Txiv Hmab Txiv Ntoo (Olea Europaea L) Extract Loaded Lipid Nanoparticles: Optimization Of Processing Parameters By Box-Behnken Statistical Design, In-vitro Characterization, Thiab Kev Ntsuam Xyuas Ntawm Anti-oxidant Thiab Anti-microbial Activity

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

Abstract:Txoj kev tshawb fawb tam sim no tsom los npaj thiab tshuaj xyuas cov khoom lipid nanoparticles (SLNs) ntawm cov txiv ntseej eaves extract hmoov (OLP) uas muaj ntau cov tshuaj tiv thaiv oxidant thiab tshuaj tua kab mob xws li oleuropein, ntuj polyphenol. Qhov teeb meem loj txog OLP yog qhov tsis ruaj khov vim ib puag ncig thiab yog li cuam tshuam txog bioactivity. Yuav kom kov yeej qhov teeb meem no, SLNs tau tsim los ntawm kub homogenous ua raws li cov txheej txheem sonication los tiv thaiv cov tshuaj thiab txhim kho nws cov tshuaj tua kab mob antioxidant thiab tshuaj tua kab mob. Lipids zoo li compritol 888ATO thiab surfactants zoo li tween 80 tau siv rau kev txhim kho thiab ruaj khov ntawm SLNS thiab kev ua kom zoo dua qub tau ua tiav los ntawm Box-Behnken statistical design (3x3).Qhov optimized batch (F9) pom particle loj, kev ua haujlwm zoo, PDI, thiab zeta muaj peev xwm 277.46 nm, 80.48 feem pua, 0.275. thiab -23.18 mV raws. Optimized formulation (F9) tau nthuav tawm cov qauv tso tawm ntev txog 24 teev nrog kev txiav txim thawj zaug tso tawm kinetic (R²=0.9984) thiab cov txheej txheem ntawm cov tshuaj tso tawm tau pom tias yog Fickian diffusion hom (n=0) .441). Raws li kev tshawb fawb txog kev ruaj ntseg, nws tuaj yeem pom tias cov qauv SLNs ruaj khov. Anti-oxidation thiab anti-microbial kev tshawb fawb tau ua los ntawm optimized formulation thiab kev tshawb pom pom tias SLNs pom ib qho kev txhim kho radical scavenging kev ua si thiab anti-microbial kev tiv thaiv Gram-positive (Staphylococcus aureus) thiab Gram-negative (Pseudomonas aeruginosa) kab mob. Thaum kawg, nws tau xaus lus tias tsim SLNs tuaj yeem tiv thaiv thiab tsim nyog rau kev xa OLP.

Ntsiab lus:Olea europaea, oleuropein,khoom lipid nanoparticles, txiv ntseej nplooj extract,anti-oxidant kev ua haujlwm, anti-microbial kev ua haujlwm

Thov nias ntawm no kom paub ntxiv

1 Kev Taw Qhia

Tsev neeg Dicotyledons Oleaceae muaj txog 30 genera ntawm deciduous nroj tsuag nrog rau cov ntoo txiv ntseej thiab nws muaj txog li 600 hom. Cov tswv cuab Oleaceae tau loj hlob zoo nyob rau hauv cov tshuaj pleev thiab sub-temperate vim li cas ntawm Asia?. Txiv roj roj (Olea europaea) roj tau los ntawm ntau qhov chaw xws li txiv hmab txiv ntoo, nplooj, thiab lwm yam. Kev lig kev cai nws tau siv los ua cov tshuaj pej xeem los kho ntau hom kev ua npaws thiab lwm yam teeb meem cuam tshuam nrog lub cev2).

Txiv ntseej nplooj extract tau nthuav tawm ntau yam kev kho mob xws li tshuaj tiv thaiv hypertensive, hypoglycemic, tshuaj tua kab mob, hypouricemic, thiab lwm yam. Tsis tas li ntawd, oleuropein (ib qho zoo ib yam secoiridoid) tau txais los ntawm cov txiv ntseej cog tau nthuav tawm hypocholesterolemic thiab hypoglycemic kev ua haujlwm nrog lub zog tiv thaiv oxidant thiab nrog rau kev ua haujlwm tiv thaiv. Degradation khoom ntawm oleuropein ie. hydroxytyrosol kuj tau nthuav tawm tag nrho cov haujlwm saum toj no hais nrog rau cov peev xwm dawb radical scavenging action5.bioflavonoidsKev koom nrog tag nrho cov kev kho mob no cuam tshuam rau cov txiv ntseej ua rau nws yog ib qho tseem ceeb hauv kev noj qab haus huv · 4).

Hauv qhov project no, cov kws sau ntawv muaj kev txhawj xeeb nrog cov tshuaj tiv thaiv kab mob thiab tshuaj tiv thaiv oxidant ntawm extract ntawm Olive nplooj. Cov tshuaj tiv thaiv kab mob ntawm cov txiv ntseej extract yog vim p-hydroxybenzoic acid, cyclotrisiloxane hexamethyl, Cyclopentasiloxane octamethyl, thiab Cyclopentasiloxane desmethyl, vanillic, caffeic, protocatechuic, syringic, gallic acid, thiab lwm yam khoom xyaw, xws li querosiloxane. oleanolic acid kuj yog lub luag haujlwm rau kev tiv thaiv kab mob. Cov tshuaj tiv thaiv kab mob ntawm tag nrho cov tshuaj no tau tshaj tawm yav dhau los los ntawm ntau tus kws tshawb fawb tiv thaiv kab mob, poov xab, fungi, kab mob, retroviruses, thiab lwm yam kab mob, 2).

Cistanche tuaj yeem tiv thaiv kev laus

Kev noj zaub mov kom zoo ntawm cov khoom siv tshuaj tiv thaiv oxidant zoo li txiv hmab txiv ntoo thiab nplooj tau txais kev saib xyuas ntau thiab pom muaj txiaj ntsig zoo rau kev noj qab haus huv ntawm tib neeg. Cov txiaj ntsig ntawm ntau qhov kev tshawb fawb txog kev kis mob tau tshawb pom tias kev noj cov zaub mov uas muaj polyphenol-nplua nuj txo qhov tshwm sim ntawm tus kab mob coronary artery (CHD). CHD zoo li MI (myocardial infarction), IS ( ischemic stroke), thiab lwm yam cuam tshuam nrog athero-sclerosis "Cov kev tshawb fawb tsis ntev los no tau tshawb pom tias kev puas tsuaj oxidative yog ib qho tseem ceeb rau kev nce qib ntawm atherosclerosis, tshwj xeeb tshaj yog, raws li txoj kev xav ntawm oxidative stress, Kev hloov pauv oxidative ntawm low-density lipoprotein (LDL) yog xav tias ua lub luag haujlwm tseem ceeb hauv kev txhim kho atherosclerosis. Yog li ntawd, qhov inhibition ntawm cov txheej txheem no yog suav tias yog ib qho tseem ceeb ntawm kev kho mob, ntau yam tseem ceeb xws li Oleuropein (hydroxytyrosol, tyrosol). , verbascoside (tseem hu ua ac-toeside lossis Kalinin), ligstroside, apigenin-7-glucoside, di-cosmetic-7-glucoside, luteolin, catechin, thiab lwm yam tau txais rau nplooj ntawm txiv ntseej thiab nthuav tawm cov tshuaj tiv thaiv oxidant Hydroxytyrosol yog lub ntsiab lus tseem ceeb ntawm cov nplooj txiv ntseej oleuropein uas muaj patent los tiv thaiv oxidant zog).

Dhau li ntawm qhov kev ua no, txiv ntseej nplooj extract tau nthuav tawm lwm cov haujlwm tshuaj xws li tshuaj tiv thaiv kab mob, 12), tiv thaiv kab mob qog noj ntshav 8.14), tiv thaiv kab mob 5), hypoglycemicl4), hypolipidemic17, tiv thaiv platelet aggregation. Lub koom haum zoo li EFSA (Euro-pean Food). Lub Chaw Haujlwm Saib Xyuas Kev Nyab Xeeb) thiab EMA (European Medicine Agency) tau muab lawv qhov kev ntsuam xyuas ntawm kev siv tshuaj kho mob ntawm cov txiv ntseej nplooj extractants. Nrog rau qhov tseem ceeb, cov khoom extraction ntawm cov txiv ntseej nplooj raug kev txom nyem los ntawm cov teeb meem tsis ruaj khov nyob rau hauv atmospheric tej yam kev mob (kub, teeb, thiab oxygen). nrog rau cov kab mob lom thiab yog li nthuav tawm tsis zoo bioactivity20,21).muab cistancheTxhawm rau txhim kho kev ruaj ntseg, ntau yam nano-based formulations tau tsim los tshawb txog ntau yam kev ua ntawm cov txiv ntseej nplooj extract. Cov formulations muaj xws li nano-emulsion (W / O thiab ntau hom) 2), inclusion complexes, tshuaj tsuag qhuav khoom?4), electrostatic extrusion25, bipolar complexes), nano-liposomes20, Nano-emulsion, micro -encapsulation, PLA nanoparticles2, thiab NLCs0.Ntau yam teeb meem cuam tshuam nrog cov formulations xws li cov nqi qis, cov txheej txheem siv sij hawm, kev cuam tshuam kev ua haujlwm, kev sib kis hnyav, thiab qhov loj me me. Yog li, txhawm rau tswj cov kev tsis zoo no, lipid nanoparticles tuaj rau hauv lub neej. Lipid nanoparticles es-tshwj xeeb yog cov khoom lipid nanoparticles (SLNs) muaj ntau yam zoo xws li kev ua haujlwm zoo dua, tswj cov khoom me me, biocompatibility, biodegradability, thiab yooj yim ntawm kev npaj. Ntxiv mus, lipids siv rau kev npaj ntawm SLNs muaj qee qhov kev sib koom ua ke ntawm kev tiv thaiv kev ua haujlwm nrog rau cov tshuaj entrapped. Txog rau tam sim no, tsis muaj kev tsim raws li cov khoom lipid nanoparticles (SLNs) rau kev tshawb nrhiav nws cov tshuaj tiv thaiv kab mob thiab cov tshuaj tiv thaiv oxidant zoo li txhim kho kev ruaj ntseg ntawm cov txiv ntseej tawm extract khoom tau tsim.

Yog li, txoj haujlwm tshawb fawb tau tsom mus rau "kev txhim kho ntawm cov khoom lipid nanoparticles (SLNs) rau cov nplooj txiv ntseej (Olea europaea L.) rho cov hmoov (OLP) . tshem tawm los ntawm kev ziab kom tau cov khoom siv hmoov qhuav. Txiv ntseej nplooj extract fais fab (OLP) tau muab tso rau hauv SLNs (OLP-SLNs) thiab kev ua kom zoo tau ua tiav los ntawm cov kws tshaj lij tsim software (Model 8.0.7.1) siv Box-Behnken Peb yam xws li tshuaj (OLP) rau lipid ratio, surfactant concentration (feem pua ​​​​, Tween 80), thiab homogenization ceev (rpm) raug coj los ua tus tsis muaj kev ywj pheej, thiab yam xws li particle loj, kev ua haujlwm zoo, thiab polydispersity index (PDI) Cov txheej txheem ua kom zoo tau raug soj ntsuam rau kev tshawb fawb morphological, DSC txoj kev tshawb fawb, kev tshawb fawb hauv vitro, thiab kev tshawb fawb ruaj khov. ntawm tus tsim S LNs formulation.

2 Cov ntaub ntawv thiab cov txheej txheem

Cov nplooj txiv ntseej tau txais los ntawm lub vaj hauv zos. Cov nplooj tshiab tau qhuav thiab hmoov rau extraction. Ntau yam lipids zoo li muaj nuj nqis ATO5, compritol 888 ATO tau txais los ntawm Gattefosse (Lub Tebchaws Yelemees). Glyceryl monostearate (GMS), palmitic acid, stearic acid tween 80, thiab lwm yam tau yuav los ntawm central drug house ltd (New Delhi, India).2, 2-Diphenyl-1-Picryl hydroxyl-hydrate dawb radicals (DPPH) ) tau txais los ntawm Merck (Darmstadt, Lub Tebchaws Yelemees). Ascorbic acid, potassium dihydrogen phosphate, ammonium acetate, methanol, sodium hydroxide, poloxamer 188, ethanol, nutrient agar, thiab dialysis bag (M.Wt txiav tawm 12, 000 kD) tau txais los ntawm Sigma Aldrich (St Louis, USA). Tag nrho lwm cov tshuaj siv rau kev kawm yog qib ntsuas.

2.1 Extraction ntawm phenolic Cheebtsam

Cov nplooj tshiab ntawm txiv ntseej tau qhuav thiab hmoov rau ex-traction. Cov ntsiab cai nquag tau muab rho tawm los ntawm lub zog nrog kev pab ntawm cov kuab tshuaj (ethanol: dej, 4: 1 v / v). Ib tug txaus kom muaj nuj nqis ntawm hmoov (250 mg) tau muab coj mus rau hauv lub extraction lub raj mis thiab cov kuab tshuaj ntxiv (1000 mL) ethanol: dej, 4: 1 v /) thiab cov sib tov yog tsis tu ncua agitated rau lub sij hawm ntawm 24 h. Tom qab cov kauj ruam no, lub cev sib tov tau lim thiab cov kuab tshuaj tau evaporated rau dryness ntawm 40 degree kom tau cov hmoov).

2.2 Kev Npaj thiab kev ua kom zoo ntawm SLNs: Ua ntej

Kev tshuaj xyuas kev kawm thiab ua ntej optimization ntawm formulation variables Rau kev xaiv ntawm haum lipid, surfactant, homogenization ceev & sij hawm, sonication lub sij hawm, thiab lwm yam., ua ntej kev tshawb fawb tau ua. Kev xaiv cov lipid tsim nyog nyob ntawm cov tshuaj solubility hauv lipid thiab kev xaiv ntawm surfactant yog nyob ntawm seb qhov solubility ntawm lipid hauv surfactant.

Qhov kev txhawj xeeb lipid (100 mg) tau yaj saum nws cov ntsiab lus melting (nyob ib ncig ntawm 10 degree saum toj no) nyob rau hauv lub vial ua raws li kev koom ua ke ntawm cov tshuaj (OLP) nrog tas li co. Cov tsos ntawm lub teeb daj ntseg qhia txog qhov kawg. Cov kev sim zoo sib xws tau ua rau kev xaiv cov surfactants tsim nyog.

2.3 Kev sim tsim

Hauv kev tshawb fawb tam sim no, BBD ntawm cov kws tshaj lij tsim software nrog peb yam thiab peb theem tau siv rau kev ua kom zoo. Hauv kev tshawb fawb, peb yam xws li tshuaj rau lipid ratio (A,1:3-1:6), surfactant concentration (B, feem pua ​​,1.5-4.5 feem pua), thiab homogenization ceev (C , rpm, 3000-6000 rpm, rau ob h) raug coj los ua qhov tsis muaj kev ywj pheej thiab par-title loj (nm, Y1), kev ua haujlwm zoo (feem pua ​​​​, Y2) thiab PDI (Y3) raug coj los ua qhov tsis sib xws. Lub hom phiaj ntawm kev siv cov qauv no yog txhawm rau txhim kho cov lus hais saum toj no peb tus kheej tsis muaj kev ywj pheej thiab ua tiav qhov zoo tshaj plaws particle loj, qhov siab tshaj plaws entrapment efficiency, thiab tsawg kawg tau PDI. Cov ntaub ntawv tau haum rau hauv cov kws tshaj lij tsim software rau BBD15). Tag nrho ntawm 17 batch tau tsim nrog 5 batch muaj qhov sib xws (tsib lub ntsiab lus) (Table 1). Qhov kev txiav txim thib ob ntawm qhov sib npaug polynomial tau siv los ua kom pom qhov cuam tshuam ntawm ntau yam tsis muaj kev ywj pheej ntawm qhov loj me, kev ua haujlwm zoo thiab PDI.

2.4 Kev txhim kho ntawm SLNs

OLP-loaded SLNs tau npaj los ntawm kub homogenization ua raws li txoj kev sonication. Qhov sib txawv ntawm OLP-loaded SLNs batches yog muab nyob rau hauv Table 1. Ua ntej ntawm tag nrho cov, lub lipid (compritol 888 ATO) tau yaj nyob ib ncig ntawm 10 degree ntawm nws melting point thiab qhov xav tau kom muaj nuj nqis ntawm OLP tau ntxiv (1:{{6}). }: 6 tshuaj rau lipid ratio). Lub caij no, qhov xav tau kom muaj nuj nqis (1.5-4.5 feem pua) ntawm cov surfactant tau yaj hauv dej deionized thiab qhov kub ntawm cov theem no tau khaws cia zoo ib yam li cov theem lipid. Cov theem aqueous tau muab tso rau hauv lipid theem maj mam thiab raug kub homogenization ntawm lub nplhaib sib txawv ceev (rpm, 3000-6000 rpm, ob h) kom tau txais cov chav kawm SLNs dispersion. Cov emulsion tau tso cai rau

txias thiab ces sonicated rau 10 min ntawm 100 feem pua ​​​​amplitude nrog kev pab los ntawm ib tug sonicator (Probe type, Vibra-Cell'VCX 130; Sonics, CT, USA) kom tau qhov kawg SLNs dispersion. Cov dispersion tau sau rau hauv lub khob vial thiab khaws cia rau hauv lub tub yees rau kev kawm ntxiv. Ib yam li ntawd, SLNs dawb paug tau tsim tsis muaj API (tshuaj).

2.5 Tus yam ntxwv ntawm OLP-SLNs

2.5.1 Kev ntsuas particle loj, PDI, thiab zeta muaj peev xwm

Photon Correlation Spectroscopy (PCS) siv lub tshuab zeta sizer (Malvern, nano ZS 90, Malvern Instruments, UK) tau siv rau kev ntsuas qhov nruab nrab particle loj thiab polydispersity index (PDI) ntawm ntau yam khoom siv. Qhov kub tau khaws cia ntawm 25 degree thiab lub kaum sab xis tawg tau teem rau ntawm 90 degree.cistanchDeionized dej yog siv rau dilution ntawm thawj OLP-SLNs dispersion. Zeta muaj peev xwm uas qhia tau hais tias tus nqi saum npoo tau txiav txim siab nrog kev pab ntawm tib lub cuab yeej nrog lub zog hluav taws xob ntawm ib puag ncig 20 V / cm27

2.5.2 Kev kawm Morphological

Lub tshuab hluav taws xob xaim hluav taws xob (TEM, Fei electron optics, Nyiv) siv tooj liab daim phiaj coated carbon yog siv los tshuaj xyuas cov morphology thiab cov duab ntawm npaj OLP-loaded SLNs (optimized batch F9). Qhov tsis zoo staining nrog phosphotungstic acid stain (2 feem pua ​​w/w, ntev 20-30 vib nas this) tau ua rau stain cov qauv OLP-SLNs tom qab ziab ntawm chav tsev kub. Thaum kawg, tus qauv tau kuaj pom los ntawm TEM.

2.5.3 Kev ntsuam xyuas kev ua haujlwm ntawm kev nkag mus

Entrapment efficiency (EE) tau txiav txim siab los ntawm kev tshuaj xyuas cov tshuaj uas tsis muaj nyob hauv cov tshuaj muaj nyob hauv OLP-SLNs dispersion. Cov txheej txheem centrifugation tau siv rau lub hom phiaj no. Kev ntsuas kom muaj nuj nqis (10 mL) ntawm OLP-SLNs tau muab coj los rau hauv lub raj centrifuge thiab tso cai rau cov xuab zeb nrog kev pab los ntawm lub tshuab cua txias (Remi, Is Nrias teb) ntawm 12000rpm rau 15 feeb. Lub xub ntiag ntawm cov tshuaj uas tsis tau nkag mus tau raug tshuaj xyuas los ntawm UV-pom spectrophotometer (Model 1800, Shimadzu . Nyiv) ntawm 230 nm. Qhov feem pua ​​​​ntawm cov tshuaj tau raug txiav txim los ntawm cov qauv hauv qab no (kev sib npaug 1):

2.5.4 Sib txawv scanning calorimetry (DSC) kev kawm

Lub hom phiaj ntawm txoj kev tshawb no yog los soj ntsuam tus cwj pwm thermal ntawm ntau yam formulations viz ntshiab OLP, compritol 888 ATO, thiab optimized OLP-SLNs formulation (F9) siv txawv scanning calorimetry (Mettler, Toledo, USA). Txhua tus qauv tau ntim rau hauv lub lauj kaub aluminium thiab luam tawm ntawm qhov ntsuas kub ntawm 20-350 degree (tus nqi 10 degree / min) siv lub laujkaub khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob khoob. . 2.5.5 Txoj kev tshawb nrhiav tshuaj tso tawm (feem pua)

Cov tshuaj tso tawm kev kawm ntawm optimized batch (F9) yog ua los ntawm Franz diffusion cell siv dialysis membrane. Ua ntej daim ntawv thov ntawm daim nyias nyias nyias, nws tau tsim los ntawm kev kho mob 0.35 feem pua ​​w/v sodium sulfite tov ntawm 80 degree rau 1-2 min tom qab acidification nrog HSO({{ 13}}.2 feem pua, v/v) thiab tom qab ntawd muab cia rau 12 teev hauv dej distilled. Saline phosphate buffer (pH 7.4) tau ntim rau hauv lub thawv receptor thiab SLN dispersion (F9,1 mL) tau muab tso rau hauv qhov chaw pub dawb. Qhov kev sim tau ua nyob rau ntawm 37 ± 0.5 degree nrog nruam nplawm (50 rpm).Aliquots (1 mL) raug coj los ntawm lub receptor compartment ntawm lub sijhawm teem tseg thiab cov tshuaj muaj nyob rau hauv txhua tus qauv raug kuaj pom los ntawm UV-pom spectrophotometer ntawm 230nm ua. Qhov tsim nyog tso tawm kinetic los ntawm optimized SLNs tau kuaj pom los ntawm kev haum cov ntaub ntawv tso tawm rau hauv xoom, ua ntej, tus qauv Higuchi thiab Korsmeyer-Peppas qauv raws li tau muab rau hauv Table 2, thiab tus nqi ntawm R (Correlation coefficient) tau txiav txim siab. Tus qauv nrog tus nqi siab tshaj R-tus nqi tau suav tias yog tus qauv zoo8).

Kab lus no yog muab rho tawm los ntawm J. Oleo Sci. 70, (10) 1403-1416 (2021)