Amafutha e-Saposhnikovia divaricata ahlanzekile ekhandlela nensipho enza i-wholesale diffuser uwoyela obalulekile omusha wama-diffuser okushisa umhlanga

incazelo emfushane:

 

2.1. Ukulungiswa kwe-SDE

Ama-rhizome e-SD athengwe njengekhambi elomisiwe kwa-Hanherb Co. (Guri, Korea). Izinto zokutshala ziqinisekiswe ngokwe-taxonomically nguDkt. Go-Ya Choi we-Korea Institute of Oriental Medicine (KIOM). Isampula yevawusha (inombolo 2014 SDE-6) yafakwa ku-Korean Herbarium of Standard Herbal Resources. Ama-rhizomes omisiwe e-SD (320 g) akhishwa kabili nge-ethanol engu-70% (ene-reflux engamahora angu-2) futhi ukukhishwa kwabe sekugxilwa ngaphansi kwengcindezi encishisiwe. I-decoction ihlungiwe, i-lyophilized, futhi igcinwe ku-4 ° C. Isivuno se-extract ezomisiwe ezisetshenziswa zokuqalisa ezingahluziwe kwaba ngu-48.13% (w/w).

 

2.2. Ukuhlaziywa Kwe-Quantitative High-Performance Liquid Chromatography (HPLC).

Ukuhlaziywa kweChromatographic kwenziwa ngesistimu ye-HPLC (Waters Co., Milford, MA, USA) kanye nomtshina we-photodiode array. Ukuhlaziywa kwe-HPLC kwe-SDE, i-prim-O-izinga leglucosylcimifugin lathengwa eKorea Promotion Institute for Traditional Medicine Industry (Gyeongsan, Korea), kanyeisekhondi-O-glucosylhamaudol futhi 4′-O-β-D-glucosyl-5-O-methylvisamminol yahlukaniswa ngaphakathi kwelabhorethri yethu futhi ikhonjwe ngokuhlaziywa kwe-spectral, ngokuyinhloko yi-NMR ne-MS.

Amasampula e-SDE (0.1 mg) ahlakazwa ku-70% ethanol (10 mL). Ukuhlukaniswa kwe-Chromatographic kwenziwe ngekholomu ye-XSelect HSS T3 C18 (4.6 × 250 mm, 5μm, Waters Co., Milford, MA, USA). Isigaba seselula sasihlanganisa i-acetonitrile (A) kanye ne-acetic acid engu-0.1% emanzini (B) ngesilinganiso sokugeleza esingu-1.0 mL/min. Uhlelo lwe-multistep gradient lusetshenziswe kanje: 5% A (0 min), 5–20% A (0–10 min), 20% A (10–23 min), kanye 20–65% A (23–40 min ). Ubude begagasi bokutholwa buskenwe ku-210–400 nm futhi barekhodwa ku-254 nm. Umthamo womjovo wawuyi-10.0μIzixazululo ezijwayelekile ze-L. zokunquma ama-chromone amathathu zalungiswa ekuhlanganiseni kokugcina okungu-7.781 mg/mL (prim-O-glucosylcimifugin), 31.125 mg/mL (4′-O-β-D-glucosyl-5-O-methylvisamminol), kanye ne-31.125 mg/mL (isekhondi-O-glucosylhamaudol) ku-methanol futhi igcinwe ku-4°C.

2.3. Ukuhlolwa Komsebenzi Wokulwa NokuvuvukalaKu-Vitro

2.3.1. I-Cell Culture kanye nokwelashwa okuyisampula

Amaseli angu-RAW 264.7 atholwe ku-American Type Culture Collection (ATCC, Manassas, VA, USA) futhi atshalwa endaweni ye-DMEM equkethe ama-antibiotic angu-1% kanye no-5.5% FBS. Amaseli afakwe endaweni enomswakama ongu-5% CO2 ku-37°C. Ukuze kukhuthazwe amaseli, okuphakathi kwathathelwa indawo yi-DMEM medium entsha, kanye ne-lipopolysaccharide (LPS, Sigma-Aldrich Chemical Co., St. Louis, MO, USA) ngo-1μg/mL yengezwe kukhona noma ingekho i-SDE (200 noma 400μg/mL) amahora angama-24 engeziwe.

2.3.2. Ukunqunywa kwe-Nitric Oxide (NO), i-Prostaglandin E2 (PGE2), i-Tumor Necrosis Factor-α(TNF-α), kanye ne-Interleukin-6 (IL-6) Production

Amaseli aphathwe nge-SDE futhi avuselelwe nge-LPS amahora angama-24. AKUKHO ukukhiqizwa okuhlaziywe ngokulinganisa i-nitrite kusetshenziswa i-reagent ye-Griess ngokocwaningo lwangaphambilini [12]. Ukugcinwa kwe-cytokines evuthayo PGE2, TNF-α, futhi i-IL-6 yanqunywa kusetshenziswa ikhithi ye-ELISA (amasistimu e-R&D) ngokuya ngemiyalelo yomkhiqizi. Imiphumela ye-SDE ku-NO kanye nokukhiqizwa kwe-cytokine kunqunywe ku-540 nm noma ku-450 nm kusetshenziswa i-Wallac EnVision.™umfundi we-microplate (PerkinElmer).

2.4. Ukuhlolwa komsebenzi we-AntiosteoarthritisKu-Vivo

2.4.1. Izilwane

Amagundane e-Sprague-Dawley abesilisa (amasonto angu-7 ubudala) athengwe kwa-Samtako Inc. (Osan, Korea) futhi ahlaliswa ngaphansi kwezimo ezilawulwayo ngomjikelezo wokukhanya/umnyama ongu-12-h°C kanye% umswakama. Amagundane anikezwa ukudla kwaselabhorethri namanzii-ad libitum. Zonke izinqubo zokuhlola zenziwe ngokuhambisana neziqondiso Zezikhungo Zezempilo Zikazwelonke (NIH) futhi zagunyazwa IKomidi Lokunakekelwa Kwezilwane Nokusetshenziswa lasenyuvesi yase-Daejeon (Daejeon, republic of Korea).

2.4.2. Ukufakwa kwe-OA nge-MIA kumagundane

Izilwane zazingahleliwe futhi zabelwe amaqembu okwelapha ngaphambi kokuqala kocwaningo (iqembu ngalinye). Isixazululo se-MIA (3 mg/50μL ye-0.9% saline) ijovwe ngokuqondile endaweni ye-intra-articular yedolo elingakwesokudla ngaphansi kwe-anesthesia eyenziwe ngengxube ye-ketamine ne-xylazine. Amagundane ahlukaniswa ngokungahleliwe abe amaqembu amane: (1) iqembu le-saline elingenayo i-MIA injection, (2) iqembu le-MIA elinomjovo we-MIA, (3) iqembu eliphathwe nge-SDE (200 mg / kg) ngomjovo we-MIA, kanye (4) ) iqembu elilashwa nge-indomethacin- (IM-) (2 mg/kg) ngomjovo we-MIA. Amagundane aphathwa ngomlomo nge-SDE kanye ne-IM 1 ngesonto ngaphambi kokujova kwe-MIA kumaviki angu-4. Umthamo we-SDE ne-IM osetshenziswe kulolu cwaningo wawusekelwe kulabo abaqashwe ezifundweni ezedlule [10,13,14].

2.4.3. Izilinganiso ze-Hindpaw Weight-Bearing Distribution

Ngemuva kokungeniswa kwe-OA, ibhalansi yasekuqaleni kumandla okuthwala isisindo wama-hindpaws yaphazamiseka. Umhloli we-incapacitance (i-Linton instrumentation, Norfolk, UK) wasetshenziselwa ukuhlola izinguquko ekubekezeleleni isisindo. Amagundane afakwa ngokucophelela ekamelweni lokulinganisa. Amandla athwala isisindo asetshenziswa isitho sangemuva alinganiswa phakathi nenkathi ye-3 s. Isilinganiso sokusabalalisa isisindo sibalwa ngesibalo esilandelayo: [isisindo esithweni sangemuva esingakwesokudla/(isisindo esigangeni sangemuva kwesokudla + isisindo esithweni sangemuva kwesokunxele)] × 100 [15].

2.4.4. Izilinganiso zeSerum Cytokine Levels

Amasampula egazi ayeyi-centrifuged ku-1,500 g imizuzu engu-10 ku-4 ° C; bese kuthi i-serum iqoqwe futhi igcinwe ku-−70°C ize isetshenziswe. Amazinga we-IL-1β, IL-6, TNF-α, kanye ne-PGE2 ku-serum kukalwe kusetshenziswa amakhithi e-ELISA avela ku-R&D Systems (Minneapolis, MN, USA) ngokwemiyalelo yomkhiqizi.

2.4.5. Ukuhlaziywa Kwe-RT-PCR Yesikhathi Sangempela

Isamba se-RNA sakhishwa esiculweni esihlangene samadolo kusetshenziswa i-TRI reagent® (Sigma-Aldrich, St. Louis, MO, USA), eguqulelwe emuva ku-cDNA kanye ne-PCR-akhuliswe kusetshenziswa ikhithi ye-TM One Step RT PCR ene-SYBR eluhlaza (Applied Biosystems , Grand Island, NY, USA). I-PCR yobuningi besikhathi sangempela yenziwe kusetshenziswa isistimu ye-PCR ye-Applied Biosystems 7500 Real-Time (Applied Biosystems, Grand Island, NY, USA). Ukulandelana kwe-primer kanye nokulandelana kwe-probe kukhonjisiwe kuThebula1. Ama-aliquots amasampula e-cDNAs kanye nenani elilinganayo le-GAPDH cDNA akhuliswe ngengxube eyinhloko ye-TaqMan® Universal PCR equkethe i-DNA polymerase ngokuya ngemiyalo yomkhiqizi (Applied Biosystems, Foster, CA, USA). Izimo ze-PCR zaziyi-2 min ku-50 ° C, i-10 min ku-94 ° C, i-15 s ku-95 ° C, ne-1 iminithi ku-60 ° C emijikelezweni ye-40. Ukuhlangana kofuzo oluqondiwe kwanqunywa kusetshenziswa indlela yokuqhathanisa i-Ct (inombolo yomjikelezo we-threshold endaweni enqamula phakathi kwesakhiwo sokukhulisa nomkhawulo), ngokuya ngemiyalelo yomkhiqizi.

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