Kev hloov pauv hauv Amino Acid Profiles Thiab Bioactive Compounds Ntawm Thai Silk Cocoons As Cuam Tshuam Los Ntawm Dej Extraction Part 2

Thov hu rauoscar.xiao@wecistanche.comyog xav paub ntxiv

2.8. Kev sib txheeb

Kev txheeb xyuas kev sib raug zoo (Pearson test) tau siv los ua kev sib piv (raws li kev sib txheeb coefficients, r) ntawm thiab nruab nrab ntawm qhov tseem ceeb rau tag nrho cov ntsiab lus phenolic, tag nrho cov ntsiab lus flavonoid, DPPHradical-scavenging kev ua, ABTS ntxiv, FRAP assay, anti- -amylase , anti- -glucosidase, thiab anti-glycation ntawm SPE hauv Nangsew thiab Eri hom (Tables 5 thiab 6). Txhua qhov ntsuas tau los ntawm txoj kev tshawb fawb pom tau tias muaj kev sib raug zoo ntawm TPC, TFC, DPPH, ABTS, FRAP anti- -amylase, anti- -glucosidase, thiab anti-glycation. Qhov no tej zaum yuav yog vim hais tias cov bioactive compounds nyob rau hauv txhob lo lo ntxhuav extract muaj peev xwm ntawm scavenging ntau dawb radicals thiab anti- -amylase, anti- -glucosidase, thiab anti-glycation. Peb qhov kev tshawb pom kuj tau pom tias muaj kev sib raug zoo heev ntawm cov txiaj ntsig rau ob hom kab mob. Cov txiaj ntsig no tau pom zoo nrog cov Benzie thiab Stezo [29], uas pom muaj kev sib raug zoo ntawm tag nrho cov ntsiab lus phenolic thiab FRAP kev soj ntsuam. Cov txiaj ntsig zoo sib xws kuj tau pom nyob nruab nrab ntawm TPC thiab DPPH(R2=0.711) thiab FRAP (R{15}}.948), ntsig txog hauv iab gourd raws li qhia los ntawm Kubola [30]. Kumar et al.[31] ua qhov kev ntsuam xyuas ntawm kev ua haujlwm antioxidant thiab tag nrho phenol hauv ntau hom Lantana Camara nplooj. Cov ntawv rho tawm tau pom muaj kev sib raug zoo ntawm cov ntsiab lus phenolic thiab DPPH, FRAP, thiab ABTSscavenging kev ua ub no. Qhov kev sib raug zoo tshaj plaws (r=0.998, R2=0.997) tau pom ntawm tag nrho cov ntsiab lus phenolic thiab ABTS scavenging assay. Tsis tas li ntawd, kev sib raug zoo ntawm ABTSand TPC(R2=0.885) thiab DPPH(R{25}}.999), feem, hauv Gynura procumbens nplooj tau raug tshaj tawm los ntawm Kaewseejan li al. [32].

Cov kev sib raug zoo ntawm cov khoom siv roj ntsha thiab cov haujlwm inhibitory tau pom nyob rau hauv kev tshawb fawb ntawm Quan li al.[33] leej twg tshawb xyuas cov tshuaj antioxidant thiab muaj peev xwm tiv thaiv kab mob ntshav qab zib ntawm C.tramdenum bark extracts. Lawv cov txiaj ntsig kuj pom tiasantioxidantkev soj ntsuam (xws li DPPH, ABTS, txo lub zog, thiab nitric oxide) tau sib raug zoo nrog -amylase thiab -glucosidase inhibitory assay ntawm p<>

Thov nias ntawm no kom paub ntxiv

3. Cov ntaub ntawv thiab cov txheej txheem 3.1.Silk Coc00ns

Nangsew thiab Eri hom ntawm Thai silk cocoons tau sau. CovNangsew strain(daj cocoons) tau los ntawm Baan Hua Saban, Phutthaisong District, Buriram, Thaib teb. Tus kab mob Eri (dawb cocoons) tuaj ntawm Nong Ya Plong Community, Mancha Khiri, Khon Kaen, Thaib teb.

3.2. Tshuaj thiab Reagents

Txhua tus qauv tau txais los ntawm Sigma-Aldrich Co.(St. Louis, MO, USA), suav nrog cov amino acids tseem ceeb: phenylalanine, valine,tryptophan, threonine, isoleucine, methionine, histidine, arginine, lysine, thiab leucine. Cov amino acids uas tsis tseem ceeb tau kawm yog glycine, glutamic acid, aspartic acid, glutamine, serine, tyrosine, alanine, thiab asparagine. Cov tshuaj tiv thaiv antioxidant kev txiav txim siab reagents tau txais los ntawm Sigma-Aldrich Co: Folin-Ciocalteu reagent,2,2-diphenyl-1-picrylhydrazyl(DPPH), 2,4,6-tripiridyl-s-triazine (TPTZ), 2,2'-and-bis(3-thylbenzthiazoline-6-sulphonic acid)(ABTS). Cov kuab tshuaj thiab cov tshuaj reagents siv hauv HPLC tsom xam tau yuav los ntawm Merck (Darmstadt, Lub teb chaws Yelemees). Cov -amylase (los ntawm Aspergillus oryzae), -Glucosidase (los ntawm Bacillus stearothermophilus), p-nitrophenyl- -D-glucopyranoside (PNP-G), bovine serum albumin (BSA), thiab sodium azide tau muas los ntawm Sigma-Aldrich. Co.

Cistanche tuaj yeem txhim kho kev tiv thaiv

3.3. Extraction nrog Distilled dej

Extraction tau nqa tawm siv txoj kev ntawm Sangwong li al. [34]. Silk cocoons tau txiav rau hauv me me pieces. Ob gram ntawm cov khoom no tau muab rho tawm nrog 200 mL ntawm cov dej distilled ntawm 100 degree rau lub sijhawm rho tawm. Cov extracts tau centrifuged ntawm 5000 × g rau 10min los ntawm centrifuge (Universal 320/320R, Hettich, Boucherville, QC, Canada). Cov extracts tau lim los ntawm Whatman No.1 daim ntawv nyob rau hauv lub tshuab nqus tsev kom tshem tawm cov khoom insoluble, yog li tsim cov silk protein extract (SPE).

3.4. Kev txiav txim siab protein

Kev txiav txim siab ntawm cov protein tau txiav txim siab piav qhia los ntawm Bradford assay [35]. SPE (0.5 mL) tau ntxiv rau 1.0mL ntawm Bradford tov thiab incubated ntawm chav tsev kub rau 5 min. Bovine serum albumin (BSA) tau siv los ua tus qauv siv protein. Lub absorbance ntawm cov qauv raug ntsuas ntawm 595 nm nrog ib tug pom spectrophotometer (DR 2700rM Portable Spectrophotometer, Hach, Loveland, CO, USA).

3.5.Amin0-Acid Cov ntsiab lus los ntawm LCMS/MS

Cov amino-acid tsom xam koom nrog LC-MS-MS (Shimadzu LCMS-8030) triple-quadrupole mass spectrometer, hom electrospray ionization (ESI), ua raws li kev tshuaj xyuas hauv Shimadzu HPLC system (Shimadzu, Kyoto, Nyiv) (Chumroenphat et al. [36). Kev tshuaj xyuas HPLC suav nrog tus nqi ntws ntawm 0.2 mL/min; Qhov kub ntawm lub qhov cub yog 38 degree thiab autosampler yog ntawm 4 degree. Cov theem txawb tau npaj (A) aqueous formic acid {{10}}}.1 feem pua ​​(o/o) thiab (B) methanol-dej (50:50); ces lub tov yog formic acid 0.1 feem pua ​​(o/v).Lub koob autosampler tau purged nrog methanol ua ntej thiab tom qab aspiration ntawm tus qauv. Cov kev ntsuam xyuas tau ua nyob rau hauv triplicate.

3.6. Tag nrho cov ntsiab lus Phenolic (TPC)

Tag nrho cov ntsiab lus phenolic nyob rau hauv silk protein extract tau soj ntsuam raws li txoj kev Folin-Ciocalteu [30]. Luv luv, 0.3 mL ntawm SPE yog tov nrog 2.25 mL ntawm 10 feem pua ​​​​Folin-Ciocalteu reagent yaj hauv dej distilled. Tom qab 5 min incubation, 2.25 mL ntawm 6 feem pua ​​​​sodium carbonate (NazCO) daws tau ntxiv thiab cov mixs tau tso tseg rau sawv ntsug rau 90 min ntawm chav tsev kub. Kev nqus ntawm cov qauv tshuaj tau ntsuas ntawm 725 nm siv lub spectrophotometer. TPC hauv taum tau qhia tias yog mg gallic acid sib npaug (GAE) ib g qhuav hnyav (mg GAE / g DW). Cov kev ntsuam xyuas tau ua nyob rau hauv triplicate.

3.7. Tag nrho cov ntsiab lus Flavonoid (TFC)

Tag nrho cov ntsiab lus flavonoid ntawm SPE raug txiav txim raws li Kubota thiab [30], hloov kho. Luv luv, 0.5 mL ntawm txhua tus qauv tov yog tov nrog 2.25 ml ntawm distilled dej thiab 0.15 ml ntawm 5 feem pua ​​NaNO, tov (w/v). Cov tshuaj tau tso cai sawv ntsug rau 6 min thiab tom qab ntawd 0.3 mL ntawm 10 feem pua ​​AlCl3(w/v) tau ntxiv rau cov tshuaj. Tom qab 5 min, 0.1 mL ntawm 1 M NaOH (w / ø) kev daws teeb meem tau ntxiv thiab tom qab ntawd qhov nqus tau ntsuas ntawm 510 nm siv lub spectrophotometer. Cov txiaj ntsig tau qhia raws li mg rutin sib npaug (RE) ib g qhuav qhov hnyav (mg RE / g DW).

3.8. DPPH Radical Scavenging Activity

Cov kev ua haujlwm antioxidant ntawm SPE tau txiav txim siab raws li kev ua haujlwm ntawm qhov ruaj khov 1,1-diphenyl-2-picrylhydrazyl (DPPH) dawb radical raws li Brand-Williams li al.[37]. Silk protein extract 0.1 mL tau ntxiv rau 3 ml ntawm a 0.001 M DPPHin methanol. Cov sib tov tau khaws cia rau 30 min nyob rau hauv qhov tsaus ntuj ntawm chav tsev kub. Absorbance tau txiav txim siab ntawm 517 nm siv lub spectrophotometer. Qhov sib npaug ntawm kev suav cov DPPH inhibition yog raws li hauv qab no:

(1) Inhibition (%)=[(Abs. control- Abs.sample)/Abs. tswj] × 100)

qha Abs. tus qauv yog qhov nqus ntawm cov qauv thiab Abs. tswj yog absorbance ntawm kev tswj.

3.9.Antioxidant Activity los ntawm ABTS Assay

ABTS radical cation method ntawm Re et al. (1999) [22] tau hloov kho los ntsuas qhov cuam tshuam dawb radical-scavenging. Cov tshuaj ATBS ua haujlwm tau npaj los ntawm kev sib txuas ntawm 7.4 mM ABTS thiab 2.6 mM poov tshuaj persulfate ntawm qhov sib piv ntawm 1: 1 (v / v), tso cai rau nws tawm tsam hauv qhov tsaus ntuj rau 12 teev ntawm chav tsev kub, thiab tom qab ntawd diluting nrog dej deionized. . Lub absorbance tau txiav txim siab ntawm 734 nm siv lub spectrophotometer kom txog thaum nws mus txog 1.1 ±0.02.Tom qab ntawd 150 μL ntawm SPE thiab 2850 μL ntawm ATBS ua haujlwm daws tau tshwm sim hauv qhov tsaus ntuj rau 2 h nrog kev sib tov, thiab cov absorbance ntawm 734 nm siv lub spectrophotometer; 100 feem pua ​​​​methanol tau siv los tswj. Qhov sib npaug rau xam qhov inhibition yog raws li Equation(1).

3.10. Ferric Txo / Antioxidant Power Assay (FRAP)

Kev txo hwj chim ntawm cov extracts yog raws li FRAP assay [23]. Cov tshiab FRAP reagent tau npaj los ntawm kev sib xyaw 100 mL ntawm acetate tsis (0.3 mol LI, pH 3.6), 10 mL ntawm TPTZsolution yaj hauv 10 mL ntawm 40 mmol L-1HCland 10 mL ntawm 20mmolL -1FeCl; Nyob rau hauv ib tug ratio ntawm 10∶1∶1 thiab, 120 ml ntawm distilled dej ntawm 37 degree. Luv luv, 60 μL ntawm SPE thiab 180uL ntawm cov dej deionized tau ntxiv rau 1.8 mL ntawm FRAP reagent. Cov tshuaj tau shaken los ntawm vortex mixing. Tom qab cov tshuaj tau incubated nyob rau hauv ib da dej ntawm 37 degree rau 4 min, lub absorbance yog ntsuas ntawm 593 nm tiv thaiv ib tug tswj. FRAPvalues ​​tau tshwm sim raws li mg FeSO4 ib g qhuav hauv paus (mg FeSO4 / g DW).

3.11.Inhibitory Activity tiv thaiv cov Enzyme -Amylase

-Amylase enzyme inhibitory kev ua haujlwm tau ntsuas los ntawm txoj kev Xiao li al. (2006)[38] nrog kev hloov kho me ntsis. Lub substrate yog npaj los ntawm boiling 100 mg qos starch nyob rau hauv 5 mL phosphate tsis (pH 7.0) rau 5 min, ces txias rau chav tsev kub. SPE (50μL), substrate (300μL) thiab 5ug / mL -amylase tov (20μL) tau tov thiab cov tshuaj tau incubated ntawm 37 degree rau 15 min. Cov tshuaj tiv thaiv tau nres los ntawm kev ntxiv 50 μL 1 M HCl, thiab tom qab ntawd ntxiv 50μL iodine tov. Lub absorbance tau ntsuas ntawm 650nm los ntawm tus nyeem ntawv microplate. Acarbose tau siv los ua kev tswj xyuas zoo. Qhov feem pua ​​​​ inhibition ntawm x-amylase raug soj ntsuam raws li Equation (1).

3.12.Inhibitory Activity tiv thaiv cov Enzyme -Glucosidase

Kev ntsuam xyuas -glucosidase enzyme inhibition tau ua raws li txoj kev ntawm Wang thiab Zhao [39].SPE (2mL yaj hauv dimethylsulfoxide; DMSO) thiab 0.5 U/mL a-glucosidase(40 mL) tau sib xyaw hauv 120 mL ntawm 0.1 M phosphate buffer (pH 7.0). Tom qab 5 feeb ua ntej incubation, 5 mM p-nitrophenyl{16}}D-glucopyranoside tov (40 mL) tau ntxiv, thiab cov tshuaj tau incubated ntawm 37 degree rau 30 min. Lub absorbance tau ntsuas ntawm 405 nm los ntawm UV / Vis absorbance spectrophotometer microplate nyeem ntawv. Qhov feem pua ​​​​ inhibition ntawm -glucosidase raug soj ntsuam raws li Equation (1).

3.13.Kev ntsuam xyuas ntawm Anti-AGEs Tsim Kev Ua Haujlwm

Lub peev xwm inhibitory ntawm AGEs tsim ntawm cov protein ntau thiab nws cov feem tau raug ntsuas los ntawm kev siv Vinson thiab Howard [40]. Tag nrho cov ntim ntawm cov tshuaj tiv thaiv glycation (2.5 mL) tau npaj los ntawm kev sib xyaw 5{{10}}0 μL ntawm SPE, 500 μL ntawm 20 mg / mL BSA hauv phosphate tsis, 500 μL ntawm 0.5 M qabzib hauv phosphate buffer thiab 1mL ntawm 0.1 M phosphate tsis nyob ntawm pH 7.4 muaj 0.02 feem pua ​​(w/v) sodium azide. Qhov sib tov no tau tsim nyob rau ntawm 37 degree rau 5 hnub nyob rau hauv qhov tsaus ntuj thiab tom qab ntawd tus naj npawb ntawm fluorescent AGEs tsim tau txiav txim siab siv lub fluorescent spectrometer nrog ib qho excitation wavelength ntawm 330 nm thiab emission wavelength ntawm 410 nm. Qhov feem pua ​​​​ntawm kev tsim tawm los tiv thaiv AGEs raug xam raws li qhov tshwm sim ntawm fluorescent siv (FI) siv cov kab zauv hauv qab no: (2) n =[1-(FIsample - FIsample blank)/(control-control blank )] × 100 qhov twg Flsample yog fluorescent siv ntawm cov qauv, qauv dawb paug yog cov fluorescent siv ntawm cov qauv dawb paug, tswj yog fluorescent siv ntawm kev tswj thiab FIcontrol blank yog fluorescent siv ntawm cov qauv tswj dawb paug.

3.14. Kev txheeb cais

Tag nrho cov ntaub ntawv tau txheeb xyuas los ntawm SPSS software (IBM SPSS, Chicago, IL, USA). Cov txiaj ntsig tau tshaj tawm raws li qhov nruab nrab ± tus qauv sib txawv (SD) ntawm peb qhov sib txawv thiab cov ntaub ntawv tau txheeb xyuas siv ib txoj kev tsom xam ntawm qhov sib txawv (ANOVA) nrog qhov sib txawv tsawg kawg nkaus (LSD) kuaj los txiav txim qhov tseem ceeb ntawm kev tswj hwm thiab Pearson qhov kev sib raug zoo. sim ntsuas qhov sib txheeb ntawm cov txhais tau tias. Nyob rau hauv txhua rooj plaub, p<0.05 was="" considered="">

4. Cov lus xaus

Peb txoj kev tshawb fawb txog cov txiaj ntsig ntawm kev rho tawm cov protein ntau cuam tshuam nrog cov dej distilled ntawm 100 degree rau rau teev; Cov txheej txheem no tau muab cov txiaj ntsig zoo tshaj plaws ntawm cov ntsiab lus protein, amino acids, tag nrho cov ntsiab lus phenolic (TPC), tag nrho cov ntsiab lus flavonoid (TFC), DPPH radical-scavenging kev ua, ABTS radical scavenging peev xwm soj ntsuam, FRAP assay, anti- -amylase, anti-a-glucosidase, thiab anti-glycation thaum piv rau lwm lub sijhawm rho tawm. Tsis tas li ntawd, SPE ntawm Nangsew hom tau muab cov ntsiab lus siab dua ntawm tag nrho cov khoom noj muaj txiaj ntsig zoo, thaum piv nrog cov extracts ntawm Eri hom. Cov txiaj ntsig tau qhia tias Nangsew yuav yog hom nyiam tshaj plaws rau peb cov phiaj xwm txuas ntxiv los tsim cov khoom siv ntuj tsim rau cov khoom noj muaj txiaj ntsig, raws li sericin rho tawm thaum kho cocoon, uas muab sijhawm rau lwm txoj hauv kev tsim cov dej qab zib ua haujlwm.

Tus sau kev koom tes:Conceptualization, CBand SS; Txoj kev, CBand SS; Validation, CBand SS; Kev Tshawb Fawb Kev Tshawb Fawb, CBand SS; Kev Tshawb Fawb, CBand SS; Kev Sau Ntawv—Kev Npaj Ua Ntej, CBand SS; Kev Sau Ntawv-Tshawb xyuas thiab Hloov Kho, CBand SS; Visualisation, CBandS.S. 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 Thaib Kev Tshawb Fawb Txog Kev Tshawb Fawb thiab Kev Tsim Kho Tshiab (TSRI) thiab Siam Natural Products Co., Ltd.

Institutional Review Board Statement:Tsis siv tau.

Cov Lus Qhia Txog Kev Pom Zoo:Tsis siv tau. Cov Lus Qhia Muaj Cov Ntaub Ntawv: Tsis siv tau.

Kev lees paub:Lub Laboratory Equipment Center ntawm Mahasarakham University rau kev koom tes thiab kev tshawb fawb tau muab. Ua tsaug rau Colin Wrigley, University of Queensland, Australia, rau kev nyeem ntawv Askiv.

Kev tsis sib haum xeeb ntawm kev txaus siab:Cov kws sau ntawv tshaj tawm tsis muaj kev sib cav txog kev txaus siab.

Kab lus no yog muab rho tawm los ntawm Molecules 2021, 26, 2033. https://doi.org/10.3390/molecules26072033 https://www.mdpi.com/journal/molecules