescent sim los ntawm D-galactose, nrog rau txo cov ntsiab lus ntawm MDA thiab collagen hauv lub ntsws thiab ntshav, thiab nce cov ntsiab lus ntawm elastin, muaj Cov nyhuv scavenging zoo ntawm DPPH, ncua lub sijhawm ntawm hypoxia hauv cov nas senescent, txhim kho cov haujlwm ntawm SOD hauv cov ntshav, thiab ncua lub physiological degeneration ntawm lub ntsws hauv kev sim cov nas nrog cellular morphological degeneration, kev sim tau pom tias Cistanche muaj peev xwm antioxidant zoo. thiab muaj peev xwm los ua ib qho tshuaj tiv thaiv thiab kho cov kab mob ntawm daim tawv nqaij laus. Nyob rau tib lub sijhawm, echinacoside hauv Cistanche muaj lub peev xwm tseem ceeb txhawm rau txhawm rau DPPH dawb radicals thiab muaj peev xwm tshem tawm cov pa oxygen reactive thiab tiv thaiv dawb radical-induced collagen degradation, thiab kuj muaj kev kho zoo ntawm thymine dawb radical anion puas.

Nyem rau Cistanche ntsiav tshuaj Cov txiaj ntsig

【Yog xav paub ntxiv: david.deng@wecistanche.com / WhatApp:86 13632399501 】

1. Taw qhia

Mandarins, C. reticulata, yog qee qhov zoo tshaj plaws citrus qoob loo rau kev siv tshiab [1]. Lawv nquag qhia ua 'tangerines;' Txawm li cas los xij, 'Mandarin' yog lub npe nrov dua. Ob peb hybrids thiab ntau yam ntawm mandarin hom yog faib nyob rau hauv xwm [1]. Cov hom nto moo tshaj plaws muaj xws li C. unshiu Marcovitch, C. nobilis Loureiro, C. deliciosa Tenore, thiab C. reticulata Blanco [1,2]. Mandarins yog ib qho tseem ceeb ntawm Citrus txiv hmab txiv ntoo sau nyob rau hauv ntau lub teb chaws. Txawm hais tias cov qoob loo feem ntau yog ua haujlwm rau khoom noj qab zib, lawv muaj kev lag luam tseem ceeb vim lawv cov roj tseem ceeb [3]. Citrus flavors yog siv rau hauv dej haus, confectionery, ncuav qab zib, thiab khoom qab zib [4]. Tsis tas li ntawd, C. reticulata tev yog siv rau flavorings ntawm cawv [4].

Inverse docking yog ib hom kev tshuaj ntsuam xyuas virtual uas tuaj yeem siv los ua kom pom tias muaj peev xwm molecular lub hom phiaj rau cov qauv sib xyaw. Yav dhau los, peb tau siv qhov kev tshuaj ntsuam xyuas zoo li no los txiav txim siab cov hom phiaj zoo tshaj plaws rau ntau cov khoom lag luam cais tawm [5]. Txawm li cas los xij, cov qhab nia docking uas tau tshwm sim los ntawm qhov kev tshuaj ntsuam ua ntej no tsuas yog muab qee cov ntaub ntawv hais txog cov qauv kev sib xyaw ua ke ntawm cov hom phiaj protein thiab cov lus nug cov qauv sib xyaw [6]. Raws li qhov no, cov kev sim molecular dynamics simulation (MDS) thwmsim, suav nrog kev ruaj khov tsis muaj zog (∆Gbinding) muab cov txiaj ntsig zoo dua thiab ntseeg siab uas tuaj yeem xaiv rau hauv vitro kuaj. Ntawm no, peb raug C. reticulata cov noob rau kev sib cais ntawm cov txheej txheem chromatographic kom tau qee lub tswv yim txog cov phytochemical loj hauv cov khoom pov tseg no. Tom qab ntawd, peb tau tshawb xyuas seb cov phytochemicals puas muaj qee yam tshuaj muaj txiaj ntsig los ntawm kev ua rau lawv mus rau ib kauj ruam hauv kev tshuaj xyuas silico-raws li tau pib los ntawm kev sib txuas lus sib txawv thiab xaus nrog ntau qhov kev sim MDS. Lub peev xwm ntawm bioactive phytochemicals muab tau los ntawm C. reticulata noob los ntawm lub peev xwm siab ntawm cov txiv hmab txiv ntoo uas siv tau cov khoom pov tseg raws li lub pas dej loj loj rau cov neeg ua haujlwm txhawb kev noj qab haus huv.

2. Cov ntaub ntawv thiab cov txheej txheem

2.1. Cov khoom cog

C. reticulata txiv hmab txiv ntoo tau sau thaum Lub Ib Hlis 2021 los ntawm ib cheeb tsam hauv cheeb tsam hauv Beni-Suef, tim lyiv teb chaws, thiab tau lees paub los ntawm tus kws tshaj lij botanist, Prof. Dr. Abd El-Halim A. Mohammed Department of Flora thiab Phytotaxonomy Research; Lub koom haum tshawb fawb Horticultural; Dokki; Cairo; Egypt). Ib qho piv txwv ntawm cov txiv hmab txiv ntoo tau khaws cia nyob rau hauv daim npav naj npawb 2021-BuPD 80 ntawm Pharmacognosy Department; Tsev Kawm Ntawv Pharmacy; University of Beni-Suef; Egypt).

2.2. Tshuaj thiab Reagents

Ethyl acetate (EtOAc), methanol (MeOH), n-Hexane (n-Hex), ethanol (EtOH), dichloromethane (DCM), thiab n-butanol (n-BuOH) tau txais los ntawm El-Nasr Company, Egypt. Sigma Aldrich, Saint Louis, MO, USA, muab cov kuab tshuaj deuterated rau NMR tsom xam nrog rau cov kuab tshuaj purity siab rau HPLC, spectroscopic, thiab chromatographic tsom xam (xws li, methanol-d4 (CD3OD), dimethyl sulfoxide-d6 (DMSO), chloroform. -d (CDCl3), methanol rau HPLC, HPLC-qib dej, thiab HPLC-qib acetonitrile). Rau chromatographic cais, Sephadex LH20 (0.25–0.1 mm, GE Healthcare, Sigma-Aldrich; CA, USA) thiab Silica gel (E-Merck, Sigma-Aldrich, CA, USA) tau siv. Rau lub tshuab nqus tsev kua chromatography, silica gel rau TLC (Tsev tshuaj thiab tshuaj; El-Nasr tuam txhab; Cairo, Egypt) tau siv (VLC). Pre-coated silica gel (E-Merck, Darmstadt, Lub teb chaws Yelemees) tau siv rau kev tshawb fawb TLC. Cov chaw tsim khoom raug txheeb xyuas los ntawm para anisaldehyde tom qab cua sov ntawm 110 ◦C [7]. Tag nrho cov ntaub ntawv rau kev tshawb nrhiav lom neeg los ntawm Sigma Chemical Company (SCC; CA, USA).

2.3. Spectral Analyzes

NMR signals tau txais ntawm 400 rau 1H-NMR thiab 100 MHz rau 13C-NMR. Lub CDCl3-d, CD3OD-d4, thiab DMSO-d6 hnyav peaks tau ua haujlwm raws li cov cim qhia. NMR teeb liab tau txais los ntawm kev siv NMR tshuab Bruker Advance-III 400 MHz (Bruker AG-Billerica, California, MA, USA).

Cov ntaub ntawv daws teeb meem siab tau sau tseg siv Acquity-Ultra-Performance-LiquidChromatography-tshuab txuas rau Quadrupole-Time-of-Flight-Hybrid-Mass-Spectrometer (Waters, Milford, MA, USA). Agilent/1260-Infinity-preparative-HPLC tau siv los ua kom tiav HPLC chromatographic sib cais.

2.4. Kev npaj ntawm C. reticulata Noob Extract

C. reticulata noob (1 kilogram) tau muas tshiab thiab tom qab ntawd cua qhuav hauv qhov ntxoov ntxoo. Tom qab ntawd, cov noob tau grated siv lub tshuab sib tsoo (Henan, Mainland Tuam Tshoj). Tom qab ntawd cov hmoov nplej tau muab tshem tawm kom huv si los ntawm 70 feem pua ​​EtOH (1 L × 5). Lub resulting kua extract yog concentrated siv ib tug Rotary Evaporator (Buchi-Rotavapor-R-300, ColeParmer, Vernon Hills, IL, USA) nyob rau hauv lub nqus tsev vacuum ntawm 45 ◦C los muab 100 g crude qhuav extract, uas yog ces muab faib siv ib tug series ntawm nce polarity solvents: n-Hex-DCMEtOAc-n-BuOH. Fractions I (24.0 g), II (5.0 g), III (3.0 g), thiab IV (6.0 g) tau txais los ntawm evaporating cov organic theem sib cais ntawm kev poob siab. Tag nrho cov khoom rho tawm thiab cov seem tau khaws cia ntawm 4 ◦C rau kev tshuaj xyuas phytochemical thiab biochemical tom qab.

2.5. Kev rho tawm ntawm C. reticulata Noob 'Major Natural Products 

TLC-silica gel kem tau siv los nthuav tawm feem I (20 g) rau VLC li qub. EtOAc thiab n-Hex successive elution (0–100% , 5% nce) tau siv. Peb ntu ntu (I1–I3) tau txais los ntawm kev sib sau cov feem zoo ib yam thiab evaporating lawv nyob rau hauv kev txo qis. Nyob rau lwm kem silica gel, subfraction I1 (1.0 g) yog feem ntau fractionated. Chromatographic elution tau ua los ntawm n-Hex rau EtOAc, gradient mixes (0–10 feem pua ​​, 1 feem pua ​​​​nce, 100 mL txhua) los muab cov compound 13. (10 mg).

Sub-fraction I2 (50 mg) tau ntxiv chromatographed ntawm silica-gel-60 (100 × 1 cm, 20 g). Cov txheej txheem chromatographic tau ua tiav siv DCM: MeOH, isocratic sib tov (0.5 feem pua ​​, 500 mL) los muab cov tshuaj 8 (20 mg) thiab 9 (15 mg). Sub-fraction I3 yog me ntsis fractionated ntawm silica gel 60 (100 × 1 cm, 20 g). Elution ua hauj lwm DCM: MeOH, isocratic sib tov (0.5 feem pua, 500 mL) kom them taus 10 (20 mg).

TLC silica-gel kem tau siv los nthuav tawm feem II (2.5 g). DCM: EtOAc (0–100 feem pua, nce 5 feem pua) tau siv los hais cov qauv. Cov kua dej tsim tau tau txais hauv 250 mL qhov chaw. Cov feem zoo tib yam tau txais thiab txo mus rau ob ntu ntu (II1–II2) ntawm kev txo qis. Nyob rau lwm kem silica gel, subfraction I1 (1.0 g) yog feem ntau fractionated. Cov tshuaj 5 tau eluted siv DCM: EtOAc (0-10 feem pua, 1 feem pua ​​​​nce, 100 mL txhua) (10 mg).

Sub-fraction I2 (50 mg) tau ntxiv chromatographed ntawm silica gel60 (100 × 1 cm, 20 g). Chromatographic elution tau siv los ntawm kev siv DCM rau MeOH, (isocratic elution; 0.5 feem pua ​​, 500 mL) los muab cov tshuaj 11 (20 mg) thiab 12 (15 mg).

Ntawm VLC ib txwm, feem IV (6.0 g) tau sib cais siv silica gel. DCM: EtOAc (0–100 feem pua ​​, 5 feem pua ​​​​nce, 250 mL txhua) tau siv los qhia cov qauv. Chromatographic effluent tau txais nyob rau hauv 250 mL increments.

Cov feem zoo tib yam tau muab faib ua pawg thiab txo qis hauv kev txo qis kom tawm tau tsib feem (IV1–V5); sub-fractions IV1, IV2, IV3, thiab IV4 tsuas yog me ntsis chromatographed ntawm Sephadex-LH20 kem eluted nrog MeOH kom yield 4 (30 mg), 6 (50 mg), 7 (70 mg), thiab 1 (15 mg, Raws li txoj cai, thaum lub sij hawm ntu ntu IV5 yog qhov muaj txiaj ntsig zoo (10 mg).

2.6. Hauv Silico Study

2.6.1. Inverse Docking

Qhov yuav tshwm sim molecular lub hom phiaj rau cov flavonoids sib cais tau xaus los ntawm kev muab lawv cov qauv mus rau qhov kev tshuaj ntsuam virtual nyob ntawm qhov kev sib hloov docking tawm tsam yuav luag txhua cov protein uas tau muab tawm hauv Protein-Data-Bank; PDB.

IdTarget online-based platform tau ua haujlwm rau cov kauj ruam no. Cov txheej txheem virtual paltry kev tshuaj ntsuam xyuas no siv ib txoj hauv kev rov qab docking, uas yog DivideConquer-Docking, uas tsim cov kev sib tshooj sib tshooj, ua rau cov protein tshawb nrhiav kev txwv ntau dua. Yog li ntawd, nws tuaj yeem ua tau ntau qhov chaw nres nkoj hauv lub sijhawm luv luv [8]. Cov qhab nia tshwm sim tau piav qhia raws li cov npe ntawm cov kev ntaus nqi xws li qhov phem tshaj plaws mus rau qhov zoo tshaj plaws. Txhawm rau txheeb xyuas cov txiaj ntsig zoo tshaj plaws rau txhua tus qauv, peb siv tus qhab nia ntawm −10 kcal / mol raws li tus nqi txiav tawm (Table 1).

Molecular dynamics simulation (MDS) thwmsim tau ua raws li yav dhau los qhia los ntawm Alhadrami li al. 2021 [6]. Lub tshuab MDS ntawm Maestro software (ie, Desmond v. 2.2; Hamburg, Lub teb chaws Yelemees) tau ua haujlwm rau kev ua haujlwm MDS thwmsim [9–11]. OPLS-Force-Field tau siv rau kev simulation khiav.

Kev npaj cov protein-structures tau ua los ntawm System-Builder algorithm, uas lawv tau ntim rau hauv lub thawv dej orthorhombic (20 Å3) siv cov qauv dej TIP3P ua ke nrog Na plus , thiab Cl- ions, {{ 16}}.15 M rau txhua tus ntawm lawv. Tom qab ntawd, cov qauv ua qauv zoo tshaj plaws tau raug rau qhov sib npaug ua ntej rau 10 ns. Ligand cov qauv tau txiav txim siab thaum lub sijhawm npaj cov qauv raws li OPLSForce-Field. Cov hlau uas muaj cov protein zoo li MMPs uas koom nrog histidine-Zn ntxiv rau 2 txoj hauv kev ua haujlwm zoo yuav tsum raug ntsuas thaum lub sijhawm tsim cov protein. Txhawm rau mus, lub xeev hetero yuav tsum tau teeb tsa rau hetero atoms zoo li Zn (Tsim Hetero States). Txoj haujlwm no yog ib feem ntawm maestro's Protein Formation wizard. Cov kauj ruam no yuav pab txhawb txoj kev loj hlob ntawm lub xeev hetero los yog koom ua ke covalent lub xeev, rau lub heteroatom (ie, Zn ntxiv 2) nyob rau hauv lub network nrog cov protein kom lub zog teb xws li OPLS tuaj yeem soj ntsuam cov zinc atom sai. Rau kev luam tawm los ntawm Nano-scale Molecular Dynamics software (NAMD 3.0) [12], lub topologies thiab tsis ntawm cov qauv qauv tau txiav txim siab los ntawm online platform Ligand Reader thiab Modeler. Harmonic Tcl rog tau ua haujlwm los tuav Zn ntxiv 2 hauv qhov chaw.

Kev suav ntawm kev khi dawb zog (∆Gbinding) tau ua raws li tau piav qhia dhau los los ntawm Alhadrami li al. 2021 [6]. ∆ Kev sib tsoo tau ua los ntawm cov txheej txheem pub dawb zog perturbation (FEP) [12], uas tau piav qhia hauv Kim et al cov ntawv tshaj tawm. Nyob rau hauv ib tug nutshell, cov txheej txheem no xam cov uas yuav tsum tau siv tau lub zog ∆Gbinding siv cov kab zauv ∆Gbinding=∆GComplex − ∆GLigand. Tus nqi ntawm txhua ∆ Sib tsoo yog suav nrog NAMD program hauv kev sim cais.

Txhua cov ntaub ntawv tawm tswv yim tuaj yeem npaj tau ntawm lub vev xaib CharmmGUI. Cov ntaub ntawv tau raug siv los tsim cov kev npaj ∆Gbinding suav nrog siv FEP raws tu qauv hauv NAMD. Qhov sib npaug tom qab tau teeb tsa hauv NPT (300 K, 1 atm, Langevin-Piston-Pressure "Complex"-"Ligand") nyob rau hauv lub neej ntawm TIP3P qauv dej. Tom qab ntawd, 10 ns FEP simulations tau tsim rau txhua tus qauv, thiab qhov kawg 5 ns ntawm cov nyiaj muaj zog tau suav rau cov nqi hluav taws xob ua tiav [12]. Thaum kawg, qhov kev sib txuas ua ke tau xav txog thiab ntsuas los ntawm kev ua haujlwm VMD program [13].

2.7. Hauv Vitro Assay ntawm Hyaluronidase Kev Ua Haujlwm

Cov kev ua ntawm hyaluronidase inhibitory tau tsim raws li cov txheej txheem tsim ua ntej tso cai rau Chaiyana li al. 2019 [14]. Cov nyhuv inhibitory ntawm hyaluronidase kev ua haujlwm tau kwv yees los ntawm qhov sib npaug hauv qab no: hyaluronidase inhibition (%)=(1 − A / B) × 100, qhov twg A thiab B yog cov cim hyaluronic acid tom qab kev kho mob hauv lub neej lossis tsis muaj. tus qauv, cais. Raws li kev tswj hwm zoo, 6-O-palmitoyl-Lascorbic acid tau siv. Qhov kev sim tau ua tiav hauv triplicate.

2.8. Hauv Vitro Assay ntawm Xanthine Oxidase Kev Ua Haujlwm

Xanthine oxidase (XO) inhibitory muaj peev xwm tau ua tiav raws li txoj kev tau piav qhia yav dhau los [15]. Cov xim uas tsim los ntawm hydrogen peroxide tso tawm thaum lub sij hawm oxidation ntawm XO yog ntsuas ntawm 570 nm siv lub phaj nyeem EPOCH ™ "MICROPLATE READER (BIOTEK)". Plaub caug-plaub microliters ntawm xanthine oxidase tsis yog tov nrog 2 µL ntawm xanthine substrate tov thiab 2 µL ntawm XO. L-mimosine tau siv los ua kev tswj kom zoo. Qhov kev sim no tau ua hauv triplicate.

2.9. Hauv Vitro Assay ntawm Tyrosinase Kev Ua Haujlwm

Cov kev ua ntawm tyrosinase inhibitory ntawm txhua flavonoid tau pom los ntawm kev siv DOPAchrome txheej txheem ua raws li lub tswv yim ntawm Momtaz li al. 2008 [16], uas tau hloov kho los ntawm Nerya li al. 2003 [17], thiab Curto et al. Xyoo 1999 [18]. Lub tyrosinase inhibitory kev ua haujlwm tau ntsuas los ntawm qhov sib npaug hauv qab no: inhibitory kev ua haujlwm (%)=(1 − A / B) × 100, (9) qhov twg A thiab B yog qhov nqus ntawm qhov sib tov nyob rau hauv lub xub ntiag lossis tsis muaj. kuaj flavonoid, feem. Txhua qhov kev sim tau ua tiav hauv triplicate.

3. Cov txiaj ntsig thiab kev sib tham

3.1. Phytochemical Kev Tshawb Fawb ntawm C. reticulata Noob

Siv UV, 1H, thiab DEPT-Q NMR, ntxiv rau kev sib piv nrog cov ntaub ntawv thiab siv qee cov qauv qhabnias, cov paub cov tshuaj kaempferol 1 [19], kaempferol-40 -}O- -Dglucopyranoside 2 [19], kaempferol -7-O- -D-glucopyranoside 3 [19], 2-hydroxy genistein 4 [19], hesperetin 5 [19], 3000 (1000-O- - D-glucopyranosyl)-sucrose 6 [20], 6(1000 -O- -Dfructofuranosy)-30 (1"-O- -}D-glucopyranosyl)-sucrose 7 [20 ], 1-O-elaidoyl-glycerol 8 [21], 1- O-oleoyl-2-palmitoyl-glycerol 9 [22], 1-O-oleoyl-glycerol 10 [ 21], (E)-3-((4-methyl heptyl oxy) carbonyl)acrylic acid 11 [23], (E)-3-(6-methyl nonyl oxy) carbonyl)acrylic acid 12 [23] thiab -sitosterol 13 [24] (Daim duab 1) tau txheeb xyuas los ntawm crude ethanolic extract ntawm C. reticulata noob. Daim duab 1 thiab S1–S28). Nws yog ib qho tsim nyog sau cia tias, tsis zoo li cov flavanones thiab flavones nyob rau hauv C. reticulata aerial qhov chaw, nrog rau cov txiv hmab txiv ntoo, flavonols tau tshwm sim yog hom flavonoids nyob rau hauv cov noob.

3.2. Hauv Kev Tshawb Fawb Silico

Txhawm rau ua kom muaj kev ua haujlwm lom neeg ntawm cov tshuaj sib cais, peb tau muab lawv rau hauv cov kev tshuaj ntsuam hauv-silico-based. Ua ntej, peb tau tsom mus rau cov flavonoids cais (1–5) rau peb qhov kev tshuaj ntsuam xyuas thawj zaug vim tias flavonoids yog cov paub zoo txog bioactive cog theem nrab metabolites. Tsis tas li ntawd, cov khoom sib cais tseem tshuav (6-13) yog cov metabolites tseem ceeb uas yuav tsis muaj txiaj ntsig kho.

Cov qauv ntawm flavonoids 1-5 tau raug kuaj xyuas raws li kev sib tw virtual tawm tsam yuav luag txhua cov protein uas tuav hauv PDB. Lub platform online, uas yog idTarget tau ua haujlwm rau qhov kev tshuaj ntsuam xyuas virtual ua ntej no [8]. Cov qhab nias rov qab tau muab rov qab ua ib daim ntawv teev npe los ntawm tus nqi tsis zoo tshaj plaws mus rau qhov tsawg tshaj plaws. Peb tau teeb tsa qhov qhab nia ntawm −10 kcal / mol raws li tus nqi txiav tawm los ua ib lub hom phiaj zoo rau txhua qhov sib cais flavonoid (1-5). Ob peb lub hom phiaj zoo molecular tau pom tias yog lub hom phiaj zoo rau cov khoom sib txuas, suav nrog cov protein kinases thiab synthases (Table 1).

Interestingly, peb lub hom phiaj molecular tau tshaj tawm ntawm cov flavonoids, hyaluronidase, xanthine oxidase, thiab tyrosinase enzymes. Cov enzymes no muaj feem xyuam rau kev tswj xyuas cov tawv nqaij noj qab haus huv [25–30], thiab yog li qhov kev tshuaj ntsuam xyuas ua ntej ua ntej tau txheeb xyuas cov flavonoids no ua cov tshuaj tiv thaiv tawv nqaij.

3.3. Molecular Dynamics Simulation thiab Kev Sib Txuas Tsis Muaj Kev Sib Tw Dawb Zog

Txhawm rau ntxiv dag zog rau kev soj ntsuam ua ntej docking-based, peb kwv yees qhov kev sib khi tsis muaj zog (∆Gbinding) ntawm cov tebchaw 1-5 nrog hyaluronidase, xanthine oxidase, thiab tyrosinase enzymes siv MDS-dependent txoj kev, uas yog txoj kev pub dawb lub zog perturbation ( FEP) [26,31].

Raws li pom nyob rau hauv Table 2, tsuas yog cov tebchaw 2, 3, thiab 5 muaj peev xwm ua kom tiav qhov siab tshaj plaws affinity ntawm hyaluronidase, xanthine oxidase, thiab tyrosinase enzymes nrog ∆ Sib tsoo cov nqi xws li −8.1 txog −9.9 kcal / mol. Cov khoom sib txuas ntxiv tau txais ∆ Gbinding tus nqi siab dua -6 kcal / mol.

Ntxiv 50 ns MDS cov kev sim tau ua los tshawb xyuas cov kev sib cuam tshuam thiab kev ruaj ntseg ntawm cov tebchaw 2, 3, thiab 5 nrog hyaluronidase, xanthine oxidase, thiab tyrosinase enzymes. Raws li tau piav nyob rau hauv daim duab 2, ob qho tib si compound 3 thiab cov co-crystalized ligand ua tiav zoo sib xws ruaj khov nyob rau hauv lub enzyme txoj kev khi qhov chaw thoob plaws hauv MDS nrog RMSD ntawm ~ 2.8 Å los ntawm qhov pib docking poses. Compound 2 0 s RMSDs kuj zoo li cov compound 3 txog 28.7 ns thaum nws pib deviate me ntsis siab dua los ntawm compound 3 thiab cov co-crystalized ligand kom ua tiav qhov nruab nrab RMSD ntawm 3.8 Å thoob plaws hauv MDS. Qhov kawg snapshot muab los ntawm MDS ntawm cov tebchaw 2, thiab 3 thiab cov cocrystallized ligand qhia tias TYR-75, ASP-129, thiab GLU-131 yog cov sib cuam tshuam cov amino acid residues ntawm H- kev sib raug zoo.

Hais txog xanthine oxidase, compound 3 tau ua tiav ruaj khov nyob hauv nws qhov chaw nquag thoob plaws hauv MDS, kaw qhov nruab nrab RMSD ntawm 2.6 Å uas tau sib koom ua ke ntawm cov co-crystalized inhibitor (RMSD ~ 2.4 Å). Xws li kev ruaj khov ruaj khov ntawm compound 3 tau pom tseeb vim yog H-bonds network uas nws tsim nrog LYS-771, PHE-798, GLU-802, SER-876, ARG{{ 12}}, PHE-911, ARG-912 and THR-1010 (Figure 3).

Thaum kawg, compound 5 kuj tseem nyob ruaj khov hauv tyrosinase qhov chaw ua haujlwm thoob plaws 50 ns MDS nrog qhov nruab nrab RMSD tus nqi ntawm 4.9 Å, uas yog siab dua li ntawm co-crystalized inhibitor (RMSD ~ 3.8 Å). Raws li pom nyob rau hauv lub xeem MDS-los ntawm snapshot, compound 5 muaj peev xwm cuam tshuam nrog ob Zn2 ntxiv ions zoo ib yam li cov co-crystalized inhibitor. Tsis tas li ntawd, nws tau ruaj khov nrog ntau H-bonds nrog LYS-198, GLY-209, TYR-362, ARG-374, thiab THR-391 (Daim duab 4 ).

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