I-oyile ye-Saposhnikovia ecocekileyo ye-divaricata yekhandlela kunye nesepha yenza i-oyile ethengisayo idiffuser ebalulekileyo entsha kwizitshisi zeengcongolo.

inkcazelo emfutshane:

 

2.1. Ukulungiswa kwe-SDE

I-rhizomes ye-SD yathengwa njengemifuno eyomileyo evela ku-Hanherb Co. (Guri, Korea). Izixhobo zezityalo zaqinisekiswa nge-taxonomically nguDkt. Go-Ya Choi weKorea Institute of Oriental Medicine (KIOM). Isampuli yevawutsha (inombolo ye-2014 SDE-6) yafakwa kwi-Korean Herbarium ye-Standard Herbal Resources. I-rhizomes eyomileyo ye-SD (320 g) ikhutshwe kabini kunye ne-70% ye-ethanol (nge-2 h reflux) kwaye isicatshulwa sabe sigxininiswe phantsi koxinzelelo oluncitshisiweyo. I-decoction yahluzwa, i-lyophilized, kwaye igcinwe kwi-4 ° C. Isivuno sesicatshulwa esomileyo kwimathiriyeli yokuqala ekrwada yaba ngama-48.13% (w/w).

 

2.2. Uhlalutyo lwe-Quantitative High-Performance Liquid Chromatography (HPLC).

Uhlalutyo lweChromatographic lwenziwa ngenkqubo ye-HPLC (Waters Co., Milford, MA, USA) kunye ne-photodiode array detector. Kuhlalutyo lwe-HPLC ye-SDE, i-prim-OUmgangatho weglucosylcimifugin wathengwa kwiZiko laseKorea lokuKhuthaza iZiko loShishino loNyango lweSintu (Gyeongsan, Korea), kunyeumzuzwana-O-glucosylhamaudol kunye ne-4′-O-β-D-glucosyl-5-O-i-methylvisamminol yayiye yodwa ngaphakathi kwibhubhoratri yethu kwaye ichongiwe ngokuhlalutya kwe-spectral, ngokuyinhloko yi-NMR kunye ne-MS.

Iisampuli ze-SDE (0.1 mg) zachithwa kwi-70% ethanol (10 mL). Ukwahlulwa kweChromatographic kwenziwa ngekholamu ye-XSelect HSS T3 C18 (4.6 × 250 mm, 5μm, Waters Co., Milford, MA, USA). Isigaba esihambahambayo sasiquka i-acetonitrile (A) kunye ne-0.1% i-acetic acid emanzini (B) kwinqanaba lokuhamba kwe-1.0 mL / min. Inkqubo yokuthambeka kwamanyathelo amaninzi isetyenziswe ngolu hlobo lulandelayo: 5% A (0 imiz), 5–20% A (0–10 min), 20% A (10–23 min), kunye 20–65% A (23–40 min. ). I-wavelength yokufumanisa ihlolwe kwi-210-400 nm kwaye irekhodwe kwi-254 nm. Umthamo wokutofa wawuyi-10.0μIzisombululo ezisemgangathweni ze-L. zokumiselwa kweechromones ezintathu zalungiswa kwinqanaba lokugqibela le-7.781 mg / mL (prim-O-glucosylcimifugin), 31.125 mg/mL (4′-O-β-D-glucosyl-5-O-methylvisamminol), kunye ne-31.125 mg / mL (umzuzwana-O-glucosylhamaudol) kwi-methanol kwaye igcinwe kwi-4 ° C.

2.3. Uvavanyo lwe-Anti-Inflammatory ActivityI-In vitro

2.3.1. Inkcubeko yeSeli kunye noNyango lweSampuli

Iiseli ze-RAW ze-264.7 zifunyenwe kwi-American Type Culture Collection (ATCC, Manassas, VA, USA) kwaye ikhulile kwi-DMEM medium equkethe i-1% ye-antibiotics kunye ne-5.5% ye-FBS. Iiseli zafukanywa kwi-atmosfera efumileyo ye-5% CO2 kuma-37°C. Ukuvuselela iiseli, i-medium yatshintshwa nge-DMEM entsha, kunye ne-lipopolysaccharide (LPS, Sigma-Aldrich Chemical Co., St. Louis, MO, USA) kwi-1μI-g/mL yongezwa xa kukho okanye ukungabikho kwe-SDE (200 okanye i-400μg/mL) ukongeza i-24 h.

2.3.2. Ukumiselwa kweNitric oxide (NO), iProstaglandin E2 (PGE2), iTumor Necrosis Factor-α(TNF-α), kunye ne-Interleukin-6 (IL-6) Ukuveliswa

Iiseli zaphathwa nge-SDE kwaye zivuselelwe nge-LPS kwi-24 h. AKUKHO mveliso yahlalutywa ngokulinganisa i-nitrite usebenzisa i-reagent ye-Griess ngokwesifundo sangaphambili [12]. Imfihlo ye-cytokines evuthayo PGE2, TNF-α, kunye ne-IL-6 yagqitywa ngokusebenzisa i-ELISA kit (iinkqubo ze-R & D) ngokwemiyalelo yomenzi. Iziphumo ze-SDE kwi-NO kunye nokuveliswa kwe-cytokine zinqunywe kwi-540 nm okanye i-450 nm usebenzisa i-Wallac EnVision.™umfundi we-microplate (PerkinElmer).

2.4. Uvavanyo lwe-Antiosteoarthritis ActivityKwiVivo

2.4.1. Izilwanyana

Iigundane ze-Sprague-Dawley zamadoda (iiveki ezi-7 ubudala) zathengwa kwi-Samtako Inc. (Osan, Korea) kwaye zigcinwe phantsi kweemeko ezilawulwayo kunye nomjikelezo we-12-h ukukhanya / ubumnyama°C kunye% ukufuma. Iimpuku zanikwa ukutya kwaselabhoratri kunye namanzikunye ne-libitum. Zonke iinkqubo zovavanyo zenziwa ngokuhambelana nezikhokelo zeNational Institutes of Health (NIH) kwaye zivunyiwe yiKomiti yoLondolozo lweZilwanyana kunye nokuSetyenziswa kweYunivesithi yaseDaejeon (iDaejeon, iRiphabhliki yaseKorea).

2.4.2. Ukuqaliswa kwe-OA nge-MIA kwiimpuku

Izilwanyana zazingahleliwe kwaye zabelwa kumaqela onyango ngaphambi kokuba kuqaliswe isifundo (ngeqela ngalinye). Isisombululo se-MIA (3 mg/50μL ye-0.9% ye-saline) ifakwe ngokuthe ngqo kwindawo ye-intra-articular yedolo lasekunene phantsi kwe-anesthesia eyenziwe ngomxube we-ketamine kunye ne-xylazine. Iigundane zahlulwe ngokungaqhelekanga zibe ngamaqela amane: (1) iqela le-saline elingenayo i-MIA injection, (2) iqela le-MIA kunye ne-MIA injection, (3) iqela eliphathwa nge-SDE (200 mg / kg) kunye ne-MIA injection, kunye (4) ) iqela le-indomethacin- (IM-) eliphathwayo (2 mg / kg) ngenaliti ye-MIA. Iigundane zilawulwa ngomlomo kunye ne-SDE kunye ne-IM 1 iveki ngaphambi kokujova kwe-MIA kwiiveki ze-4. Idosi ye-SDE kunye ne-IM esetyenziswe kwesi sifundo yayisekelwe kwabo baqeshwe kwizifundo zangaphambili [10,13,14].

2.4.3. Imilinganiselo ye-Hindpaw Weight-Bearing Distribution

Emva kokungeniswa kwe-OA, ibhalansi yokuqala kwi-weight-bearing capability of hindpaws yaphazamiseka. Umhloli we-incapacitance (i-Linton instrumentation, i-Norfolk, e-UK) yayisetyenziselwa ukuvavanya utshintsho lokunyamezela ubunzima. Iimpuku zazibekwe ngobunono kwigumbi lokulinganisa. Amandla okuthwala ubunzima okwenziwa lilungu elingasemva aye alinganiselwa kwisithuba se-3 s. Umlinganiselo wolwabiwo lobunzima ubalwa ngolu hlobo lulandelayo: [ubunzima kwilungu elingasemva lasekunene/(ubunzima kwilungu elingasemva lasekunene + ubunzima kwilungu elingasemva lasekhohlo)] × 100 [15].

2.4.4. Imilinganiselo yeSerum Cytokine Levels

Iisampuli zegazi zaziyi-centrifuged kwi-1,500 g ye-10 min kwi-4 ° C; emva koko i-serum iqokelelwe kwaye igcinwe ku-−70 ° C de isetyenziswe. Amanqanaba e-IL-1β, IL-6, TNF-α, kunye ne-PGE2 kwi-serum yalinganiswa ngokusebenzisa iikiti ze-ELISA ezivela kwii-R & D Systems (Minneapolis, MN, USA) ngokwemiyalelo yomenzi.

2.4.5. Uhlalutyo lwe-RT-PCR lweXesha lokwenyani

I-RNA iyonke ikhutshwe kwi-knee joint tissue isebenzisa i-TRI reagent® (i-Sigma-Aldrich, iSt. Louis, MO, eU.SA), iguqulelwe-iguqulelwe kwi-cDNA kunye ne-PCR-amplified usebenzisa i-TM One Step RT PCR kit kunye ne-SYBR eluhlaza (i-Applied Biosystems , Grand Island, NY, USA). I-PCR yexesha langempela le-quantitative quantitative kusetyenziswa i-Applied Biosystems 7500 ye-Real-Time PCR inkqubo (i-Applied Biosystems, i-Grand Island, i-NY, i-USA). Ulandelelwano lwe-primer kunye nolandelelwano lweprobe lubonisiwe kwiThebhile1. Ii-Aliquots zesampulu ye-cDNAs kunye nesixa esilinganayo se-GAPDH cDNA zandiswe ngomxube we-TaqMan® Universal PCR oqulethe i-DNA polymerase ngokwemiyalelo yomenzi (Applied Biosystems, Foster, CA, USA). Iimeko ze-PCR ziyi-2 min kwi-50 ° C, i-10 min kwi-94 ° C, i-15 s kwi-95 ° C, kunye ne-1 min kwi-60 ° C kwi-40 imijikelezo. Ukugxininiswa kwejene ekujoliswe kuyo kunqunywe ngokusebenzisa i-Ct yokuthelekisa (inani lomjikelo we-threshold kwindawo yokuwela phakathi kweploti yokukhulisa kunye ne-threshold) indlela, ngokwemiyalelo yomenzi.

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