Ioyile ecocekileyo yeSaposhnikovia divaricata yokwenza ikhandlela kunye nesepha ngobuninzi ioyile ebalulekileyo entsha kwi-reed burner diffusers

inkcazo emfutshane:

 

2.1. Ukulungiswa kwe-SDE

Iirhizomes ze-SD zithengwe njengemifuno eyomileyo kwiHanherb Co. (eGuri, eKorea). Izinto zesityalo ziqinisekiswe ngokwe-taxonomically nguGqr. Go-Ya Choi weKorea Institute of Oriental Medicine (KIOM). Isampuli yevoucher (inombolo 2014 SDE-6) ibekwe kwiKorean Herbarium of Standard Herbal Resources. Iirhizomes ezomileyo ze-SD (320 g) zikhutshwe kabini nge-70% ye-ethanol (ene-reflux yeeyure ezi-2) kwaye i-extract yaxutywa phantsi koxinzelelo oluphantsi. I-decoction yahluzwa, yaguqulwa yaba yi-lyophilized, yaza yagcinwa kwi-4°C. Isivuno se-extract eyomileyo kwizinto zokuqala ezikrwada yayiyi-48.13% (w/w).

 

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

Uhlalutyo lweChromatographic lwenziwe ngenkqubo ye-HPLC (Waters Co., Milford, MA, USA) kunye nesixhobo sokubona uluhlu lwe-photodiode. Kuhlalutyo lwe-HPLC lwe-SDE, i-prim-OUmgangatho we-glucosylcimifugin uthengwe kwi-Korea Promotion Institute for Traditional Medicine Industry (eGyeongsan, eKorea), kwayeisek-O-glucosylhamaudol kunye ne-4′-O-β-D-glucosyl-5-O-methylvisamminol zahlulwe kwilebhu yethu zaza zachongwa ngohlalutyo lwe-spectral, ikakhulu yi-NMR kunye ne-MS.

Iisampulu ze-SDE (0.1 mg) zanyibilikiswa kwi-70% ye-ethanol (10 mL). Ukwahlulwa kwe-chromatographic kwenziwe ngekholamu ye-XSelect HSS T3 C18 (4.6 × 250 mm, 5).μm, Waters Co., Milford, MA, USA). Isigaba esihambayo sasine-acetonitrile (A) kunye ne-0.1% ye-acetic acid emanzini (B) kwisantya sokuhamba se-1.0 mL/min. Inkqubo ye-gradient enezinyathelo ezininzi isetyenziswe ngolu hlobo lulandelayo: 5% A (0 min), 5–20% A (0–10 min), 20% A (10–23 min), kunye ne-20–65% A (23–40 min). Ububanzi bokukhanya bokubona buskeniwe kwi-210–400 nm kwaye barekhodwa kwi-254 nm. Umthamo we-injection wawuyi-10.0μL. Izisombululo ezisemgangathweni zokumisela iikhromones ezintathu zalungiswa kuxinzelelo lokugqibela lwe-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 (isek-O-glucosylhamaudol) kwi-methanol kwaye igcinwe kwi-4°C.

2.3. Uvavanyo lomsebenzi wokulwa nokudumbaI-In vitro

2.3.1. Ukuphuhliswa kweeSeli kunye noNyango lweSampuli

Iiseli zeRAW ezingama-264.7 zifunyenwe kwi-American Type Culture Collection (ATCC, Manassas, VA, USA) zaze zakhuliswa kwi-DMEM medium equlethe i-1% yee-antibiotics kunye ne-5.5% FBS. Iiseli zafakwa kwindawo enomswakama ye-5% CO2 kuma-37°C. Ukuvuselela iiseli, i-medium yatshintshwa yi-DMEM medium entsha, kunye ne-lipopolysaccharide (LPS, Sigma-Aldrich Chemical Co., St. Louis, MO, USA) kwi-1μg/mL yongezwe xa kukho okanye kungekho i-SDE (200 okanye 400μg/mL) iiyure ezingama-24 ezongezelelweyo.

2.3.2. Ukugqitywa kweNitric Oxide (NO), iProstaglandin E2 (PGE2), iTumor Necrosis Factor-α(I-TNF-α), kunye neMveliso ye-Interleukin-6 (IL-6)

Iiseli ziphathwe nge-SDE zaza zavuselelwa nge-LPS kangangeeyure ezingama-24. AKUKHO mveliso ihlaziyiweyo ngokulinganisa i-nitrite kusetyenziswa i-Griess reagent ngokwesifundo sangaphambili [12Ukukhutshwa kwee-cytokines ezivuthayo ze-PGE2, TNF-α, kwaye i-IL-6 yachongwa kusetyenziswa i-ELISA kit (iinkqubo ze-R&D) ngokwemiyalelo yomenzi. Iziphumo ze-SDE kwi-NO kunye nemveliso ye-cytokine zachongwa kwi-540 nm okanye kwi-450 nm kusetyenziswa i-Wallac EnVision.™umfundi we-microplate (PerkinElmer).

2.4. Uvavanyo lomsebenzi we-AntiosteoarthritisEVivo

2.4.1. Izilwanyana

Iimpuku eziziinkunzi zeSprague-Dawley (ezineeveki ezisi-7 ubudala) zithengwe kwiSamtako Inc. (e-Osan, eKorea) zaza zagcinwa phantsi kweemeko ezilawulwayo kunye nomjikelo wokukhanya/ubumnyama weeyure ezili-12 e-°C kunye% yomswakama. Iimpuku zinikwe ukutya kwaselabhoratri kunye namanziukukhululwa kwintengisoZonke iinkqubo zovavanyo zenziwe ngokuhambelana nezikhokelo zeNational Institutes of Health (NIH) kwaye zavunywa yiKomiti yoKhathalelo lweZilwanyana kunye nokusetyenziswa kweyunivesithi yaseDaejeon (eDaejeon, kwiriphabliki yaseKorea).

2.4.2. Ukuqaliswa kwe-OA kunye ne-MIA kwiigundane

Izilwanyana zacwangciswa ngokungacwangciswanga zaza zabelwa kumaqela onyango ngaphambi kokuba kuqaliswe uphando (ngeqela ngalinye). Isisombululo se-MIA (3 mg/50μI-L ye-0.9% saline) yafakwa ngqo kwindawo yangaphakathi kwedolo lasekunene phantsi kwe-anesthesia eyenziwe ngumxube we-ketamine kunye ne-xylazine. Iimpuku zahlulwe ngokungacwangciswanga zaba ngamaqela amane: (1) iqela le-saline elingenayo inaliti ye-MIA, (2) iqela le-MIA eline-injection ye-MIA, (3) iqela elinyangwa yi-SDE (200 mg/kg) eline-injection ye-MIA, kunye (4) iqela elinyangwa yi-indomethacin- (IM-) (2 mg/kg) eline-injection ye-MIA. Iimpuku zanikwa ngomlomo nge-SDE kunye ne-IM iveki e-1 ngaphambi kwe-injection ye-MIA iiveki ezi-4. Idosi ye-SDE kunye ne-IM esetyenzisiweyo kolu phononongo yayisekelwe kwezo zisetyenziswe kwizifundo zangaphambili [10,13,14].

2.4.3. Ukulinganiswa koSasazo lokuthwala ubunzima beHindpaw

Emva kokungeniswa kwe-OA, ibhalansi yokuqala kumandla okuthwala ubunzima eempundu zangasemva yaphazamiseka. Isixhobo sokuvavanya ukungakwazi ukuthwala ubunzima (iLinton instrumentation, eNorfolk, e-UK) sasetyenziswa ukuvavanya utshintsho kunyamezelo lokuthwala ubunzima. Amagundane afakwa ngononophelo kwigumbi lokulinganisa. Amandla okuthwala ubunzima asetyenziswa lilungu langasemva alinganiswa ngokwexesha lemizuzwana emi-3. Umlinganiselo wokusasazwa kobunzima ubalwe ngale equation ilandelayo: [ubunzima kwilungu langasemva lasekunene/(ubunzima kwilungu langasemva lasekunene + ubunzima kwilungu langasemva lasekhohlo)] × 100 [15].

2.4.4. Ukulinganiswa kwamanqanaba e-Serum Cytokine

Iisampulu zegazi zifakwe kwi-centrifuge kwi-1,500 g imizuzu eli-10 kwi-4°C; emva koko i-serum yaqokelelwa yaza yagcinwa kwi-−70°C de isetyenziswe.β, IL-6, TNF-α, kunye ne-PGE2 kwi-serum zilinganiswe kusetyenziswa iikhithi ze-ELISA ezivela kwi-R&D Systems (eMinneapolis, MN, e-USA) ngokwemiyalelo yomenzi.

2.4.5. Uhlalutyo lwe-RT-PCR yoBuninzi beXesha langempela

I-RNA iyonke ikhutshwe kwizicubu zedolo kusetyenziswa i-TRI reagent® (Sigma-Aldrich, St. Louis, MO, USA), yaguqulelwa ngasemva kwi-cDNA yaza yandiswa yi-PCR kusetyenziswa i-TM One Step RT PCR kit ene-SYBR green (Applied Biosystems, Grand Island, NY, USA). I-Real-time quantitative PCR yenziwe kusetyenziswa inkqubo ye-Applied Biosystems 7500 Real-Time PCR (Applied Biosystems, Grand Island, NY, USA). I-primer sequences kunye ne-probe-sequence ziboniswe kwiTheyibhile1Ii-aliquots zeesampuli ze-cDNA kunye nenani elilinganayo le-GAPDH cDNA zandiswa ngomxube we-TaqMan® Universal PCR master oqulethe i-DNA polymerase ngokwemiyalelo yomenzi (Applied Biosystems, Foster, CA, USA). Iimeko ze-PCR zaziyimizuzu emi-2 kwi-50°C, imizuzu eli-10 kwi-94°C, imizuzwana eli-15 kwi-95°C, kunye nomzuzu o-1 kwi-60°C kwimijikelo engama-40. Uxinzelelo lwe-gene ekujoliswe kuyo lumiselwe kusetyenziswa indlela yokuthelekisa i-Ct (inombolo yomjikelo womda kwindawo enqamlezileyo phakathi kweploti yokukhulisa kunye nomda), ngokwemiyalelo yomenzi.

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