Anti-Oxidative Thiab Anti-Aging Activities Of Porcine By-Product Collagen Hydrolysates Ua Part 2

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2.2. Ultrafiltration nyhuv ntawm Collagen Hydrolyzate Properties

Cov txheej txheem ultrafiltration tej zaum yuav yog ib qho tseem ceeb, muaj txiaj ntsig zoo rau kev tsim cov peptide feem me me nrog qhov xav tau molecular loj thiab siab bioactivity, nyob ntawm seb muaj pes tsawg leeg ntawm qhov pib hydrolyzate thiab cov haujlwm tau kawm [19]. Solubility, pub dawb amino pawg cov ntsiab lus, thiab cov txiaj ntsig ntawm CHs tom qab lyophilization tau qhia (Table 1). Solubility thiab pub dawb amino pawg cov ntsiab lus ntawm kev tswj yog 11.95 feem pua ​​thiab 0.79 feem pua, feem. Tom qab enzymatic hydrolysis thiab ultrafiltration nrog 3 kDa molecular hnyav txiav tawm, qhov siab tshaj solubility (21.17 feem pua), thiab cov amino pawg dawb cov ntsiab lus (14.17 feem pua) tau pom hauv CH-Alcalase.<3 kda.="" however,=""><3 kda="" was="" the="" lowest="" yield="" (12.42%)observed.="" therefore,="" although="" ultrafiltration="" is="" useful="" in="" separating="" chs="" with="" a="" low="" molecular="" weight="" it="" may="" cause="" a="" reduction="" in="">

Qhov nruab nrab molecular hnyav ntawm cov protein hydrolysates yog ib qho tseem ceeb uas txiav txim siab lawv cov khoom lom neeg [19]. Feem ntau, qhov nruab nrab feem nrog MW<3 kda="" represents="" a="" collagen="" hydrolysate;="" an="" average="" fraction="" with="" mw="">50 kDa represents gelatin, and an average fraction with MW>300 kDa sawv cev rau collagen [20,21]. Qhov txheeb ze molecular hnyav faib ntawm kev tswj (cov qauv ua ntej), CH-Alcalase, thiab CH-Alcalase<3 kda="" is="" depicted="" in="" figure="" 4.="" the="" molecular="" weight="" distribution="" was="" over="" 20,100="" da="" for="" the="" control,="" which="" did="" not="" include="" the="" collagen="" hydrolysate=""><3 kda).="" this="" could="" not="" be="" numerically="" provided="" in="" this="" study,="" as="" the="" detection="" limit="" of="" the="" index="" detector="" system="" only="" ranged="" from="" 106="" to="" 20,100="" da.="" however,="" ch-alcalase="" showed="" detectable="" values="" in="" a="" higher="" range="" of="" relative="" molecular="" weight="" distribution,="" which="" ranged="" from="" 20,100="" da="" to="" 4270="" da="" (maximum="" peak:12,600="" da).="" in=""><3 kda,="" the="" molecular="" weight="" distribution="" mainly="" showed="" three="" peaks:="" one="" with="" an="" mw="" of="" approximately="" 4270="" da="" (maximum="" peak),="" one="" with="" an="" mw="" of="" approximately="" 424="" da,="" and="" one="" with="" an="" mw="" of="" approximately="" 222="" da="" and="" 102="" da.="" ultrafiltration="" is="" an="" effective="" purification="" method="" used="" to="" obtain="" low="" molecular="" weight="" peptides="" from="" crude="" hydrolysates.="" results="" indicated="" that="" enzymatic="" hydrolysis="" by="" alcalase="" clearly="" reduced="" the="" high="" mw="" of="" the="" control="" (either="" collagen="" or="" gelatin),="" and="" that="" ultrafiltration="" was="" an="" effective="" purification="" method="" that="" can="" be="" used="" to="" obtain="" low="" molecular="" weight=""><3 kda)="" from="" crude="" collagen="" hydrolysates.="" reportedly,="" low="" molecular="" weight="" peptides="" (2-20="" amino="" acids)="" are="" more="" biologically="" active="" compared="" to="" their="" parent="" polypeptide/proteins,="" which="" are="" larger="">

Thov nias ntawm no kom paub ntxiv

Amino acid muaj pes tsawg leeg hauv CHs tau qhia (Table 2). CHS los ntawm ntau cov proteases muaj cov amino acid sib txawv thiab cov khoom antioxidant [23]. Cov amino acid muaj pes tsawg leeg ntawm collagen yog nplua nuj nyob rau hauv glycine (Gly), proline (Pro), thiab glutamic acid (Glu). Cov ntsiab lus amino acid ntawm CHs (CH-Alcalase thiab CH-Alcalase<3 kda)="" increased="" more="" than="" that="" of="" the="" control,="" following="" enzymatic="" hydrolysis="" with="" or="" without="" ultrafiltration.="" in="" particular,="" the="" content="" of="" gly,="" pro,="" and="" glu="" was="" much="" higher="" in=""><3 kda(gly="" 218="" mg/g,="" pro="" 152="" mg/g,="" and="" glu="" 120="" mg/g)="" than="" ch-alcalase(gly149="" mg/g,="" pro="" 95="" mg/g,="" and="" glu78="" mg/g).="" an="" increase="" in="" the="" content="" of="" these="" amino="" acids="" is="" strongly="" related="" to="" enhanced="" antioxidant="" capabilities="" [16,20].="" gly="" and="" pro="" contain="" hydrophobic="" amino="" acid="" groups,="" and="" glu="" contains="" negatively="" charged="" amino="" acid="" groups.="">cistanche qiaCov amino acids no tau tshaj tawm los txhim kho kev ua haujlwm antioxidant vim tias lawv cov solubilities ntau dua hauv lipids lossis los ntawm cov tshuaj tiv thaiv dawb radical [1,24].

2.3. Antioxidant thiab Anti-aging Activities ntawm Collagen Hydrolysates

Antioxidant kev ua ub no ntawm CHs tau ntsuas siv 2,2'-and-bis-(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) radical-scavenging kev soj ntsuam thiab txo lub zog soj ntsuam. ABTS radical-scavenging kev ua ntawm kev tswj (collagen suspension), CH-Alcalase, thiab CH-Alcalase<3 kda="" at="" different="" concentrations="" is="" shown="" (figure="" 5a).abts="" radical="" scavenging="" activity="" of="" peptides="" assay="" is="" important="" to="" exclusively="" measure="" the="" ability="" of="" an="" antioxidant="" peptide="" to="" induce="" a="" hydrogen="" atom="" transfer="" [25].="" abts="" radical-scavenging="" effects="" of="" all="" treatments="" increased="" in="" a="" concentration-dependent="" manner=""><0.05). ch-alcalase="" and=""><3 kda="" showed="" much="" higher="" abts="" radical-scavenging="" activity="" values,="" with="" 41.4%-88.2%="" compared="" to="" the=""><8.5%. both="" ch-alcalase="" and=""><3 kda="" had="" high="" abtsradical-scavenging="" abilities="" of="" ~60%="" when="" concentrations="" were="" greater="" than="" 2.5="" mg/ml.=""><3 kda="" showed="" a="" significantly="" higher="" abts="" radical-scavenging="" activity="" value="" than="" ch-alcalase.="">cistanche salsa extractFeem ntau, kev ua haujlwm antioxidant ntawm peptides tuaj yeem cuam tshuam los ntawm lawv cov amino acid sequences, tus naj npawb ntawm cov amino acids dawb tam sim no, qib ntawm hydrolysis, thiab qhov hnyav molecular ntawm peptides [26,27]. Hauv particular, tsis tshua muaj molecular hnyav hydrolysates muaj zog antioxidant zog piv rau siab molecular hnyav hydrolysates [2,26].

Daim duab 5. Antioxidant kev ua ub no ntawm collagen hydrolysates (CH) nyob rau hauv 2,2'-and-bis-(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) radical scavenging(A) thiab txo hwj chim (B) kev soj ntsuam. Cov ntaub ntawv qhia los ntawm cov ntawv sib txawv (ac) qhia qhov sib txawv tseem ceeb nyob ntawm kev kho mob sib txawv (p<0.05).>cistanche tubulosa cov txiaj ntsig thiab kev phivCov ntaub ntawv qhia los ntawm cov ntawv sib txawv (AD) qhia qhov sib txawv tseem ceeb nyob ntawm qhov xav tau (p < 0.05).

Cistanche tuaj yeem tiv thaiv kev laus

Kev txo hwj chim ntawm kev tswj, CH-Alcalase, thiab CH-Alcalase<3 kda="" are="" shown="" (figure="" 5b).="" the="" reducing="" power="" of="" peptides="" may="" also="" serve="" as="" a="" significant="" indicator="" of="" their="" antioxidant="" potential="" [28,29].="" the="" reducing="" power="" of="" chs="" ranged="" from="" 0.074="" to="" 0.424="" in="" a="" dose-dependent="" manner=""><0.05). the="" control="" group="" had="" the="" lowest="" reducing="" power,="" which="" did="" not="" change="" significantly="" with="" increasing="" concentrations="" of="" the="" control.="" in="" contrast="" to="" abts="" radical-scavenging="" activity,="" the="" reducing="" power="" of="" ch-alcalase="" was="" higher="" than="" that="" of=""><3 kda.="">cistanche tubulosa extractCov kev soj ntsuam zoo sib xws tau qhia tias crude collagen hydrolyzate tuaj yeem ua tau zoo dua hauv kev txo hwj chim tshaj ultrafiltrated collagen peptides [30].

Inhibition ntawm tyrosinase, collagenase, thiab elastase kev ua haujlwm tau siv los txheeb xyuas lawv cov teebmeem hauv vitro los tiv thaiv kev laus [20]. Cov txiaj ntsig ntawm inhibition ntawm tyrosinase, collagenase, thiab elastase kev ua haujlwm ntawm kev tswj, CH-Alcalase, thiab CH-Alcalase<3 kda="" are="" summarized="" (table="" 3).="" tyrosinase,="" collagenase,="" and="" elastase="" inhibitors="" have="" been="" used="" as="" important="" ingredients="" of="" cosmetics="" for="" skin="" whitening,="" anti-aging,="" and="" anti-wrinkling,="" respectively.="" collagenase="" and="" elastase,="" especially,="" are="" known="" to="" be="" major="" enzymes="" responsible="" for="" dehydration="" and="" wrinkle="" formation="" on="" the="" skin="" surface[31].="" the="" results="" indicated="" that="" the="" tyrosinase="" inhibition="" effect="" of="" vitamin="" c(95.50%,1="" mg/ml,="" positive="" control)="" was="" higher="" than="" that="" of="" the="" other="" treatments(table="" 4).="" tyrosinase="" inhibition="" effects="" of="" control,="" ch-alcalase,="" and=""><3 kda="" groups="" were="" 28.21%,="" 15.44%,="" and="" 30.20%,="" respectively.="" thus,="" the="" chs="" did="" not="" show="" a="" better="" skin="" whitening="" effect="" compared="" to="" the="" control.="" collagenase="" inhibition="" by="" the="" control,="" ch-alcalase,="" and=""><3 kda="" groups="" were="" 6.45%,54.37%,="" and="" 61.90%,="" respectively.="" in="" addition,="" ch-alcalase="" and=""><3 kda="" inhibition="" of="" collagenase="" corresponded="" to="" that="" of="" vitamin="" cat="" 1="" mg/ml.="" thus,="" chs="" obtained="" from="" this="" study="" may="" be="" effective="" collagenase="" inhibitors="" that="" may="" possibly="" play="" an="" important="" role="" in="" anti-aging="" activities.="" however,="" all="" collagen="" samples="" did="" not="" display="" elastase="" inhibition="" effects,="" which="" may="" result="" from="" a="" lack="" of="" skin="" elasticity.="" overall,="" vitamin="" c(1="" mg/ml)="" inhibited="" various="" activities="" of="" the="" enzymes="" in="" the="" following="" order:="" tyrosinase(95.50%)="">collagenase(48.09)> elastase(2/.00%o).CH-Alcalase(5 mg/mL)inhibited various activities of the enzymes in the following order: collagenase (54.37%)> tyrosinase (15.44%)>elastase (tsis muaj kev ua haujlwm). CH-Alcalase<3 kda="" (5="" mg/ml)="" inhibited="" these="" activities="" in="" the="" following="" order:="" collagenase="" (61.90%)="">tyrosinase (30.20%)>elastase (tsis muaj kev ua haujlwm). Cov qauv zoo sib xws tau tshaj tawm hauv lwm txoj kev tshawb fawb qhia tias collagen hydrolysates tau pom muaj peev xwm los ua cov tshuaj tiv thaiv kev laus thiab collagenase inhibitors [20].

3. Cov ntaub ntawv thiab cov txheej txheem

3.1. Porcine Skin Pretreatment

Porcine tawv nqaij tau yuav los ntawm ib lub chaw muag khoom hauv zos (Seoul, Kauslim), thiab tag nrho cov pom rog thiab cov ntaub so ntswg ntawm daim tawv nqaij porcine raug tshem tawm siv rab chais hniav. Cov tawv nqaij porcine siv nyob rau hauv txoj kev tshawb no yog tau los ntawm ib tug porcine thiaj li yuav txo tau lom variation. Trimmed porcine daim tawv nqaij raug ntxuav hauv dej ntawm 90 degree rau 1 min plaub zaug kom tshem tawm cov rog thiab cov khoom seem. Tom qab ntawd daim tawv nqaij raug txiav mus rau hauv 1 cm square seem thiab pulverized nyob rau hauv dej distilled rau 3 min siv plaub-tis hniav blender (CNHR-26, Bosch, Hong Kong, Tuam Tshoj). Pulverized porcine daim tawv nqaij yog homogenized ntawm high-ceev (25, 000rpm) rau 5 min siv ib tug Ultra Turrax (T25, IKA Labotechnik, Staufen, lub teb chaws Yelemees). Kwv yees li ntawm 100g ntawm porcine daim tawv nqaij sib tov (50 feem pua ​​​​ntawm cov khoom kawg) tau ntim hauv lub tshuab nqus tsev thiab khov ntawm -20 degree thiab khaws cia rau siv li ntawm 1 lub hlis.

3.2. Kev lag luam Proteases thiab Reagents

Alcalase, Flavorzyme, Neutrase, thiab Protamex tau yuav los ntawm Novozymes (Bagsvaerd, Denmark). Bromelain thiab Papain tau yuav los ntawm Daesong Sangsa (Seoul, Kauslim). Tag nrho cov tshuaj tua kab mob antioxidant thiab tiv thaiv kev laus tau yuav los ntawm Sigma-Aldrich Chemical Company (St.Louis, MS, USA). Tag nrho lwm cov tshuaj reagents thiab cov kuab tshuaj siv nyob rau hauv txoj kev tshawb no yog ntawm qib analytical.

3.3. Enzyme Hydrolysis

Enzymatic hydrolysis tau tsim rau cov khoom noj khoom haus qib kev lag luam enzymes siv (raws li cov chaw tsim khoom pom zoo; Table 4). Cov npaj porcine daim tawv nqaij sib tov yog diluted nrog dej distilled mus rau ib tug kawg khoom ntsiab lus ntawm 5 feem pua. Qhov concentration no tau raug xaiv los xyuas kom meej tus cwj pwm ntws vim nws cov viscosity tsawg. Qhov 5 feem pua ​​​​porcine daim tawv nqaij sib xyaw yog hu ua collagen suspension (los yog tswj). Lub collagen raug tshem tawm yog hydrolyzed nyob rau hauv reactors siv rau cov khoom noj-qib kev lag luam enzymes ntawm ib qho enzyme: substrate ratio ntawm 1: 100. Cov qauv aliquots (5 mL) tau kos ntawm 1, 3,6, 12, thiab 24 h ntawm hydrolysis thiab tam sim ntawd rhuab ntawm 100 degree rau 10 min kom inactivate lub enzyme, tom qab ntawd txias rau 0 degree siv dej khov. Thaum lub sijhawm ua piv txwv, pH tau tswj tau siv 1 M NaOH raws li qhov tsim nyog. Tom qab txias, cov qauv tau centrifuged ntawm 4000 × g rau 15 min, thiab cov supernatant (collagen hydrolysate; CH) tau sau. CH tau concentrated ntxiv los ntawm ultrafiltration, siv AmiconStirred Cells system (Catalog No. UFSC 20001, EMD Millipore Corporation, Burlington, MA, USA) nrog rau 3 kDa molecular hnyav txiav tawm (MWCO) (UltracelMembrane, EMD Millipore Corporation, Burlington, MA, USA) ntawm 60 psi nitrogen gas, ntawm 20 degree .cistanche tubulosa tshuaj xyuasCov CH npaj tau khov-qhuav thiab khaws cia rau hauv cov thawv cua nruj ntawm 20 degree kom txog thaum kev soj ntsuam.

3.4. Kev txiav txim siab ntawm pH, Protein Recovery, Solubility Free Amino Group Content, thiab Production Yield Lub pH ntawm cov qauv (tswj thiab CHs) tau txiav txim siab siv pH meter (Model S220, Mettler Toledo GmbH, Columbus, OH, USA). Cov protein rov qab los yog solubility tau txiav txim siab los ntawm kev kwv yees cov ntsiab lus ntawm cov protein uas siv bicinchoninic acid (BCA) protein assay, raws li cov chaw tsim khoom cov lus qhia (Sigma-Aldrich, St. Louis, MS, USA) nrog serum albumin raws li tus qauv. Cov ntsiab lus ntawm cov amino pawg dawb tau txiav txim siab los ntawm 2,4,6-trinitrobenzene sulfonic acid (TNBSA) assay raws li cov chaw tsim khoom cov lus qhia (Thermo Fisher Scientific, Waltham, MA, USA) siv L-leucine raws li tus qauv. Ob qho tib si ntub thiab qhuav qhov hnyav tau ntsuas los ntsuas qhov chaw tsim khoom.

3.5. Molecular Weight Distribution

3.5.1.Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS-PAGE cov qauv ntawm cov qauv (tswj thiab CHs) tau ntsuas raws li txoj kev qhia yav dhau los [10]. Cov qauv tau diluted nrog 8 M urea (qhov kawg ntawm cov protein ntau, 4 mg / mL). Txhua tus qauv tau muab sib xyaw nrog ib feem ntawm KTG 020 qauv tsis (10 feem pua ​​​​ntawm glycerol, 2 feem pua ​​​​ntawm SDS, 0.003 feem pua ​​​​ntawm bromophenol xiav, 5 feem pua ​​​​ntawm -mercaptoethanol, thiab 63 mM Tris-HCl, pH6.8) los ntawm KOMA Biotech Inc ., (Seoul, Kauslim), thiab boil rau 2 min. Cov qauv sib xyaw (20 μL) tau ntim rau hauv EzWayTM PAG 6% acrylamide gels (KOMA Biotech Inc., Seoul, Kauslim). Tom qab electrophoresis, cov gel tau tsau, stained, thiab de-stained. Qhov hnyav molecular tau txiav txim siab siv cov qauv dav dav ntawm cov qauv molecular ntawm 10 thiab 210 kDa.

3.5.2. Gel Permeation Chromatography (GPC)

Qhov hnyav molecular faib ntawm cov qauv tau txiav txim raws li ib txoj kev qhia yav dhau los [3]. Gel permeation chromatography (GPC) tau ua tiav siv YL9100 high-performance kua chromatography (HPLC)system(YL9100, Youngling Instrument Co., Ltd, Gyeonggi-do, Kauslim) nruab nrog peb Ultrahydrogel TM 120 kab ( 7.8 × 3000 mm) los ntawm Waters (Milford, MA, USA). Cov theem ntawm lub xov tooj ntawm tes yog distilled / deionized dej ntawm tus nqi ntws ntawm 1.0 mL / min, thiab qhov hnyav molecular faib ntawm collagen peptides tau saib xyuas siv YL 9100 refractive index detector ntawm 40 degree. Cov khoom siv ntsuas molecular hnyav (106-67,500 Da, Polymer Standards Service, Mainz, Lub teb chaws Yelemees) ua tus qauv.

3.6.Amin0 Acid Composition

Cov amino acid muaj pes tsawg leeg ntawm cov qauv tau soj ntsuam los ntawm derivatization nrog {{0}} fluorenyl ethoxy carbonyl (FMOC)-chloride thiab o-phthalic dialdehyde (OPA) ntawm qhov kawg 3000 HPLC system ( Dionex, Idstein, Lub teb chaws Yelemees) tau nruab nrog ob lub ntsuas hluav taws xob (lub ntsuas hluav taws xob fluorescence thiab UV ntes) thiab VDSpher 100 C18-E (4.6 mm × 150 mm, 3.5 μm particle loj, VDS Optilab, Berlin, Lub teb chaws Yelemees). Lub ntim ntim yog 1.0 L, thiab lub sijhawm txawb tau tsim los ntawm ob lub ntsiab lus: 40 mM sodium phosphate dibasic (pH 7) thiab 45 feem pua ​​(o / v) acetonitrile / 45 feem pua ​​(v / v) methanol tov. Los ntawm kev sib txuas ntawm UV detector thiab fluorescence detector, ultraviolet rays tau kuaj pom ntawm 338 nm, OPA derivative tau kuaj pom ntawm 450 nm ntawm emission wavelength thiab 340 nm ntawm excitation wavelength, FMOC derivative tau kuaj pom ntawm 305 nm6 ntawm 2 emission wavelength. nm ntawm ib qho excitation wavelength. Ib qho amino acid sib xyaw (1.0 nmol mL-I rau txhua cov amino acid) tau siv rau kev ntsuas.

qhov twg Atotalamino acid yog cov ntsiab lus tom qab hydrolysis siv 6 M HCl (ntawm 130 degree rau 24 h), thiab Afree amino acid yog cov ntsiab lus tom qab solubilizing hauv dej distilled.

3.7. Kev ntsuam xyuas ntawm Antioxidant Activity

3.7.1.2,2'-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)Radical-Scavenging Activity

ABTS radical-scavenging kev ua ntawm CH tau ntsuas raws li ib txoj kev qhia yav dhau los [20]. ABTSradical cation tau tsim los ntawm kev sib xyaw ABTS cov tshuaj tov (7.0 mM) nrog cov poov tshuaj persulfate (2.45 mM) thiab incubating cov resultant sib tov nyob rau hauv qhov tsaus ntuj ntawm chav tsev kub thaum hmo ntuj. ABTS radical tov yog diluted hauv 5. Ib mL ntawm diluted ABTS radical tov yog tov nrog 1 mL ntawm txhua tus qauv. Kaum feeb tom qab, cov qauv (Ib tus qauv, nrog rau cov qauv) thiab kev tswj (A tswj tsis muaj qauv) absorbances tau ntsuas ntawm 734 nm. Lub ABTS radical-scavenging kev ua si (feem pua) tau xam raws li:

3.7.2.Txo hwj chim

Qhov txo hwj chim ntawm CH tau ntsuas raws li txoj kev qhia yav dhau los [20]. Ib mL ntawm txhua tus qauv yog tov nrog 1 mL ntawm 0.2 M phosphate buffer (pH6.6) thiab 1 mL ntawm 1 feem pua ​​​​poov tshuaj ferricyanide. Qhov sib tov yog incubated ntawm 50 degree rau 20 min, thiab ces 1 mL ntawm 10 feem pua ​​trichloroacetic acid tau ntxiv. Ib qho aliquot ntawm 2 mL los ntawm qhov sib tov incubation yog tov nrog 2 ml ntawm distilled dej thiab 0.4 mL ntawm 0.1 feem pua ​​​​ferric chloride. Tom qab 10min, qhov absorbance ntawm cov kev daws teeb meem tau ntsuas ntawm 700 nm ntawm lub spectrophotometer (OPTIZEN, Mecasys Co., Daejeon, Kauslim) Ntau dua absorbance (ntawm 700 nm) ntawm cov tshuaj tiv thaiv sib tov tau txiav txim siab los qhia txog kev txo qis zog.

3.8. Kev ntsuam xyuas ntawm Anti-Aging Effect

3.8.1 ib. Inhibition ntawm Tyrosinase Activity

Tyrosinase inhibition tau txiav txim siab los ntawm txoj kev piav qhia yav dhau los [20]. Cov tshuaj premixture uas muaj 70 μL ntawm 0.1 M phosphate buffer (pH6.8), 30uLof nceb tyrosinase (167U / mL; Sigma-Aldrich, Tebchaws Asmeskas), thiab 20 L ntawm cov qauv raug tsim rau 5 min ntawm 30 degree. Kwv yees li ntawm 100μL ntawm 3,4-dihydroxy phenyl-L-alanine (L-DOPA) tau ntxiv los pib cov tshuaj tiv thaiv enzymatic. Absorbance ntawm 492 nm tau ntsuas rau 20 feeb los saib xyuas L-DOPA tsim. Ascorbic acid (1 mg / mL) tau txais kev tswj hwm zoo, uas tau siv rau kev sib piv. Qhov piv txwv inhibition tau xam raws li hauv qab no:

qhov twg A yog sib xyaw nrog tyrosinase yam tsis muaj qauv; B yog ib qho sib xyaw tsis muaj qauv thiab tyrosinase; C yog sib xyaw nrog cov qauv thiab tyrosinase, thiab D yog sib xyaw nrog cov qauv tab sis tsis muaj tyrosinase.

3.8.2.Inhibition ntawm Collagenase Kev Ua Haujlwm

Collagenase inhibition tau txiav txim siab los ntawm txoj kev piav qhia yav dhau los [20]. Luv luv, 50 mM tricine buffer (pH7.5) uas muaj 10 mM calcium chloride thiab 400 mM sodium chloride tau npaj. Tom qab ntawd, 50mLof a 1.0mM N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala kua thiab 0.2 mg/ml collagenase (los ntawm Clostridium histolyticum, Hom IA, 0.{ {19}} FALGPA U/mg khoom; Sigma-Aldrich, USA) tau ntxiv nyob rau hauv lub xub ntiag thiab tsis muaj cov qauv. Cov tshuaj tiv thaiv tau nres los ntawm kev ntxiv citric acid (6 feem pua). Cov tshuaj tiv thaiv sib xyaw tau sib cais los ntawm kev ntxiv ethyl acetate. Lub absorbance ntawm lub supernatant yog ntsuas ntawm 345 nm. Ascorbic acid (1 mg / mL) tau txais kev tswj hwm zoo thiab tau siv rau kev sib piv. Qhov feem pua ​​ntawm inhibition yog xam raws li:

qhov twg A yog sib xyaw nrog collagenase tsis muaj qauv; B yog ib qho sib xyaw tsis muaj qauv thiab collagenase; C yog sib xyaw nrog cov qauv thiab collagenase, thiab D yog sib xyaw nrog cov qauv tab sis tsis muaj collagenase.

3.8.3.Inhibition ntawm Elastase Kev Ua Haujlwm

Elastase inhibition tau txiav txim siab los ntawm ib txoj kev piav qhia yav dhau los [20]. N-succinyl-Ala-Ala-Ala-p-nitroanilide (Suc-Ala-Ala-Ala-pNA) ua lub substrate, thiab tso tawm p-nitroaniline tau saib xyuas rau 2{{20}} min ntawm 25 degree .A ib feem (1 ug) ntawm hom IV porcine pancreatic elastase (PPE) tau yaj hauv 1 mL ntawm 0.2 M Tris-HCl buffer (pH 8.0). Cov tshuaj tiv thaiv sib xyaw muaj 0.2 M Tris-HCl tsis muaj (pH8.0), 1 ppm PPE, 0.8 mM Suc-Ala-Ala-Ala-pNA, cov qauv, thiab cov lus hais saum toj no. Absorbance ntawm 214 nm tau ntsuas. Ascorbic acid (1 mg / mL) pab raws li kev tswj zoo siv rau kev sib piv. Qhov piv txwv inhibition tau xam raws li hauv qab no:

qhov twg A yog qhov sib xyaw nrog elastase thiab tsis muaj qauv; B yog ib qho sib xyaw uas tsis muaj qauv thiab elastase; C yog qhov sib xyaw nrog cov qauv thiab elastase, thiab D yog sib xyaw nrog cov qauv tab sis tsis muaj elastase.

3.9.Kev txheeb xyuas qhov tseeb

Cov ntaub ntawv raug nthuav tawm raws li qhov nruab nrab ± tus qauv sib txawv (SD). Qhov tseem ceeb ntawm qhov sib txawv ntawm cov pab pawg tau raug soj ntsuam los ntawm kev siv ntau qhov kev sib piv thiab kev soj ntsuam ntawm qhov sib txawv (ANOVA), tom qab los ntawm Tukey qhov sib txawv tseem ceeb (HSD). Qhov sib txawv nrog p tus nqi tsawg dua 0.05 tau suav tias yog qhov tseem ceeb.

4. Cov lus xaus

Hauv txoj kev tshawb no, collagen hydrolysates, cov khoom noj muaj txiaj ntsig, tau ua tiav los ntawm enzymatic hydrolysis, tom qab ntawd ultrafiltration thiab purification. Ntau yam kev lag luam proteases tau sim rau lawv cov txiaj ntsig zoo hauv kev tsim cov collagen hydrolysates. CHS hydrolyzed los ntawm Alcalase yog qhov ua tau zoo tshaj plaws hydrolyzed CHs, thiab ultrafiltration ua raws li kev lim dej tau zoo hauv kev tsim cov peptides uas muaj qhov hnyav molecular. Cov txiaj ntsig tau pom tias CHs tau nthuav tawm cov tshuaj tiv thaiv antioxidant thiab collagenase inhibition zoo heev. Yog li, CHs tau txais los ntawm txoj kev tshawb no tuaj yeem siv rau hauv cov khoom noj, tshuaj pleev ib ce, lossis kev lag luam tshuaj ua cov khoom siv ntuj tsim, muaj cov khoom tiv thaiv oxidative thiab tiv thaiv kev laus. Cov kev tshawb fawb ntxiv uas cuam tshuam nrog kev ntsuam xyuas hauv vivo ntawm kev laus ntawm kev ua haujlwm peptides hauv tib neeg cov tawv nqaij yuav ua pov thawj tias muaj txiaj ntsig.

Kab lus no yog muab rho tawm los ntawm Molecules 2019, 24, 1104; doi: 10.3390/molecules24061104 www.mdpi.com/journal/molecules