Citral-Containing Essential Oils Raws li Muaj Peev Xwm Tyrosinase Inhibitors: Ib Txoj Kev Qhia Txog Kev Ua Haujlwm Bio-Guided Fractionation

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

Abstract:

Kev tsim cov melanin ntau dhau ua rau mob hnyav rau cov kab mob dermatological nrog rau cov teeb meem minoraesthetic (piv txwv li, freckles thiab hnub ci lentigo). Kev txo qis ntawm tyrosinase yog ib qho kev nthuav dav rau kev kho mob ntawm cov kab mob xws li cov kab mob, thiab cov nroj tsuag rho tawm feem ntau tau ua pov thawj tias muaj txiaj ntsig zoo.tyrosinaseinhibitors. Citral (ib qho sib xyaw ntawm neral thiab geranial) yog ib qho tseem ceeb fragranceingredient uas tau pom tias muaj peev xwm tiv thaiv tyrosinase. Nws yog cov concentrated hauv cov roj yam tseem ceeb (EOs) ntawm Cymbopogon schoenanthus (L.) Spreng., Litsea cubeba (Lour.) Pers., Melissa officinalis L., thiab Verbenaofficinalis L. Txawm li cas los xij, tsuas yog L. cubeba EO tau tshawb xyuas rau siv raws li lub peev xwm ntawm daim tawv nqaij-whiteningagent. Qhov kev ua haujlwm no ntsuas lub hauv vitrotyrosinaseinhibitory kev ua ntawm cov EOs thiab cov kev tshawb fawb, siv bio-assay oriented fractionation, seb lawv cov tshuaj muaj pes tsawg leeg sib txawv cuam tshuam rau tag nrho EO inhibitory kev ua ub no los ntawm kev ua tau zoo, ntxiv, thiab / lossis kev sib tw ntawm EOs. Qhov inhibitory kev ua ntawm C. schoenanthus EO thiab ntawm M. officinalis EOs, nrog tsis txaus (ntxiv rau )-citronellal npaum li cas, yog nyob rau hauv-kab nrog lawv cov ntsiab lus citral. Ntawm qhov tod tes, L. cubebaand V. officinalis EOs inhibitedtyrosinasekom muaj txiaj ntsig ntau dua li lawv muaj -myrcene, uas tau pab txhawb rau tag nrho cov haujlwm EO. Cov kev soj ntsuam zoo sib xws tau ua rau M. officinalis EO, uas dais siab (ntxiv rau)-citronellal cov ntsiab lus uas nce citral kev ua haujlwm.

Ntsiab lus:tyrosinaseinhibition; cov roj tseem ceeb; citral

cistanche muaj kev ua kom dawb

Taw qhia

Tyrosinaseyog qhov tseem ceeb enzyme hauv biosynthesis ntawm melanin pigments nyob rau hauv ntau yam kab mob, fungi, nroj tsuag, tsiaj txhu, thiab tib neeg. Hauv tib neeg, tyrosinase catalyzes tus nqi txwv cov kauj ruam hauv melanin biosynthetic txoj hauv kev. Qhov biosynthesis no yog tus yam ntxwv los ntawm ntau cov tshuaj enzymatic thiab tshuaj lom neeg uas ua rau melanin tsim los ntawm theamino acid L-tyrosine, nrog tyrosinase catalyzing nws hydroxylation rau o-dopaquinone viaits mycophenolate thiab diphenols kev ua ub no. Txawm hais tias muaj lwm cov enzymes koom nrog hauv melanogenesis, tsuas yog cov tshuaj tiv thaiv tyrosinase-catalyzed tsis tuaj yeem tshwm sim ntawm nws tus kheej, whereas cov kauj ruam ntxiv tuaj yeem ua mus yam tsis muaj kev ua enzyme ntawm lub cev pH [1].Vim li no, tyrosinase downregulation yog ib qho kev nthuav dav heev rau kev txo qis melanin ntau dhau. ntau lawm, thiab kev siv tyrosinase inhibitors raws li daim tawv nqaij-whiteningagents tau pom qhov tseem ceeb ntawm kev kho mob thiab tshuaj pleev ib ce [2].

Hauv kev lag luam EU, lubtyrosinaseinhibitors uas ua hauj lwm raws li daim tawv nqaij-dawb tus neeg sawv cev tuaj yeem muab faib ua ob pawg tseem ceeb: cov neeg txwv tsis pub nyob hauv EU kev cai tshuaj pleev ib ce 1223/2009 (ie, hydroquinone thiab mono benzyl ether hydroquinone) vim lawv cov kev mob hnyav, tab sis tseem siv los kho hyperpigmentation raws li kev kho mob. kev saib xyuas; thiab tyrosinase inhibitors uas tau tso cai siv rau hauv cov khoom siv tshuaj pleev ib ce (ie, arbutin, aloesin, kojic acid) [2,3]. Cov pab pawg thib ob no, txawm li cas los xij, tseem muaj qhov tshwm sim los ntawm qhov muaj feem cuam tshuam loj heev; Cov kev tshawb fawb soj ntsuam ntawm kojic acid tau qhia tseeb txog cov teeb meem ntawm erythema, stinging hnov, thiab tiv tauj eczema tom qab thov. Ib yam li ntawd, EuropeanScientific Committee ntawm Consumer Safety tau tsa kev txhawj xeeb txog kev siv arbutinas cov khoom xyaw kom zoo nkauj [2], vim muaj peev xwm hydrolysis ntawm nws cov glycosidic daim ntawv cog lus uas tso tawm hydroquinone. Yog li yuav tsum muaj cov qauv tshiab molecule thiab / ormixtures ntawm bioactive compounds los kho hyperpigmentation.

Cov nroj tsuag tau muaj txiaj ntsig zoo ntawm cov neeg ua kom tawv nqaij dawb, thiab peb ntawm tsib ntawm lawv cov neeg ua haujlwm feem ntau, ob qho tib si kho mob thiab tshuaj pleev ib ce, cog cov metabolites tshwj xeeb (ie, hydroquinone, -arbutin, aloesin). Txog niaj hnub no, phenolic tebchaw tau tshawb xyuas qhov muaj peev xwmtyrosinaseinhibitors, thiab cov no suav nrog flavonoids (piv txwv li, quercetin [4]), stilbenes (piv txwv li, resveratrol [1]), phenylpropanoids (xws li, cinnamaldehyde [5], thiab eugenol [6]), thiab phenolic acids (xws li, anisic acid thiab benzoic acid [7]. Cov paj laum forterpenoids tau qis dua thiab lawv tau raug soj ntsuam raws li cov tshuaj tiv thaiv tyrosinase.

Citral yog ib qho txwv ntawm cov terpenoid derivatives nrog cov khoom tiv thaiv tyrosinase uas tau kawm. Nws yog kev sib xyaw ntawm ob isomers, cis- thiab trans-3, 7-dimethyl-2, 6-octadienal (ie, neral thiab geranial), uas tau raug pov thawj los thaiv nyob rau hauv vitro enzymaticactivity ntawm ncebtyrosinase[8]. Ntxiv rau nws qhov tseem ceeb raws li cov khoom muaj ntxhiab tsw, khoom noj, thiab tshuaj pleev ib ce, citral tau pom tias muaj kev cog lus hauv vitro lom cov dej num xws li tshuaj tiv thaiv kab mob, tshuaj tua kab mob, tshuaj tua kab mob, thiab tshuaj tiv thaiv kab mob [9–11].Ntxiv mus, cov kev tshawb fawb tsis ntev los no muaj. tseem ceeb tias citral muaj peev xwm kho tau qhov tseem ceeb raws li cov leeg nqaij du thiab cov tshuaj loog hauv zos, vim nws txhawb kev so hauv tracheal, uterine, thiab aortic du nqaij thiab inhibits paj excitability hauv cov qauv tsiaj [12-15].

Citral yog tau los ntawm cov roj yam tseem ceeb (EOs) ntawm ntau hom botanical, suav nrog Cymbopogon schoenanthus (L.) Spreng., Litsea cubeba (Lour.) Pers., Melissa officinalis L., thiab Verbena officinalis L. Rau qhov zoo tshaj plaws ntawm cov kws sau ntawv ' kev paub, tsuas yog L. cubeba EO tau tshawb xyuas rau nwstyrosinaseinhibitory kev ua si [16] Yog li ntawd, txoj kev tshawb no aims los soj ntsuam cov tyrosinase inhibitory kev ua ub no ntawm C. schoenanthus, L. cubeba, M. officinalis, thiab V.officinalis EOs, siv ib tug in vitro colorimetric assay, los ntsuam xyuas seb cov tshuaj sib txawv muaj feem xyuam rau tag nrho cov EO inhibitory kev ua ub no ntawm ib yam dab tsi. ua tau synergistic, additive thiab/los sis kev sib tw kev sib raug zoo ntawm lawv cov Cheebtsam. Txoj kev tshawb no siv abioassay-guided fractionation mus kom ze los soj ntsuam cov EOs constituents thiab lawv cov enantiomers, thaum chiral, uas pab txhawb rau EO inhibitory kev ua si tiv thaiv nceb qhov chaw ntawm tyrosinase, uas yog ib tug qauv zoo rau kev soj ntsuam ua ntej ntawm.tyrosinaseinhibitors [17].

2. Cov txiaj ntsig thiab kev sib tham

2.1. Chemical Composition thiab Citral Cov ntsiab lus ntawm Kev Tshawb Fawb Cov Roj Tseem Ceeb

Hauv peb qhov kev sim ua kom pom tseeb tag nrho cov peev txheej EO uas ua rau muaj txiaj ntsig zoo rau kev ua haujlwm lom neeg, kev tshawb nrhiav EOs tau txheeb xyuas los ntawm GC, nrog rau FID thiab MS tshawb pom. Qhov feem pua ​​​​ntawm cov txheeb ze sib raug zoo (xam los ntawm cov cheeb tsam zoo li qub rau cov txheej txheem sab hauv C13 los ntawm kev siv cov lus teb [18,19]) ntawm tag nrho cov tshuaj ntsuam xyuas tau txiav txim siab thiab siv los sib piv EO compositions. Daim duab 1 qhia txog GC-MS profile ntawm EOs soj ntsuam nrog ib kab lus. Table 1 cov npe, rau txhua qhov kev tshawb xyuas EO, cov khoom sib txuas uas qhia txog qhov feem pua ​​​​ntawm cov khoom noj muaj txiaj ntsig saum toj no 0.1, thaum ua tiav EO tshuaj lom neeg tau tshaj tawm hauv Cov Khoom Siv Ntxiv (Tables S1-S5).

Tag nrho cov kev tshawb nrhiav EOs yog nplua nuj nyob rau hauv neral (cis{{{0}}},7-dimethyl-2,6-octadienal) andgeranial (trans-3,{ {5}}dimethyl-2, 6-octadienal), uas yog cov tebchaw tshaj plaws. Theneral/geranial ratio yog zoo sib xws nyob rau hauv tag nrho cov soj ntsuam EOs thiab sib raug rau 0.74 ± 0.05. C. schoenanthus thiab L. cubeba EOs tso saib cov ntsiab lus siab tshaj plaws ntawm neral thiab geranials, uas suav nrog, qhov nruab nrab, 60 feem pua ​​​​ntawm lawv cov EO tag nrho, thiab qhov twg yog 1.5-times ntau dua hauv V. officinalis EO thiab hauv peb lub M. officinalis EOs (piv txwv li, Qauv 1, 2, thiab 3). Qhov ntxiv oxygenated compounds uas muaj rau tag nrho cov EOs yog 6-methyl-5-hepten-1-ib, linalool thiab citronellal. Cov tom kawg yog qhov muaj txiaj ntsig ntau dua ntawm M. officinalis EO 1 dua li lwm qhov kev tshawb xyuas EOs, suav nrog M. officinalis EO2 thiab 3.

Qhov ntau ntawm cov hydrocarbon feem ntau sib txawv hauv qhov sib txawv EOs.M. officinalis EO 1 tsuas muaj trans- -caryophyllene thiab -humulene li sesquiterpenehydrocarbons, uas suav txog 2.7 feem pua ​​thiab 0.13 feem pua ​​ntawm tag nrho EO, raws li. Lub C.schoenanthus EO nthuav qhia me ntsis cov khoom siv hydrocarbon ntau dua li M. officinalis EO 1 (piv txwv li, 7.0 feem pua), muaj ob qho tib si monoterpenes (ie, camphene, cis{{1{20} }}}}ocimene, limonene, -pinene, trans- -ocimene, -thujene) thiab sesquiterpenes (ie, trans- -caryophyllene, -cadinene, δ-cadinene, germacrene D, -elemene) nyob rau hauv nruab nrab tus nqi. Nyob rau hauv L. cubeba thiab V.officinalis EOs, cov hydrocarbon feem nyiaj rau 20 feem pua ​​​​ntawm tag nrho EO thiab limonene yog cov khoom siv ntau tshaj plaws (piv txwv li, 15.0 thiab 10.9 feem pua, raws li), raws li -pinene, pinene , sabinene, trans- -caryophyllene, -myrcene, camphene, thiab -copaene. Thaum kawg, M.officinalis EO 2 thiab 3 yog tus cwj pwm los ntawm cov ntsiab lus siab tshaj plaws hydrocarbon feem (38.8 feem pua ​​thiab 31.8 feem pua ​​​​ntawm tag nrho EO, feem). Nyob rau hauv ob qho tib si cov qauv, cov hydrocarbon feem feem ntau muaj sesquiterpenes, uas yog trans- -caryophyllene (27.8 feem pua ​​thiab 20.0 feem pua), thiab -humulene (3.0 feem pua ​​thiab 2.6 feem pua), thiab ib tug txo monoterpene feem uas yog mas characterized los ntawm limonene ( 4.2 feem pua ​​thiab 3.2 feem pua).

Peb cov qauv ntawm L. cubeba, V. officinalis, thiab C. schoenanthus Eos ua nyob rau hauv xyoo sib txawv nrog rau peb cov qauv ntawm M. officinalis EOs los ntawm cov chaw tsim khoom sib txawv tau nqis peev gated. GC-MS kev tshuaj xyuas ntawm C. schoenanthus, L. cubeba, M. officinalis, thiab V. officinalis tsis tau nthuav tawm cov txiaj ntsig zoo thiab ntau qhov sib txawv hauv cov tshuaj muaj pes tsawg leeg ntawm peb cov qauv ntawm ntau xyoo sib txawv. Qhov no yuav raug sau tseg rau qhov kev cia siab zoo, piv txwv li, nyob rau hauv lub khob iav amber ntawm 4 ◦C hauv qhov tsaus ntuj nrog lub taub hau qhov chaw tsis txaus ntseeg. Ntawm qhov tod tes, GC-MS kev tshuaj ntsuam pom qhov sib txawv tseem ceeb hauv kev nplua nuj ntawm citronellal thiab trans{{4 }}caryophyllene hauv peb qhov kev tshawb nrhiav M. officinalis EOs. Citronellalamounted rau 19.6 feem pua ​​, 0.26 feem pua ​​, thiab 0.31 feem pua ​​hauv M. officinalis EO 1, 2, thiab 3, raws li. Ntawm qhov tsis sib xws, raws li tau piav qhia dhau los, trans- -caryophyllene yog qhov muaj txiaj ntsig ntau dua hauv M. officinalis EOs 2 thiab 3 dua li hauv M. officinalis EO 1. Cov txiaj ntsig no tau pom zoo nrog cov kev tshawb pom qhia los ntawm Seidler-Lozykawska li al., uas qhia txog qhov sib txawv tseem ceeb hauv citral, citronellal, thiab trans- -caryophyllene abundances hauv EOs tau los ntawm 22 xaiv M. officinalis genotypes los ntawm European botanical vaj [20].

Ib qho tseeb quantitation tau ua los ntawm cov txheej txheem sab nraud calibration kom raug ntsuam xyuas qhov ntau ntawm cov muaj peev xwm bioactive tshwj xeeb tebchaw (xws li, neral, geranial, limonene, -myrcene, thiab citronellal. Tables 2 thiab 3 qhia txog kev kuaj ions (m/z) siv rau SIM- MS quantitation ntawm cov cim tebchaw nyob rau hauv kev tshawb fawb ua ke nrog lub calibration ntau, lub calibration nkhaus sib npaug, correlation qhov tseem ceeb, thiab regressionstandard yuam kev ntawm txhua qhov kev ntsuam xyuas thiab qhov ntau npaum li cas, feem.

2.2. Hauv Vitro Inhibitory Kev Ua Haujlwm ntawm Kev Tshawb Fawb Cov Roj Tseem Ceeb tawm tsam Mushroom Tyrosinase

Raws li tau piav qhia dhau los, EOs ntawm C. schoenanthus, M. officinalis, L. cubeba, thiab V.officinalis tam sim no qib siab ntawm citral, uas yog tus cwj pwm los ntawm kev tsis sib tw inhibitoryactivity tawm tsam fungal qhov chaw ntawm tyrosinase [8,16,21] . Qhov kev tshawb fawb no tsom rau kev tshuaj xyuas cov hauv vitrotyrosinaseinhibitory kev ua ub no ntawm EOs los tshawb xyuas seb lawv cov inhibitoryactivity puas tuaj yeem sau rau lawv cov ntsiab lus citral nkaus xwb, lossis seb puas muaj lwm yam bioactivecompounds uas cuam tshuam rau inhibitory teebmeem ntawm EOs.

Ncebtyrosinasetau txais los ntawm no vim nws cov homology siab rau humantyrosinase, nws tus nqi qis thiab kev npaj muaj, uas ua rau nws ua qauv zoo rau kev tshuaj xyuas ua ntej ntawm tyrosinase inhibitors [17]. Lub precision ntawm thein vitrotyrosinaseinhibition test tau soj ntsuam nyob rau hauv cov nqe lus ntawm repeatability (los ntawm kev ua lub enzymatic inhibition assay tsib zaug nyob rau hauv tib hnub) thiab intermediate precision (los ntawm rov ua cov enzymatic inhibition assay tsib zaug txhua plaub lub lis piam nyob rau ib lub sij hawm ntawm rau lub hlis). Table 4 qhia txog cov coefficient ntawm variation (CV) rau inhibition kev ntsuam xyuas nqa nrog kojic acid, uas yog siv raws li ib tug zoo tswj, thiab nrog L. cubeba EO. Cov txiaj ntsig tau txais txiaj ntsig zoo li CV yeej tsis dhau 7 feem pua ​​​​rau kev rov ua dua thiab 10 feem pua ​​​​rau kev txiav txim siab nruab nrab. Table 4 qhia txog cov coefficient ntawm variation rau inhibition kev ntsuam xyuas nqa tawm nrog kojic acid, siv raws li zoo tswj, thiab nrog L. cubeba EO. Cov txiaj ntsig zoo sib xws tau txais rau txhua qhov kev sim EOs.

Citral concentration - teb nkhaus tau kawm los ntawm plotting pom inhibitoryactivity raws li kev ua haujlwm ntawm nws cov concentration hauv cov tshuaj tiv thaiv sib xyaw. Tag nrho cov EOs tau kuaj pom qhov concentration ntawm 166.7 µg / mL, uas tau muab, tsis hais txog EO, ua rau citralconcentration nyob rau hauv nws cov concentration- teb nkhaus linearity ntau yam (y=0.3956x ntxiv rau 1.8094,R{{7}) }.9951, regression yuam kev: 2.08448, linearity range: 6.7–166.7 µg/mL) thiab tsis tsim teeb meem solubility hauv cov tshuaj tiv thaiv sib tov.

Lub thawv ntawv qhia hauv daim duab 2 nthuav qhia qhov feem pua ​​​​ntawmtyrosinaseinhibition rau txhua EO. Rau L. cubeba, V. officinalis, thiab C. schoenanthus EOs, cov txiaj ntsig tau tshaj tawm hauv daim duab 2 sib raug rau cov nceb tyrosinase inhibitory kev ua ntawm EOs ntawm 2020vim qhov kev soj ntsuam ntawm qhov sib txawv qhia tsis muaj qhov sib txawv tseem ceeb ntawm EOsof sib txawv. xyoo ntawm kev tsim khoom (p> 0.05). Hais txog L. cubeba thiab C. schoenanthus EOs, cov txiaj ntsig no tau pom zoo nrog cov txiaj ntsig tau los ntawm GCMS quantitative tsom xam uas qhia txog yuav luag zoo tib yam citral npaum li cas hauv EOs ntawm ntau xyoo sib txawv. Lub batch 2020 ntawm V. officinalis EO muaj ib qho me ntsis citral ntau dua li cov batch 2019 thiab 2018. Txawm li cas los xij, raws li citral concentration-responsecurve, citral tshaj hauv batch 2020 tsis txaus los txiav txim siab qhov feem pua ​​​​ntawm cov enzymatic inhibition. yuam kev cuam tshuam nrog kev ntsuas. Yog xav paub ntxiv, saib daim duab S1 hauv Cov Khoom Siv Ntxiv. Ntawm qhov tod tes, kev tsom xam ntawm qhov sib txawv (ANOVA) ua raws li Tukey-Kramer post-hoctest qhia tias peb qhov kev sim M. officinalis EOs, muab los ntawm cov tuam txhab sib txawv, txwv tsis pub nceb.tyrosinaserau ntau qhov sib txawv, uas yuav piav qhia ntxiv hauv kab lus hauv qab no. Qhov loj tshaj inhibitory kev ua ub no tau pom rau EOs ntawm L.cubeba, M. officinalis 1, thiab V. officinalis, uas inhibited 59 ± 6 feem pua, 55 ± 7 feem pua, thiab 52 ± 6 feem pua.tyrosinasekev ua haujlwm, feem, thaum kuaj ntawm qhov concentration ntawm 166.7 μg / mL. Cov txiaj ntsig tseem ceeb (p < 0.05)="" cov="" haujlwm="" qis="" dua="" tau="" pom="" rau="" eos="" ntawm="" c.="" schoenanthus="" thiab="" m.officinalis="" 2="" thiab="" 3="" uas="" nws="" cov="" enzyme="" inhibitory="" yog="" 42="" ±="" 5="" feem="" pua,="" 40="" ±="" 5="" feem="" pua,="" thiab="" 38="" ±="" 6.="" feem="" pua,="" feem.="" table="" 5="" muab="" cov="" inhibitor="" concentration="" uas="" halved="" lub="" enzyme="" kev="" ua="" si="" nyob="" rau="" hauv="" lub="" muab="" kev="" sim="" tej="" yam="" kev="" mob="" (ic50)="" rau="" txhua="" soj="" ntsuam="" inhibitor="" (ie,="" eos,="" singlecompounds,="" thiab="" kojic="" acid).="" tag="" nrho="" cov="" eos="" tau="" zoo="" inhibited="" nceb="" tyrosinase="" thiab="" nthuav="" tawm="" cov="" haujlwm="" inhibitory="" uas="" yog,="" qhov="" nruab="" nrab,="" 100-lub="" sij="" hawm="" qis="" dua="" kojicacid,="" uas="" tau="" siv="" los="" ua="" qhov="" zoo="">

2.3. Kev txheeb xyuas ntawm Ntxiv Bioactive Cheebtsam, Dhau li ntawm Citral, los ntawm Bioassay-Guided FractionationThe histogram qhia hauv daim duab 3 piv cov feem pua ​​​​ntawm kev sim ntsuas enzymatic inhibitions rau cov txiaj ntsig uas yuav xav tau yog neral thiab geranial (considered li sum, piv txwv li, citral) yog cov tsuas yog active compounds nyob rau hauv cov kev tshawb fawb EOs. Cov txiaj ntsig no tau ntsuas los ntawm kev sib cuam tshuam los ntawm citral concentration-response nkhaus. Raws li tuaj yeem sau tseg, C. schoenanthus, M. officinalis 2, thiab M. officinalis 3 tso tawm inhibitory ac tivities uas nyob rau hauv-kab nrog lawv cov ntsiab lus citral, thaum L. cubeba, M. officinalis 1, thiab V. officinalis EOs inhibited nceb. tyrosinase mus rau qhov ntau dua li qhov xav tau. Ib txoj hauv kev bio-guided tau txais yuav los txheeb xyuas cov ntsiab lus ntxiv uas ua rau muaj txiaj ntsig rau kev ua haujlwm citral. Cov pa oxygenated thiab hydrocarbon feem ntawm L. cubeba, M. officinalis 1, thiab V. officinalis EOs raug cais los ntawm flash chromatography thiab kuaj ib tus zuj zus rau lawv cov nceb.tyrosinaseinhibitory kev ua ub no. Cov seem phytohemical kuaj rau lawv cov nceb tyrosinase inhibitory kev ua ub no. Cov fractions phytohemical fractions raug kuaj nyob rau tib qhov concentration raws li lawv cov concentration thaum kuaj ing 166.7 µg / mL ntawm EO (saib Cov Khoom Siv thiab Cov Txheej Txheem ntawm Tshooj 3.2).Table 6 qhia txog qhov concentration ntawm neral, geranial, citronellal, limonene, thiab -myrcene nyob rau hauv cov pa oxygenated thiab hydrocarbon feem ntawm cov fractionated EOs.

Raws li rau L. cubeba thiab V. officinalis EOs, ob qho tib si cov pa oxygenated thiab hydrocarbon fractions inhibited nceb tyrosinase, txawm tias muaj ntau qhov sib txawv. Cov kev ua ub no ntawm theoxygenated feem (53 ± 3 feem pua ​​​​thiab 44 ± 5, feem) suav rau feem ntau ntawm EOsanti-tyrosinasemuaj peev xwm thiab tau nyob rau hauv txoj kab nrog cov ntsiab lus citral, tawm tswv yim hais tias cov tebchaw uas ua rau citral kev ua si yog cov hydrocarbon fractions.Cov hydrocarbon feem ntawm L. cubeba thiab V. officinalis EOs tam sim no zoo ib yam tshuaj muaj pes tsawg leeg. Limonene (68.4 thiab 50.3 feem pua, raws li), trans- -caryophyllene (12.0 thiab 7.8 feem pua, raws li), -pinene (1.7 thiab 7.5 feem pua, raws li), -pinene (2.5 thiab 12.9%), sabinene (2.7 thiab 3.8, raws li), thiab -myrcene (2.0 thiab 2.4 feem pua, raws li) yog cov sib xyaw ua ke ntau tshaj plaws hauv ob feem thiab muaj nyob rau hauv cov nqi sib xws, tshwj tsis yog rau -pinene thiab -pinene, uas yeej nyob rau hauv V. officinalis EO hydro carbon feem.

The chiral recognition revealed high enantiomeric purities in favor of the (-)-configured enantiomers for trans-β-caryophyllene (>99 feem pua ​​​​hauv ob qho tib si EOs), limonene (97 thiab 94 feem pua ​​​​hauv L. cubeba thiab V. officinalis EO, feem) thiab sabinene (87 feem pua ​​​​hauv ob qho tib si EOs), thaum sib txawv ent enantiomeric ntau dhau tau pom rau -pinene ((-)- enantiomer: 38 feem pua ​​​​hauv L. cubebaEO thiab 73 feem pua ​​​​hauv V. officinalis EO) thiab -pinene ((-)-enantiomer: 67 feem pua ​​​​hauv L. cubeba EO thiab 88 feem pua ​​​​hauv V. officinalis EO). Hauv ob qho EOs, (-)-limonene tso nyiaj ntau dua 50 feem pua ​​​​ntawm tag nrho cov feem. Txawm li cas los xij, txawm hais tias cov kev tshawb fawb yav dhau los tau tshaj tawm txog kev ua haujlwm inhibitory tiv thaiv cov nceb tyrosinase vim tias nws muaj ntau heev [22,23], (-)-limonene ntawm no tsis qhia txog tyrosinase inhibitory kev ua. Cov txiaj ntsig zoo sib xws tau txais rau (ntxiv rau )-limonene, cov sib xyaw ntawm cov racemic, thiab cov tebchaw (-)-trans- -caryophyllene, (±)- -pinene, thiab (±)- -pinene . Sabinene tsis raug kuaj raws li nws twb tau ua pov thawj tias muaj qhov tsis zootyrosinaseCov tshuaj tiv thaiv kab mob [8] Hauv kev pom zoo nrog kev tshawb pom yav dhau los [8], myrcene txo cov nceb tyrosinase kev ua haujlwm. Thaum kuaj ntawm qhov concentration pom 166.7 µg / mL ntawm L. cubeba thiab V. officinalis EOs, -myrcene kev ua haujlwm txuas rau qhov sib txawv ntawm EOs 'kev cia siab inhibitory cuam tshuam yog tias citral yog tib qho kev sib xyaw ua ke thiab cov txiaj ntsig kev sim. Contrary to cov kev soj ntsuam los ntawm Matsuura li al. [8], -myrceneproved los ua cov nceb muaj zog tyrosinase inhibitor dua li citral, vim nws IC50 yuav luag qis dua (13.3 µg / mL vs. 121.8 µg / mL). Qhov sib txawv no yuav raug muab rau cov substrates sib txawv siv; Matsuura et al. tshawb nrhiav nceb tyrosinase diphenolaseactivity nkaus xwb, raws li lawv siv L-DOPA ua substrate, whereas, nyob rau hauv txoj kev tshawb no, L-tyrosine yog siv. Cov kev tshawb pom tam sim no qhia tias -myrcene yuav ua tau zoo dua ntawm inhibitingmushroom tyrosinase monophenolase kev ua si dua li diphenolase ib.

M. officinalis EO 1 qhia txog qhov me me hydrocarbon feem uas suav nrog tsawg dua 3 feem pua ​​​​ntawm tag nrho, thiab tsis muaj tyrosinase inhibitory kev ua. Txawm li cas los xij, M. officinalisEO 1 cov pa oxygenated feem inhibited nceb tyrosinase mus rau ntau dua li qhov xav tau los ntawm nws cov ntsiab lus citral (Daim duab 3). Cov feem no muaj cov nyiaj tseem ceeb ntawm citronellal ntxiv rau neral thiab geranial thiab chiral tsom xam pom tias muaj kev siab dawb huv ntawm citronellal siab ntawm (ntxiv rau ) enantiomer (98.3 feem pua). Thaum kuaj nyob ib leeg, ntawm qhov siab ntawm 166.7 µg / mL, (ntxiv rau )-citronellal inhibited mushroomtyrosinase rau qhov tsis txaus ntseeg, txawm hais tias nws cov haujlwm tau ua kom zoo dua thaum kuaj nrog citral. Cov txiaj ntsig no tuaj yeem piav qhia qhov sib txawv pom hauv feem pua ​​​​ntawm cov ncebtyrosinaseinhibition nyob rau hauv ntau yam M. officinalis EOs. M.officinalis EO 2 thiab 3 tam sim no cov ntsiab lus citronellal tsawg heev, uas tej zaum yuav yog vim li cas vim li cas lawv cov haujlwm inhibitory qis dua li ntawm M. officinalis EO 1.

3. Cov ntaub ntawv thiab cov txheej txheem

3.1. Reagents

Dimethyl sulfoxide (DMSO), nceb tyrosinase los ntawm Agaricus bisporus (JE Lange) Imbach, L-tyrosine, kojic acid, citral, citronellal, -myrcene, (-)-limonene, (±)-limonene, ( ±)- , thiab pinene tau yuav los ntawm Merck Life Science Srl (Milan, Ltalis). Litsea cubeba, Verbena officinalis, thiab Cymbopogon schoenanthus EOs tau muab los ntawm Erboristeria Magentina Srl (Poirino, Ltalis). Peb pawg ntawm cov xyoo sib txawv (piv txwv li, 2020,2019, 2018) tau sim rau txhua tus. Peb cov qauv ntawm Melissa officinalis EOs tau tshawb xyuas; ib qho yog muab los ntawm Agronatura (Spigno Monferrato, Alessandria), ib qho los ntawm ErboristeriaMagentina Srl, thaum kawg tau yuav los ntawm lub khw hauv zos thiab yog los ntawm SpecchiasolS.rl (Bussolengo, Ltalis). Hauv cov ntawv nyeem, cov kws sau ntawv xa mus rau qhov sib txawv EOs ntawm Melissaofficinalis li M. officinalis EOs 1, 2, thiab 3, raws li. Cov muab EOs tau txais ua raws li cov txheej txheem tau piav qhia hauv European Pharmacopoeia [24]. Melissa officinalisand Verbena officinalis EOs tau tsim los ntawm hydrodistillation los ntawm nplooj thiab cov nroj tsuag hauv qhov chaw, feem; Ib yam li ntawd, Litsea cubeba thiab Cymbopogon schoenanthus EOs tau txais los ntawm chav distillation ntawm cov txiv hmab txiv ntoo tshiab thiab cov chaw dav hlau tshiab, feem. Txhua EO tau soj ntsuam ib tus zuj zus los ntawm GC-MS sai li sai tau thaum nws tau yuav / muab los ntawm cov chaw tsim khoom lag luam, txhua xyoo khaws cia, thiab ua ntej kev kawm txog nws cov kev ua haujlwm ntawm mushroomtyrosinase inhibitory.

3.2. Hauv Vitro Tyrosinase Inhibitory Assay

Covtyrosinaseinhibitory kev ua ub no ntawm EOs thiab cais cov tebchaw tau tshawb xyuas hauv vitro siv lub colorimetric nyeem ntawv-raws li enzyme assay optimized los ntawm Zengh li al. [25], nrog kev hloov kho me ntsis. Lub tyrosinase inhibitory kev ua ub no ntawm EOs, nrog rau lawv cov hydrocarbon thiab oxygenated feem thiab cov ntsiab lus ntshiab tau tshawb xyuas hauv vitrousing ib colorimetric nyeem ntawv-based enzyme assay uas tau optimized los ntawm Zengh li al. [25], nrog kev hloov kho me ntsis: qhov kev ntsuam xyuas tau ua nyob rau hauv chav tsev kub thiabtyrosinaseinhibition raug ntsuas los txiav txim siab txog kev tswj hwm thiab kev nqus cov qauv tom qab 6 min ntawm incuba tion, es tsis yog tom qab 20 min, thiaj li ua haujlwm raws li cov kab tawm ntawm cov enzymaticreaction, uas muab cov txiaj ntsig inhibition ntau dua [26,27]. Mushroom tyrosinasefrom Agaricus bisporus (JE Lange) Imbach tau raug xaiv rau txoj kev tshawb no. L-Tyrosine tau siv los ua cov substrate, txhais tau hais tias tag nrho tyrosinase inhibitory kev ua haujlwm tau tshawb xyuas yam tsis muaj qhov txawv ntawm tyrosinase monophenolase thiab diphenolase kev ua haujlwm.Photometric ntsuas ntawm 475 nm tau ua ntawm Thermo spectronic Genesys6 thiab kojic acid tau siv los ua tus tswj hwm zoo. Cov kev daws teeb meem ntawm kev tshawb nrhiav muaj peev xwm inhibitors (EOs, EO cais feem, EO tus neeg sib txuas, thiab kojicacid) tau npaj hauv DMSO. Table 7 qhia txog qhov ntsuas qhov ntsuas rau txhua qhov inves tigated muaj peev xwm inhibitor. Cov ncebtyrosinasetov 200 U/mL (27.9 µg/mL) tau npaj hauv sodium phosphate buffer (pH 6.8) thiab aliquots ntawm 9 mL tau khaws cia hauv −18 ◦C thiab thawed ua ntej kev sim. Tyrosine tov 0.1 mg/mL tau npaj rau hauv sodium phosphate buffer (pH 6.8) thiab txuas ntxiv txhua hnub. Cov tshuaj tiv thaiv sib xyaw ua ke tau muab tso rau hauv lub vial hauv qhov kev txiav txim hauv qab no: 1 mL ntawm nceb tyrosinasesolution 200 U / mL; 1 mL ntawm sodium phosphate buffer tov; 10 µL ntawm EO / ib qho com pound / kojic acid tov; thiab, thaum kawg, 1 mL ntawm tyrosine tov 0.1 mg / mL. Qhov kawg DMSO feem pua ​​ntawm cov tshuaj tiv thaiv sib tov yog 0.3 feem pua. Qhov kev ntsuam xyuas tau ua nyob rau hauv lub kaw lus 4 mL vial kom tsis txhob poob ntawm ib qho EO Cheebtsam rau hauv ib puag ncig ib puag ncig thiab txo qis lawv tso rau hauv lub taub hau qhov chaw saum toj no cov tshuaj tiv thaiv sib tov. Cov tshuaj tiv thaiv yog incubated nyob rau hauv ib lub thermostatic da dej ntawm 25 ◦C rau 6 min. Tom qab ntawd, qhov absorbance ntawm 475 nm tau sau npe, raws li qhov wavelength no tso cai rau kev txheeb xyuas ntawm dopachrome. Lub absorbance sib raug rau 100 feem pua ​​​​ntawm tyrosinase kev ua haujlwm tau ntsuas los ntawm kev hloov EOs / tus kheej cov tshuaj / kojic acid tov nrog 10 µL ntawm ntshiab DMSO.Blank daws tau npaj raws li hauv qab no: 2 mL ntawm sodium phosphate buffer tov, 10 µL ntawm EO / tus kheej compound. /kojic acid / DMSO daws, thiab 1 mL ntawm tyrosinesolution 0.1 mg / mL. Qhov feem pua ​​​​ntawm tyrosinase inhibition tau ntsuas raws li qhov sib npaug hauv qab no: feem pua ​​Inhibition=∆A (Control) − ∆A (Sample) / ∆A (Control) × 100,∆A (Control) lossis (Sample) {{ 33}} A475 (Control) lossis (Sample) − A475 (Control Blank) lossis (Sample Blank).

3.3. Flash Kem Chromatography

EO fractionation tau ua tiav ntawm lub teeb ci kem chromatography PuriFlash450 los ntawm Sepachrom (Rho, Milan, Ltalis), nruab nrog ob qho tib si UV thiab ELSD ntes. Cov ntsiab lus ntawm EO fractionated: 900.0 mg. Nyob ruaj ruaj: spherical silica gel hais, 50 µm, 25 mg (Purezza®-Sphera Cartridge Stationary) yog los ntawm Sepachrom; mobile theem: petrolether (A) thiab ethyl acetate (B); ntws-nqi 25 mL / min. Linear gradient elution tau txais los ntawm 100 feem pua ​​​​ntawm A txog 80 feem pua ​​​​ntawm A thiab 20 feem pua ​​​​ntawm B tshaj 20 min.

3.4. Cov xwm txheej tsom xam

Cov kev daws teeb meem EOs thiab cov ntawm lawv cov feem cuam tshuam tau npaj rau hauv cyclohexaneat concentration ntawm 5.0 mg/mL thiab tshuaj xyuas los ntawm GC-MS. Citral, citronellal, -myrcene, thiab limonene tau raug txheeb xyuas nyob rau hauv txhua EO thiab cov khoom sib txuas sib txuas uas siv cov txheej txheem calibration sab nraud. Cov qib calibration tsim nyog tau npaj incyclohexane thiab tshuaj xyuas los ntawm GC-MS. Tridecane (C13) 1. Table 2 piav qhia txog qhov xav tau ntau npaum li cas rau txhua qhov sib xyaw kom muaj nuj nqis.

GC-MS kev tshuaj xyuas tau ua tiav siv Gerstel MPS-2 ntau hom qauv (Mülheim ib der Ruhr, Lub teb chaws Yelemees) ntsia rau ntawm Agilent 6890 N GC txuas rau 5975 MSDand nruab nrog ChemStation Version E. 02.02.1431 data processing system (AgilentTechnologies, Santa Clara, CA, USA). GC tej yam kev mob: injector kub: 250 ◦C; txhaj tshuaj: phua; ratio: 1/20; cov pa roj: helium; Tus nqi ntws tsis tu ncua: 1 mL / min; kab: Mega5 (95% polydimethylsiloxane, 5% phenyl) df 0.25 µm, dc 0.25 mm, ntev 25 m, los ntawm MEGA (Legnano, Ltalis). Kev ntsuas kub: 50 ◦C / / 3 ◦C / min / / 180 ◦C / / 10 ◦C / min / / 250 ◦C (5 min). MSD tej yam kev mob: MS ua nyob rau hauv EI hom (70 eV); scan ntau: 35 txog 350 amu; nyob 40ms; ion qhov kub: 230 ◦C; quadrupole kub: 150 ◦C; Hloov kab kub: 280 ◦C. EO cov cim tau raug txheeb xyuas los ntawm kev sib piv ob qho tib si lawv cov kab ntsuas kev ntsuas (ITs), xam piv rau C9-C25 hydrocarbon sib tov, thiab lawv cov massspectra tawm tsam cov qauv qhabnias, lossis los ntawm cov khw muag khoom loj loj (Adams, 2007). EO chiral tsom xam tau ua los ntawm kev siv cov kev ntsuas tib yam ntawm 2, 3-di-O-methyl-6-Ot-butyldimethylsilyl- -CD (2,3DM6TBDMS -CD) df 0.25 µm, dc 0.25 hli, ntev 25 m los ntawm MEGA. Kev ntsuas kub: 40 ◦C (1 min) // 2 ◦C / min / / 220 ◦C (5 min).

GC-FID tsom xam tau ua tiav ntawm tib lub ntsuas. GC tej yam kev mob: injectortemperature: 250 ◦C; txhaj hom: phua; ratio: 1/20; cov pa roj: hydrogen; Tus nqi ntws: 1 mL / min. Kev ntsuas kub: 40 ◦C (1 min) // 2 ◦C / min / / 220 ◦C (5 min).

4. Cov lus xaus

Lub hom phiaj ntawm qhov kev tshawb nrhiav no yog (1) txhawm rau tshuaj xyuas qhov tseeb hauv vitromushroomtyrosinaseinhibitory kev ua ub no ntawm Cymbopogon schoenanthus, Litsea cubeba, Melissa officinalis, thiab Verbena officinalis EOs thiab (2) los txiav txim seb lawv cov biologicalactivity yog ascribed rau lawv citral cov ntsiab lus nkaus xwb los yog yog hais tias muaj ntxiv bioactive monoterpenes uas pab rau cov kev tshawb fawb lom biological ua si los ntawm kev siv ib tug bioassay-guidedfractionation txoj hauv kev. Txoj kev tshawb no tau txheeb xyuas tias hauv L. cubeba thiab V. officinalis EOs, -myrcene pab txhawb rau EOs inhibitory kev ua si txawm tias nws tsawg thiab nws tau pom tias muaj zog inhibitory ntau dua rau citral. Qhov thib ob loj nrhiav yog tias (ntxiv rau)-citronellal txhim kho citral ncebtyrosinaseinhibitory hwj chim, muaj peev xwm viasynergistic sib cuam tshuam raws li nws tso tawm tsis muaj kev ua si ntawm nws tus kheej. Qhov kev tshawb nrhiav tom kawg tau piav qhia vim li cas hauv M. officinalis EOs uas dais tsis pom zoo (ntxiv rau )-citronellal cov nyiaj, cov haujlwm inhibitory tau nyob rau hauv-kab nrog lawv cov ntsiab lus citral thaum qhov tsis sib xws yog qhov tseeb rau M. officinalis EOwith kuj siab (ntxiv rau)-citronellal abundance.Txawm li cas los xij. Txawm hais tias cov kev tshawb fawb ntxiv tseem yuav tsum tau ua kom raug txheeb xyuas hom kev sib cuam tshuam uas tshwm sim hauv nruab nrab ntawm -myrcene thiab citral thiab nyob nruab nrab ntawm citronellal thiab citral, thiab txhawm rau txheeb xyuas cov haujlwm inhibitory ntawm cov EOs thiab cov tib neeg sib txuas ntawm tib neeg.tyrosinase, cov txiaj ntsig ntawm txoj kev tshawb no tuaj yeem pab tsim kev sib xyaw ntawm EOs lossis enrichedEOs uas txhim kho lawv cov kev ua haujlwm lom neeg thiab ua rau lawv muaj peev xwm ua kom muaj zog hauv kev kho mob hyperpigmentation.

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