Caffeoylquinic Acids Los ntawm Lepisorus Thunbergianus Thiab Lawv Melanogenesis Inhibitory Activity

Hu rau: Audrey Hu Whatsapp / hp: 0086 13880143964 Email:audrey.hu@wecistanche.com

Hak Hyun Kim, Jae Kwon Kim, Jaehyun Kim, Se-Hui Jung,* and Kooyeon Lee*

TSEEM CEEB:Hyperpigmentation tshwm sim los ntawm overactivation ntawm tyrosinase ua rau tsaus me ntsis los yog thaj ua rau thaj ntawm tib neeg tawv nqaij. Txawm hais tias cov xwm txheej no tsis muaj teeb meem, tseem muaj qhov xav tau zoo rau melanogenesis inhibitors los tiv thaiv hyperpigmentation los ntawm inhibiting tyrosinase, tus nqi txwv enzyme hauv melanogenesis. Txawm hais tias Lepisorus thunbergianus tau siv hauv kev kho mob pej xeem raws li diuretic thiab hemostatic tus neeg sawv cev, nws cov nyhuv ntawm melanogenesis tseem tsis tau tshaj tawm. Hauv txoj kev tshawb no, peb tau npaj ib qho L.thunbergianus extract thiab nws cov kuab tshuaj feem ntau thiab ntsuas lawv cov kev ua haujlwm lom neeg tawm tsam dawb radical thiab melanin synthesis. Kev rho tawm ntawm L. thunbergianus inhibited nceb tyrosinase kev ua tau zoo dua li, thiab nrog cov tshuaj antioxidant zoo sib xws rau, arbutin hauv vitro. Kev sib piv kev soj ntsuam ntawm cov tshuaj tiv thaiv melanogenesis thiab kev ua haujlwm los tiv thaiv tyrosinase ntawm L. thunbergianus hnyav fractions pom tau hais tias, los ntawm inhibiting tyrosinase kev ua, butanol feem muaj peev xwm tshaj plaws rau inhibition ntawm melanogenesisin melanoma hlwb. Peb pom los ntawm kev txheeb xyuas cov qauv siv cov kua chromatography (HPLC) thiab NMR spectroscopy tias cov ntsiab lus tseem ceeb hauv butanol feem yog peb cov caffeoylquinic acid derivatives. Peb qhov derivatives muaj qhov zoo sib xws radicalscavenging thiab anti-tyrosinase kev ua ub no hauv vitro, thaum tsuas yog 5-caffeoylquinic acid muaj cov nyhuv inhibitory ntawm -MSH-inducedmelanogenesis. Cov nyhuv inhibitory ntawm 5-caffeoylquinic acid tau txheeb xyuas los ntawm kev txiav txim siab ntawm cov ntsiab lus melanin thiab tyrosinaseactivity hauv melanoma tom qab kho cov hlwb nrog cov khoom lag luam. Tsis tas li ntawd, peb tau qhia tias 5-caffeoylquinic acid inhibitedmelanogenesis los ntawm chelating tooj liab cation los ntawm tooj liab-tyrosinase complex. Yog li, 5-caffeoylquinic acid los yog butanol fractionisolated los ntawm L. thunbergianus tej zaum yuav pab tau nyob rau hauv cov tshuaj pleev ib ce raws li ib tug tshuaj pleev ib ce.

Cistanche tubulosa: daim tawv nqaij-dawb tus neeg sawv cev.

Taw qhia

Melanin, ib pawg ntawm cov xim ntuj pom muaj nyob hauv ntau cov kab mob, ua lub luag haujlwm tseem ceeb hauv cov txiv hmab txiv ntoo, cov kab mob, thiab cov zaub thiab cov pigmentation hauv tib neeg cov tawv nqaij. 2 Inhumans, melanin pigment yog synthesized los ntawm specializeddendritic cell melanocytes nyob rau hauv hlws ris ntawm lub epidermis thiab dermis nyob rau hauv uas synthesis yog pib los ntawm raug ultraviolet (UV) rays.3 Melanin pigment yog transported rau keratinocytes los tiv thaiv daim tawv nqaij tiv thaiv UVrays; thiab dawb radicals. Yog li, cov xim ntawm daim tawv nqaij yog txiav txim siab los ntawm cov qauv kev faib tawm ntawm cov xim melanin.2 Dysregulation inmelanin synthesis ua rau ntau hom hyperpigmentation, xws li melasma, freckles, hnub nyoog me, hnub ci lentigo, postinflammatory hyperpigmentation, thiab lwm yam hyperpigmentation syndromes.4 Ntau tshaj li ib. puas proteins muaj feem xyuam rau tomelanin synthesis, thiab ntawm lawv, tyrosinase yog tus nqi-limiting enzyme uas tau lees paub tias yog lub luag hauj lwm formelanin synthesis.5 Yog li, feem ntau kev tshawb fawb ua hauj lwm los tiv thaiv hyperpigmentation yog tsom rau kev txheeb xyuas, cais, synthesis, thiab cov yam ntxwv ntawm lub zog tshiab. tyrosinase inhibitors rau kev tiv thaiv ntawm melanogenesis.1,6 Ib tug xov tooj oftyrosinase inhibitors, xws li kojic acid, arbutin, hydroquinone, azelaic acid, ellagic acid, thiab tranexamic acid, tau nrov siv nyob rau hauv lub cosmetic kev lag luam raws li anti-melanogenesisagents; Txawm li cas los xij, vim muaj kev txwv ntawm cov tshuaj chemical, uas suav nrog cytotoxicity thiab cov kev mob tshwm sim, tseem xav tau cov ntaub ntawv tshiab nrog kev txhim kho kev nyab xeeb, ruaj khov, thiab ua tau zoo.7

Cov khoom ntuj tsim, suav nrog cov nroj tsuag, cov kab mob, thiab cov fungi, tau dhau los ua lwm qhov chaw rau kev tshawb pom ntawm cov khoom siv tawv nqaij dawb vim lawv cov toxicity tsawg dua thiab zoo dua biocompatibility thiab bioavailability.1,8 Lepisorus thunbergianusis ib lub fern teej tug mus rau Polypodiaceae, uas loj hlob txuas rau pob zeb qub. los yog cov tawv ntoo ntawm cov ntoo thiab muab faib rau hauv thaj chaw sov ntawm East Asia, xws li Kaus Lim Kauslim, Nyiv, Tuam Tshoj, Philippines, thiab Indochina. L. thunbergianus tau siv los ua adiuretic, hemostatic tus neeg sawv cev thiab yog antitussive nyob rau hauv Kauslim lus teb. Cov kev tshawb fawb yav dhau los tau tshaj tawm tias L. thunbergianusextract muaj cov tshuaj tiv thaiv lipid peroxidation kev ua haujlwm hauv zos thiab antioxidant kev ua haujlwm.9 Tsis tas li ntawd, L. thunbergianus extract tau qhia qhov tseem ceeb ntawm kev loj hlob ntawm cov tshuaj tiv thaiv kab mob hauv qhov ncauj thiab cov kab mob qog noj ntshav.10 L. thunbergianusextract tuaj yeem muaj peev xwm siv tau. nyob rau hauv cosmeticindustry vim hais tias cov nroj tsuag no muaj ntau yam flavonoidcompounds thiab ntau bioactive molecules, uas muaj xws liquercetin 3-methyl ether-glucoside, vitexin, oriental, periodical-glucoside, isovitexin, orientin, Corentin, thiab caffeoylquinicacid, cov teebmeem ntawm 9. L. thunbergianus extract onmelanin synthesis nyob rau hauv melanocytes tseem tsis tau elucidated.

Daim duab 1. Kev soj ntsuam ntawm L. thunbergianus extract rau (A) ABTS radical scavenging, (B) anti-tyrosinase, thiab (C) intracellular ROS scavengingactivities

Nyob rau hauv txoj kev tshawb no, peb tau npaj cov L. thunbergianus extract thiab nws cov fractions ntawm cov serial fractionation thiab tshawb xyuas qhov cuam tshuam ntawm cov khoom hnyav ntawm melaninbiosynthesis hauv B16F10 melanoma hlwb. Peb tau txheeb xyuas cov butanol (n-BuOH) feem ua ib feem tseem ceeb uas muaj cov nyhuv inhibitor thiab ntxiv cov teebmeem ntawm caffeoylquinic acid derivatives cais los ntawm L. thunbergianus extracts ntawm inhibition ntawm melanin biosynthesis.

cistanche tshuaj xyuas: anti-oxidation

TSEEM CEEB THIAB PAUB

Ethanol Extract ntawm L. thunbergianus Scavenes 2,2′-Azinobis(3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) Radical thiab Inhibits Tyrosinase Activity.

Extraction ntawm L. thunbergianus nrog 70 feem pua ​​​​ethanol (EtOH) tau ua los ntsuas qhov muaj peev xwm inhibitory kev ua ntawm ntuj sib txuas hauv cov nroj tsuag. Cov kev ua si radical scavenging ntawm L.thunbergianus extract tau txiav txim siab nyob rau hauv qhov concentration ntawm 2-200 ug / mL. Cov ethanol extract tau pom tias muaj kev sib koom ua ke ntawm kev ua haujlwm ntawm radical scavenging nrog amaximal nyhuv ntawm 50 ug / mL nyob rau hauv uas cov txiaj ntsig tau zoo ib yam nrog cov arbutin siv los tswj qhov zoo (Daim duab 1A).

Peb kuj tau soj ntsuam cov nyhuv inhibitory ntawm extract onmushroom tyrosinase kev ua los ntawm kev ntsuas tus nqi ntawm dopachrome synthesis catalyzed los ntawm tyrosinase. Lub ethanolextract inhibited 87 feem pua ​​​​ntawm tyrosinase kev ua ub no ntawm 500 ug / mL, thaum arbutin inhibited tsuas yog 38 feem pua ​​​​ntawm tyrosinase kev ua si ntawm tib qhov concentration (Daim duab 1B). Tom qab ntawd, peb tau soj ntsuam cov kev ua ub no ntawm cov extract ntawm intracellular ROSincreased los ntawm 100 μM hydrogen peroxide. Kev kho mob nrog hydrogen peroxide-induced ib tug kwv yees li 2. Peb pom tias hydrogen peroxide-mediated nce ntawm intracellular ROS tau khawb los ntawm cov extract nyob rau hauv ib koob tshuaj raws li: qhov siab tshaj plaws scavenging kev ua si ntawm 100 ug / mL yog 78 feem pua ​​(Daim duab 1C). Cov txiaj ntsig no qhia tias ethanol extract ntawm L. thunbergianus muaj bioactiveingredients uas inhibit tyrosinase kev ua ub no nrog rau scavengingABTS radical thiab intracellular ROS.

cistanche deserticola extract: inhibits tyrosinase kev ua haujlwm.

Inhibitory Potential ntawm L. thunbergianus Fractionsagainst Melanogenesis.

A crude EtOH extract was fractionated with various solvents including hexane (Hex), methylene chloride (CH2Cl2), ethyl acetate (EtOAc), and butanol to identify and characterize ingredients inhibiting melanogenesis. To identify which solvent fractions have the potential against melanin synthesis, we performed a comparative analysis of four different L. thunbergianus solvent fractions for the radical scavenging and anti-tyrosinase activities. First, to evaluate the antioxidant activity of the four solvent fractions of the L. thunbergianus extract, we determined the ABTS radical scavenging activities in the concentration range of 2−200 μg/ mL. All fractions showed a concentration-dependent increase in ABTS radical scavenging activity (Figure 2A). In particular, EtOAc and n-BuOH fractions completely scavenged the ABTS radical at a 50 μg/mL concentration. The ABTS radical scavenging activity was in the order of n-BuOH > EtOAc >CH2Cl2> Hex, uas zoo ib yam nrog cov ntawv tshaj tawm dhau los.10bNtxiv mus, cov teebmeem inhibitory ntawm cov kuab tshuaj sib txawv ntawm cov khoom sib txawv ntawm ontyrosinase kev ua haujlwm tau nce ntxiv hauv kev ua haujlwm siab-dependentmanner: qhov siab tshaj plaws inhibitory kev ua ub no ntawm n-Hex thiab CH2Cl2fractions tau qis dua 65 feem pua, tab sis cov EtOAc thiab n-BuOHfractions siab dua 70 feem pua ​​(Daim duab 2B). Cov kev ua haujlwm inhibitory yog nyob rau hauv qhov kev txiav txim ntawm n-BuOH > EtOAc > CH2Cl2 > n-Hex.Cov txiaj ntsig no qhia tau tias ntau cov khoom siv hydrophilic, suav nrog-BuOH thiab EtOAc feem, pom tias muaj ntau dua radicalscavenging kev ua si thiab inhibitory kev ua tau zoo dua li cov lipophilic n-Hex thiab CH2Cl2. .

Tom ntej no, cov teebmeem ntawm L. thunbergianus hnyav fractions onmelanoma cell proliferation tau tshawb xyuas los ntawm kev kho B16F10 hlwb nrog 200 ug/mL txhua qhov sib txawv los yog 2mg/mL arbutin hauv cov muaj ib qho -melanocyte-stimulatinghormone (-MSH), uas paub zoo los txhawb kev tsim cov melanogenesis los ntawm microphthalmia-associated transcription factor (MITF) induction.11 Cov txiaj ntsig tau pom tias tsis muaj ib feem ntawm cov feem ntau cuam tshuam rau melanoma cellproliferation (Daim duab 2C). Tom qab ntawd peb tshawb xyuas qhov inhibitoryeffect ntawm cov kuab feem ntawm melanogenesis stimulated los ntawm -MSH hauv melanoma hlwb. Kev kho mob nrog -MSH induced anapproximately 2. Peb pom tias -MSH mediated melanogenesis tau inhibited tag nrho los ntawm n-BuOH feem (p <0.01) thiab="" ib="" feem="" inhibited="" los="" ntawm="" etoacfraction="" (40="" feem="" pua,="" p=""><0.01), thaum="" -msh-mediated="" melanogenesis="" tsis="" tau="" hloov="" pauv="" los="" ntawm="" n-hex.="" thiab="" ch2cl2fractions="" (daim="" duab="" 2d).="" tsis="" tas="" li="" ntawd,="" cov="" teebmeem="" inhibitory="" ntawm="" cov="" kuab="" tshuaj="" ntawm="" tyrosinase="" activation="" mediated="" los="" ntawm="" -msh="" tau="" txiav="" txim="" siab="" txij="" li="" tyrosinase="" plays="" lub="" luag="" haujlwm="" tseem="" ceeb="" hauv="" melanogenesis.n-buoh="" (95="" feem="" pua="" ​​,="" p=""><0.001) thiab="" etoac="" (66="" feem="" pua="" ​​,="" p="">< 0.001)="" fractions="" media="" activation="" reversed.="" los="" ntawm="" -msh,="" whereas="" n-hex="" thiab="" ch2cl2="" feem="" tsis="" tau,="" raws="" li="" qhia="" hauv="" daim="" duab="" 2e.="" tsis="" tas="" li="" ntawd,="" cov="" nyhuv="" scavenging="" ntawm="" cov="" kuab="" tshuaj="" sib="" txawv="" ntawm="" ros="" intracellular="" nce="" tau="" txhawb="" nqa="" los="" ntawm="" hydrogen="" peroxide.="" tag="" nrho="" cov="" kuab="" tshuaj="" fractions="" thim="" rov="" qab="" lub="" intracellular="" ros="" nce="" kho="" los="" ntawm="" hydrogen="" peroxide,="" raws="" li="" nyob="" rau="" hauv="" daim="" duab="" 2f.="" cov="" txiaj="" ntsig="" no="" qhia="" tau="" tias="" buohfraction="" ntawm="" l.="" thunbergianus="" inhibited="" -msh-induced="" melaninsynthesis="" los="" ntawm="" inhibiting="" intracellular="" tyrosinase="" kev="" ua="" hauv="" b16f10="" hlwb.="" tsis="" tas="" li="" ntawd,="" cov="" txiaj="" ntsig="" no="" qhia="" tau="" tias="" cov="" khoom="" xyaw="" bioactive="" inhibiting="" melanin="" synthesis="" yog="" hydrophilic="" thiab="" feem="" ntau="" pom="" nyob="" rau="" hauv="" n-buoh="">

Daim duab 2. Cov teebmeem ntawm cov kuab tshuaj feem ntau ntawm ABTS radical scavenging, anti-tyrosinase, thiab kev ua haujlwm los tiv thaiv melanogenesis.

Kev txheeb xyuas thiab cim ntawm BioactiveCompounds ntawm L. thunbergianus Extract.

Txhawm rau txheeb xyuas thiab txheeb xyuas cov khoom xyaw inhibiting melanogenesis, kev ua haujlwm siab ua kua chromatography (HPLC) kev soj ntsuam rau tag nrho cov khoom tawg tau raug ua. HPLC kev tshuaj xyuas ntawm cov kuab tshuaj tau qhia tias txhua feem muaj peb qhov tseem ceeb ntawm lub sijhawm tuav ntawm 21, 28, thiab 29 min rau cov khoom sib xyaw 1, 2, thiab 3, raws li (Daim duab 3A-D). Peb lub tebchaw loj no tau raug cais tawm ntxiv los ntawm kev npajHPLC purification. Tus nqi ntawm peb lub ntsiab lus tseem ceeb hauv cov ntu n-BuOH tau txiav txim siab siv cov khoom lag luam caffeoylquinic acid derivatives raws li cov txheej txheem sab hauv, raws li daim ntawv tshaj tawm dhau los.12

Tom qab ntawd cov khoom sib cais tau txheeb xyuas los ntawm kev sib piv ntawm lawv cov ntaub ntawv 1H thiab 13C NMR nrog cov ntaub ntawv thiab tau pom tias muaj kev pom zoo nrog cov qauv tsim (Daim duab 4).13 Daim duab 3E qhia cov qauv molecular ntawm peb cov khoom. Compound 1 (yield, 3.2 ± 0}.1 mg/100 mg n BuOH feem) tau los ua hmoov daj daj. 1HNMR (400 MHz, DMSO-d6) δ 9.66 (1H, brs), 9.20 (1H, brs), 7.49 (1H, d, J=15.9 Hz), 7.04 (1H, s), 6.99 ( 1H, d, J=8.2Hz), 6.78 (1H, d, J=8.1 Hz), 6.23 (1H, d, J=15.9 Hz), 5.20( 1H, m), 5.08 (1H, brs), 4.89 (1H, brs) 4.13 (1H, m), 3.84 (1H, m), 3.56 (1H, s), 3.35 (1H, s), 1.99 (1H, m), 1.92 (1H, m), 1.89 (1H, m), 1.79 (1H, m). 13C{1H} NMR (100 MHz, DMSO-d6) δ (176.42, 168.10, 148.14, 145.53, 144.75, 125.65, 121.07, 115.75, 114.72., 314.72. Compound 1 tau txheeb xyuas tias yog 3-caffeoylquinicacid los ntawm NMR tsom xam thiab los ntawm kev sib piv nrog cov ntaub ntawv yav dhau los.13b

Compound 2 (yield, 14.1 ± 0.1 mg/100 mg n-BuOHfraction) tau los ua hmoov daj daj. 1H NMR (400MHz, DMSO-d6) δ 9.64 (1H, brs), 9.20 (1H, brs), 7.52 (1H, d, J=15.9 Hz), 7.06 (1H, s), 7.02 ( 1H, d, J=8.1 Hz) 6.79 (1H, d, J=7.9 Hz), 6.30 (1H, d, J=15.9 Hz), 4.85 ( 1H, brs), 4.70 (1H, d, J=4.9 Hz), 4.12 (1H, brs), 3.95 (1H, m), 1.97 (2H, m), 1.88 (1H, m), 1.83 (1H, m). 13C{1H} NMR (100MHz, DMSO-d6) δ (176.02, 166.27, 148.28, 145.55, 144.77, 125.54, 121.19, 115.76, 114.65., 314.65., 114.65. Compound 2 tau txheeb xyuas tias yog 5-caffeoylquinic acid los ntawm NMR tsom xam thiab los ntawm kev sib piv nrog cov ntaub ntawv yav dhau los.13c

Compound 3 (yield, 3.9 ± 0}.2 mg/100 mg n-BuOHfraction) tau los ua hmoov daj daj. 1H NMR (400MHz, DMSO-d6) δ 9.64 (1H, brs), 9.20 (1H, brs), 7.52 (1H, d, J=15.9 Hz), 7.06 (1H, s), 7.02 ( 1H, d, J=8.1 Hz) 6.79 (1H, d, J=7.9 Hz), 6.30 (1H, d, J=15.9 Hz), 4.85 ( 1H, brs), 4.70 (1H, d, J=4.9 Hz), 4.12 (1H, brs), 3.95 (1H, m), 1.97 (2H, m), 1.88 (1H, m), 1.83 (1H, m). 13C{1H} NMR (100MHz, DMSO-d6) δ (176.02, 166.27, 148.28, 145.55, 144.77, 125.54, 121.19, 115.76, 114.65., 314.65., 114.65. Compound 3 tau txheeb xyuas tias yog 4-caffeoylquinic acid los ntawm NMR tsom xam thiab los ntawm kev sib piv nrog cov ntaub ntawv yav dhau los.13a

Daim duab 3. HPLC chromatograms ntawm (A) n-Hex, (B) CH2Cl2, (C) EtOAc, thiab (D) n-BuOH fractions ntawm L. thunbergianus thiab (E) qauv ntawm peb lub tebchaw loj.

Inhibitory Muaj peev xwm ntawm Caffeoylquinic Acid Derivatives cais tawm ntawm L. thunbergianus tawm tsam Melanogenesis.

Tom ntej no, txhawm rau txheeb xyuas cov khoom muaj roj ntsha hauv qab ntawm lub zog tiv thaiv melanogenesis, peb txiav txim siab qhov kev ua haujlwm ntawm peb caffeoylquinic acid derivativesisolated los ntawm L. thunbergianus tiv thaiv melanin synthesis inmelanoma hlwb. Ua ntej, qhov cuam tshuam ntawm peb qhov derivatives ntawm ABTS radical scavenging tau tshawb xyuas. Cov txiaj ntsig tau pom tias peb qhov derivatives muaj qhov zoo sib xws ABTS radical scavengingactivities (Daim duab 5A). Tom qab ntawd peb tau tshawb xyuas qhov inhibitoryeffect ntawm peb qhov derivatives cais los ntawm L. thunbergianus ontyrosinase kev ua haujlwm. Peb qhov derivatives tau pom qhov concentration-dependent inhibition ntawm tyrosinase kev ua haujlwm, nrog qhov siab tshaj plaws inhibition ntawm 200 μM concentration (Daim duab 5B). Tsis tas li ntawd, peb tau tshawb xyuas cov nyhuv inhibitory ntawm peb qhov kev cuam tshuam ntawm -MSH-induced melanin synthesis hauv B16F10cells. Kev kho mob nrog -MSH induced ib tug kwv yees li 2. Interestingly, cov tebchaw 1 (3-caffeoylquinicacid) thiab 3 (4-caffeoylquinic acid) nce melanin synthesis los ntawm 25 thiab 23 feem pua, raws li (Daim duab 5C, p.< 0.001),="" while="" compound="" 2="" (5-caffeoylquinic="" acid)="" completely="" reversed="" α-msh-induced="" melanogenesis="" (p="" <="" 0.001).="" these="" results="" suggest="" that="" the="" three="" caffeoylquinic="" acid="" derivatives="" with="" similar="" structures="" but="" different="" substitution="" sites="" have="" different="" biological="" activities="" on="" melanogenesis.="" furthermore,="" 5-caffeoylquinic="" acid="" has="" potent="" inhibitory="" activity="" against="">

Daim duab 4. 1H NMR spectra ntawm (A) 3-caffeoylquinic acid, (B) 4-caffeoylquinic acid, thiab (C) 5-caffeoylquinic acid thiab 13C NMR spectra ntawm (D) {{ 6}}caffeoylquinic acid, (E) 4-caffeoylquinic acid, thiab (F) 5-caffeoylquinic acid

Kev txheeb xyuas ntawm Anti-Melanogenesis Efficacy ntawm 5-Caffeoylquinic Acid.

Txhawm rau txheeb xyuas cov nyhuv inhibitory ntawm {{0}} caffeoylquinic acid tiv thaiv melanogenesis, peb tau txiav txim siab cov nyhuv inhibitory ntawm kev lag luam 5-caffeoylquinic acid onmelanin synthesis kho los ntawm -MSH. Kev kho mob nrog qis qis ntawm 0.1 thiab 0.2 mM 5-caffeoylquinicacid me ntsis nce melanin synthesis thiab intracellulartyrosinase kev ua, thaum siab dua concentrations ntawm 0.5 thiab 1 hli ntawm cov compound reversed -MSH-mediated melanogenesis thiab intracellular tyrosinase kev ua haujlwm (Daim duab 6A, B) uas cov txiaj ntsig tau ua raws li daim ntawv tshaj tawm dhau los.14

Caffeoylquinic acid yog ib qho phenolic compound tsim los ntawm theester bond ntawm caffeic acid thiab L-quinic acid. Caffeoylquinic acid derivatives tau paub tias muaj kev tiv thaiv melanogenesis thiab kev ua haujlwm tiv thaiv tyrosinase nrog rau ROS kev ua si. Tyrosinase yog ib tug multifunctionalcopper-muaj metalloenzyme nrog divalent tooj liab cations.1 Caffeoylquinic acid derivatives yuav inhibit tyrosinaseactivity los ntawm chelating tooj liab cation lub luag hauj lwm rau sustaining lub active lub xeev ntawm tyrosinase. Txhawm rau kwv yees qhov kev sib cuam tshuam ntawm 5-caffeoylquinic acid thiab tyrosinase, kev tshawb fawb txog kev sib txuas lus molecular tau ua los ntawm Maestroprogram. Qhov zoo tshaj plaws pose rau lub compound, docked totyrosinase, yog depicted nyob rau hauv daim duab 6C, D. Txoj kev tshawb no molecular docking qhia tias 5-caffeoylquinic acid cuam tshuam nrog tooj liab ntawm qhov deb ntawm 2.43 Å thiab tsim ib tug series ntawm π−π stacking nrog nws. 259, Asn 260, Nws 85, thiab Nws 263 residuesand H-bond nrog Arg 268. Tsis tas li ntawd, lub zog khi ntawm tyrosinase thiab 5-caffeoylquinic acid yog -4.425 kJ / mol. Cov txiaj ntsig no qhia tau tias 5-caffeoylquinic acid tuaj yeem cuam tshuam tyrosinase kev ua haujlwm los ntawm tooj liab chelation. Txhawm rau txheeb xyuas qhov inhibitory mechanism ntawm melanogenesis los ntawm 5-caffeoylquinicacid, peb txiav txim siab tooj liab chelating muaj peev xwm ntawm 5-caffeoylquinic acid. Lub peev xwm ntawm tooj liab chelating ntawm 5-caffeoylquinic acid tau nce nyob rau hauv ib qho concentration-dependentmanner (Daim duab 6E). Cov txiaj ntsig no qhia tau tias 5-caffeoylquinicacid inhibits melanin synthesis ntawm chelating tooj liab cationinteracting nrog tyrosinase.

Daim duab 5. Cov teebmeem ntawm peb cov caffeoylquinic acid derivatives ntawm radical scavenging, nyob rau hauv vitro anti-tyrosinase, thiab anti-melanogenesis kev ua si hauv B16F10 hlwb.

Cov khoom siv thiab cov txheej txheem

Khoom siv

L. thunbergianus tau yuav los ntawm onlinemarket www.jirisangol.com (Korea) thiab qhuav nyob rau hauv ambientconditions ua ntej siv nyob rau hauv txoj kev tshawb no. Reagents, xws li 2,2'- zinc bis (3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) thiab 3-(4,5-dimethylthiazol-2-} yl)-2,5-diphenyltetrazolium bromide(MTT), ib qho -melanocyte-stimulating hormone (-MSH), thiab enzymes, xws li nceb tyrosinase, tau txais los ntawm Sigma-Aldrich (St. Louis, MO, USA). Potassiumpersulfate (K2S2O8), pyrocatechol violet, thiab 3,4-dihydroxy-L phenylalanine (L-DOPA) tau muas los ntawm Alfa Aesar (Haverhill, MA, USA).

Extraction thiab Fractionation ntawm L. thunbergianus.

Qhuav L. thunbergianus (8{23}} g) tau pulverized siv pulverizer (HBL-3500S, Samyang Electronics, Kauslim Teb) thiab muab rho tawm nrog 7{30}} feem pua ​​EtOH peb zaug (800 mL, txhua zaus ) ntawm 70 degree rau 3 hnub. Lub resulting extract yog nqus-filtered siv Advantecfilter daim ntawv nos. 1 thiab 2 (Toyo Roshi Kaisha, Ltd., Tokyo, Nyiv) thiab nquag siv Hei-Vap Advantage rotaryevaporator (Heidolph, Lub teb chaws Yelemees) kom tau 17.2 g ntawm EtOHcrude extract. Qhov no crude extract raug tshem tawm nyob rau hauv dH2O (180mL) thiab successively fractionated nrog Hex, CH2Cl2, EtOAc, thiab n-BuOH kom tawm los Hex (1.51 g, 8.8 feem pua), CH2Cl2 (0.88 g, 5.1 feem pua), 3.293 g (3.290 g), feem pua ​​), thiab n-BuOH (3.02 g, 17.6 feem pua), raws li tau piav qhia.15 Cov txiaj ntsig tau txais txiaj ntsig thiab cov khoom seem tau khov-qhuav thiab khaws cia ntawm −80 degree ua ntej siv.

Kev txiav txim siab ntawm ABTS Dawb Radical ScavengingActivity.

Txhawm rau tshuaj xyuas cov haujlwm antioxidant ntawm L.thunbergianus extracts thiab nws cov kuab tshuaj feem ntau, ABTSradical scavenging kev ua haujlwm tau txiav txim siab, raws li tau piav qhia dhau los.16 Txhawm rau tsim cov ABTS radical, 10 mL ntawm 7 mMABTS yog tov nrog 176 μL ntawm 140 mmol. potassiumperoxy disulfate nyob rau hauv dH2O thiab incubated nyob rau hauv qhov tsaus ntuj ntawm chav tsev kub (RT) rau 16 h ua ntej siv. ABTS radicalsolution tau diluted nrog kiag li methanol kom tau txais kev nqus ntawm ze li ntawm 0.7 ntawm 734 nm. Aliquots ntawm 100 μL ntawm txhua qhov kev rho tawm lossis cov feem hauv qhov qhia pom ntau ntawm 2-200 ug / mL tau ntxiv rau 100 μL ntawm diluted ABTS radicalsolution thiab incubated rau 10 min hauv qhov tsaus ntuj ntawm RT. Theabsorbance ces ntsuas ntawm 732 nm siv SpectraMaxM5 multimode microplate nyeem ntawv (Molecular Devices, Sunnyvale, CA, USA). Lub ABTS radical scavenging kev ua si tau xam raws li hauv qab no

ABTS radical scavenging kev ua si (%) =1−(Asample/Acontrol)×100 (1)

Hauv Vitro Tyrosinase Inhibition.

Cov nyhuv inhibitory ntawm L.thunbergianus extracts thiab nws cov kuab feem ntawm tyrosinaseactivity tau soj ntsuam los ntawm tus nqi ntawm dopachromesynthesized los ntawm catalytic cov tshuaj tiv thaiv ntawm tyrosinase.2a,17Briefly, 50 μL ntawm txhua extract los yog ib feem nyob rau hauv lub qhia concentration ntau 5 μL ntawm 0. U/mLmushroom tyrosinase nyob rau hauv 50 mM phosphate-buffered saline (PBS; 8.1 mM Na2HPO4, 1.2 mM KH2PO4, pH 6.8, 2.7 mMKCl, thiab 138 mM NaCl) nyob rau hauv ib lub 96- zoo phaj thiab incubated rau.3 Tom qab ntawd, 100 μL ntawm 1 mM L-DOPA tau ntxiv cov ntiv taw zoo tom qab los ntawm incubation rau ntxiv 10 min ntawm 37 degree. Lub absorbance ntawm qhov tshwm sim yog ntsuas ntawm 475 nm siv SpectraMax M5 multimode microplatereader.

Cell Culture.

B16F10 murine melanoma cells yog culturedin Dulbecco's modified Eagle's nruab nrab (DMEM) (Gibco, Gaithersburg, USA) supplemented nrog 10 feem pua ​​​​ntawm tshav kub-inactivatedfetal bovine serum (Gibco), 100 units / mL penicillin, thiab 100ug / mL 7 degree (Geptomy) nyob rau hauv humidified 5 feem pua ​​CO2.

Cell Viability.

Kev muaj peev xwm ntawm B16F10 hlwb tau txiav txim siab siv qhov kev ntsuam xyuas MTT, raws li tau piav qhia yav dhau los.18 Luv luv, B16F10cells tau muab noob rau hauv 24-cov phiaj zoo ntawm qhov ntom ntawm 1 × 104 cellper zoo. Tom qab 24 teev, cov hlwb raug kho nrog cov ntsiab lus qhia ntawm L. thunbergianus extracts los yog feem rau 48 h.Cov hlwb tau muab tso rau hauv MTT tov rau 4 h, thiab txo cov formazan crystals tau yaj hauv DMSO. Cov tshuaj muaj txiaj ntsig tau hloov mus rau 96- cov phiaj zoo, thiab qhov nqus tau ntsuas ntawm 540 nm siv SpectraMax M5multimode microplate nyeem ntawv.

Kev txiav txim siab ntawm Intracellular Reactive OxygenSpecies (ROS).

Qib intracellular ROS tau ntsuas DCF / H2DCFDA cellular ROS kuaj cov khoom (ab113851, Abcam), raws li cov chaw tsim khoom cov lus qhia. Luv luv, B16F10 hlwb tau muab noob rau hauv 48-cov phiaj zoo ntawm qhov ntom ntawm 2 × 104 cell ib qhov dej. Tom qab 24 teev kab lis kev cai, cov hlwb raug kho rau 1 h nrog rau qhov qhia tau hais tias muaj ntau ntawm L. thunbergianus extractsor hnyav fractions hauv 0.1 feem pua ​​​​bovine serum albumin (BSA) uas muaj kab lis kev cai uas tsis muaj phenol liab. Cov hlwb raug muab tso rau hauv 100 μM hydrogen peroxide rau 30 min.Tom qab ntxuav nrog PBS, cov hlwb raug kho nrog 25 μMH2DCFDA hauv PBS rau 45 min. ROS qib tau soj ntsuam los ntawm tus nyeem ntawv microplate (Synergy H1, Biotek, Vermont, USA) nruab nrog lub fluorescence lim ntawm 485/545 nm (excitation / emission wavelength). Qhov nruab nrab txheeb ze fluorescence siv ntawm kev tswj tau sib npaug rau 100 feem pua, nrog rau kev kho mob suav nrog proportionally.

Kev txiav txim siab melanin ntsiab lus.

Cov ntsiab lus melanin tau txiav txim siab, raws li tau piav qhia yav dhau los, nrog rau qee qhov kev hloov kho.19 Cov kab mob melanoma tau coj mus rau hauv lub phaj rau lub qhov dej rau 24 teev. Lawv tau kho nrog cov qhia pom ntawm L. thunbergianus extract los yog nws cov kuab tshuaj ntxiv rau 48 teev ntxiv ntawm 100 nM -MSH.Tom qab ntxuav ob zaug nrog chilled Dulbecco's phosphate-buffered saline supplemented nrog calcium chloride thiab magnesium chloride (D-PBS, Gibco), cov hlwb uas tshwm sim tau raug tshem tawm los ntawm incubation nrog cov tshuaj trypsin-EDTA. Tom qab centrifugation ntawm 1000 rpm rau 3 min, lub cell pellet yog yaj nyob rau hauv 150 μL ntawm 1 M NaOH muaj 10 feem pua ​​​​DMSO rau 1 h ntawm 60 degree rau 1 h. Cov ntsiab lus melanin tau txiav txim siab los ntawm kev nqus ntawm 405 nm siv lub microplate nyeem ntawv.

Cistanche extract inhibits melanin.

Kev txiav txim siab ntawm Cellular Tyrosinase Kev Ua Haujlwm hauv Melanoma Cells.

Kev ua tyrosinase hauv B16F10 hlwb raug kuaj raws li tus nqi ntawm dopachrome uas tsim los ntawm cov tshuaj tiv thaiv catalytic ntawm intracellular tyrosinase.20 Luv luv, melanoma hlwb raug coj mus rau hauv ib lub phaj rau 24 hfollowed los ntawm kev kho mob nrog ntau qhov sib txawv ntawm L.thunbergianus extract los yog nws cov kuab tshuaj. fractions rau ntxiv 48 hin muaj 100 nM -MSH. Tom qab ntxuav ob zaug nrog dej txias D-PBS, cov hlwb tau lysed nyob rau hauv 200 μL ntawm radioimmunoprecipitation assay (RIPA) tsis (Sigma-Aldrich) uas muaj protease thiab phosphatase inhibitors. Tom qab centrifugation ntawm cov cell lysate sau los ntawm txhua qhov dej ntawm 15, 000g rau 15 min, 100 μL ntawm supernatant tau tov nrog 100 μL ntawm 1 mM L-DOPA hauv PBS (pH 6.8) tom qab incubation rau 30 min ntawm 37 degree . Qhov nqus ntawm dopachrome tau ntsuas ntawm 475 nm siv lub microplatereader. Cov ntaub ntawv tau normalized nrog protein ntau concentration txiav txim los ntawm bicinchoninic acid assay.

Kev rho tawm thiab ua tus yam ntxwv ntawm Bioactive Compounds siv HPLC.

HPLC tau ua tiav ntawm YL{{{0}}}(Young Lin Instrument, Kauslim Teb), nruab nrog TC-C18column (4.6 mm, 250 mm, thiab 5 μm, Agilent, USA), inconjunction nrog cov txheej txheem gradient uas muaj cov kuab tshuaj A (0.2 feem pua ​​formic acid) thiab hnyav B (MeOH). Txoj kev nqes hav solventratio tau teem rau 0-5 min, 15 feem pua ​​B; 5-10 min, 15-20 feem pua ​​B; 10-15 feeb, 20-30 feem pua ​​B; 15-30 feeb, 30-40 feem pua ​​B; 30–37 feeb, 40–60 feem pua ​​B; 37-40 feeb, 60-100 feem pua ​​B; 40-45 feeb, 100 feem pua ​​B; 45-50 feeb, 100-15 feem pua ​​B; thiab 50-55 min, 15 feem pua ​​​​B. Txhawm rau txheeb xyuas cov khoom muaj nyob hauv cov khoom hnyav, cov theem ntawm lub xov tooj ntawm tes tau xa tawm ntawm tus nqi ntws ntawm 1 mL / min, thiab kev tshawb pom ntawm eluting yog nqa tawm ntawm 330 nm. Txhawm rau sau cov khoom xyaw bioactive insolvent feem, lub sijhawm txawb tau xa tawm ntawm tus nqi ntws ntawm 15.0 mL / min, thiab kev tshawb pom ntawm eluting tau ua tiav ntawm 330 nm siv prep-HPLC (YL-9100 s, Young Lin Instrument, Kauslim Teb ), nruab nrog lub prep-C18 kem (4.6 mm, 212 mm, 10 μm, Agilent, USA), nrog rau cov gradient system uas muaj hnyav A (0.2 feem pua ​​formic acid) thiab hnyav B (MeOH). Txoj kab nqes qhov hnyav piv rau 0-10 min, 10 feem pua ​​B; 10-20 min, 10-15 feem pua ​​B; 20-40 feeb, 15% B; 40-60 feeb, 15-20 feem pua ​​B; thiab 60-70 min, 30 feem pua ​​B.

Molecular Modeling.

Cov qauv siv lead ua ntawm mushroomtyrosinase rau kev ua qauv molecular yog Agaricustyrosinase (PDB ua: 2Y9X) tau los ntawm Protein DataBank (PDB). Cov enzyme tau npaj los ntawm kev siv cov proteinwizard npaj cov haujlwm ua haujlwm hauv Maestroprogram (Maestro, version 11.9.011, Schrödinger, LLC, NewYork, NY, USA, 2019). Dej thiab tag nrho lwm cov molecules muaj nyob rau hauv cov ntaub ntawv PDB raug tshem tawm. Molecular docking tau ua tiav siv cov txheej txheem induced haum docking (IFD) raws tu qauv (Schrödinger Suite 2019 Induced Fit Docking raws tu qauv), aspreviously tshaj tawm.21

Copper Chelating Muaj peev xwm.

Lub peev xwm ntawm tooj liab chelating ntawm cov tshuaj sib cais los ntawm L. thunbergianus tau txiav txim siab raws li daim ntawv tshaj tawm dhau los.22 Txhua caffeoylquinic acidderivative (10} μL) hauv qhov qhia pom tau sib xyaw nrog 280 μL ntawm 50 mM sodium acetate buffer (pH 6.0), 6 μL ntawm 4 mM pyrocatechol violet tov npaj nyob rau hauv tib tsis, thiab 10 μL ntawm 1 ug / μL CuSO4 · 5H2O. Qhov ploj ntawm cov xim xiav tau pom los ntawm kev ntsuas qhov nqus ntawm 632nm siv SpectraMax M5 multimode microplate nyeem ntawv. Dej tau siv los ua kev tswj tsis txhob siv cov qauv. Percentcopper chelating kev ua haujlwm tau suav los ntawm qhov nqus ntawm 632 nm, uas yog raws li hauv qab no

tooj liab chelating peev xwm (%)=1- (Ib qho piv txwv/Kev tswj hwm)×100 (2)

Kev txheeb cais.

Tag nrho cov ntaub ntawv nyob rau hauv txoj kev tshawb no tau qhia tias qhov nruab nrab ± tus qauv sib txawv (SD) los ntawm peb qhov kev sim siab. Kev txheeb xyuas txheeb cais tau ua tiav siv GraphPad Prism 8.0 (GraphPad Software Inc., La Jolla, CA, USA). Qhov sib txawv ntawm qhov nruab nrab qhov tseem ceeb ntawm kev tswj hwm thiab cov pab pawg raug tshuaj xyuas tau siv ib qho kev sib txawv ntawm kev sib txawv (ANOVA) ua raws li Dunnett's post hoctest. Qhov chaw pib rau qhov tseem ceeb rau txhua qhov kev ntsuas p < 0.05="">

Cov lus xaus

Hauv txoj kev tshawb no, peb tau pom tias L. thunbergianus extractscavenes ABTS radicals thiab nthuav tawm lub zog inhibitory effecton melanin biosynthesis yam tsis muaj cytotoxicity tseem ceeb. Abioactive ingredient uas twb muaj lawm nyob rau hauv L. thunbergianus thiab inhibitingmelanogenesis yog cais nyob rau hauv lub n-BuOH feem. Tsis tas li ntawd, peb pom tias peb caffeoylquinic acidderivatives yog cov tseem ceeb compounds uas muaj nyob rau hauv lub n-BuOHfraction thiab hais tias 5-caffeoylquinic acid yog ib qho tseem ceeb ntawm cov khoom xyaw. inhibiting melanogenesis hauv melanoma hlwb los ntawm inhibiting cellular tyrosinase kev ua haujlwm. Ntawm no, peb cais anatural compound inhibitor 5-caffeoylquinic acid los ntawm L.thunbergianus extract los tiv thaiv hyperpigmentation thiab nthuav tawm nws cov txheej txheem inhibition uas underlies melanogenesis. Yog li, peb qhov kev tshawb pom qhia tias 5-caffeoylquinic acid thiab cov n-BuOH feem cais los ntawm L. thunbergianus tej zaum yuav muaj txiaj ntsig zoo hauv cov tshuaj pleev ib ce ua cov tshuaj pleev ib ce.

cistanche slices