Amafutha ahlanzekile e-Saposhnikovia divaricata okwenza ikhandlela kanye nensipho ngobuningi amafutha abalulekile amasha kuma-diffuser okushisa umhlanga

incazelo emfushane:

 

2.1. Ukulungiswa kwe-SDE

Ama-rhizome e-SD athengwe njengesitshalo esomisiwe eHanherb Co. (eGuri, eKorea). Izinto zesitshalo zaqinisekiswa ngokwe-taxonomically nguDkt. Go-Ya Choi we-Korea Institute of Oriental Medicine (KIOM). Isampula ye-voucher (inombolo 2014 SDE-6) yafakwa ku-Korean Herbarium of Standard Herbal Resources. Ama-rhizome omisiwe e-SD (320 g) akhishwe kabili nge-ethanol engu-70% (nge-reflux yamahora ama-2) kwathi i-extract yabe isigxiliswa ngaphansi kwengcindezi encishisiwe. I-decoction yahlungwa, yashintshwa yaba yi-lyophilized, yagcinwa ku-4°C. Isivuno se-extract eyomisiwe ezintweni zokuqala ezingavuthiwe sasingu-48.13% (w/w).

 

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

Ukuhlaziywa kwe-Chromatographic kwenziwe ngohlelo lwe-HPLC (Waters Co., Milford, MA, USA) kanye nesitholi se-photodiode array. Ekuhlaziyweni kwe-HPLC kwe-SDE, i-prim-OIzinga le-glucosylcimifugin lathengwa e-Korea Promotion Institute for Traditional Medicine Industry (eGyeongsan, eKorea), kanyeisekhondi-O-glucosylhamaudol kanye ne-4′-O-β-D-glucosyl-5-O-methylvisamminol zihlukaniswe ngaphakathi kwelebhu yethu futhi zahlonzwa ngokuhlaziywa kwe-spectral, ikakhulukazi yi-NMR kanye ne-MS.

Amasampula e-SDE (0.1 mg) ancibilikiswe ku-70% ethanol (10 mL). Ukuhlukaniswa kwe-chromatographic kwenziwe ngekholomu ye-XSelect HSS T3 C18 (4.6 × 250 mm, 5).μm, Waters Co., Milford, MA, USA). Isigaba esihambayo sasine-acetonitrile (A) kanye ne-acetic acid engu-0.1% emanzini (B) ngesilinganiso sokugeleza esingu-1.0 mL/min. Uhlelo lwe-gradient olunezinyathelo eziningi lusetshenziswe kanje: 5% A (0 min), 5–20% A (0–10 min), 20% A (10–23 min), kanye no-20–65% A (23–40 min). Ububanzi bokukhanya kokukhanya buskeniwe ku-210–400 nm futhi baqoshwa ku-254 nm. Umthamo womjovo wawungu-10.0μL. Izixazululo ezijwayelekile zokunquma ama-chromone amathathu zalungiswa ngokuhlushwa kokugcina okungu-7.781 mg/mL (prim-O-glucosylcimifugin), 31.125 mg/mL (4′-O-β-D-glucosyl-5-O-methylvisamminol), kanye no-31.125 mg/mL (isekhondi-O-glucosylhamaudol) ku-methanol futhi igcinwe ku-4°C.

2.3. Ukuhlolwa Komsebenzi Wokulwa NokuvuvukalaIn Vitro

2.3.1. Ukukhula Kwamaseli Nokwelashwa Kwesampula

Amaseli angu-RAW angu-264.7 atholakale ku-American Type Culture Collection (ATCC, Manassas, VA, USA) futhi akhuliswa endaweni yokwelapha ye-DMEM equkethe ama-antibiotic angu-1% kanye ne-5.5% FBS. Amaseli afakwa endaweni enomswakama engu-5% CO2 ku-37°C. Ukuze kukhuthazwe amaseli, indawo yokwelapha yathathelwa indawo indawo yokwelapha ye-DMEM entsha, kanye ne-lipopolysaccharide (LPS, Sigma-Aldrich Chemical Co., St. Louis, MO, USA) ku-1μg/mL yengezwe lapho kukhona noma kungekho khona 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(I-TNF-α), kanye nokukhiqizwa kwe-Interleukin-6 (IL-6)

Amaseli aphathwe nge-SDE futhi avuselelwa nge-LPS amahora angama-24. Akukho ukukhiqizwa okwahlaziywa ngokulinganisa i-nitrite kusetshenziswa i-Griess reagent ngokusho kocwaningo lwangaphambilini [12]. Ukukhishwa kwama-cytokines avuvukalayo i-PGE2, i-TNF-α, kanti i-IL-6 yanqunywa kusetshenziswa ikhithi ye-ELISA (izinhlelo ze-R&D) ngokwemiyalelo yomkhiqizi. Imiphumela ye-SDE ekukhiqizweni kwe-NO kanye ne-cytokine yanqunywa ku-540 nm noma ku-450 nm kusetshenziswa i-Wallac EnVision.™umfundi we-microplate (PerkinElmer).

2.4. Ukuhlolwa Komsebenzi Wokulwa Ne-OsteoarthritisE-Vivo

2.4.1. Izilwane

Amagundane amaduna eSprague-Dawley (amasonto ayi-7 ubudala) athengwe kwaSamtako Inc. (e-Osan, eKorea) futhi agcinwa ngaphansi kwezimo ezilawulwayo ngomjikelezo wokukhanya/ubumnyama wamahora ayi-12 e-°C kanye% umswakama. Amagundane anikezwe ukudla kwaselabhorethri kanye namanziukukhululeka kwesikhangisoZonke izinqubo zokuhlola zenziwe ngokuhambisana neziqondiso ze-National Institutes of Health (NIH) futhi zavunywa yiKomidi Lokunakekelwa Kwezilwane Nokusetshenziswa kweyunivesithi yaseDaejeon (eDaejeon, iRiphabhulikhi yaseKorea).

2.4.2. Ukufakwa kwe-OA nge-MIA kumagundane

Izilwane zahlelwa ngokungahleliwe futhi zabelwa emaqenjini okwelapha ngaphambi kokuqala kocwaningo (ngeqembu ngalinye). Isixazululo se-MIA (3 mg/50μI-L ye-0.9% saline) yafakwa ngqo esikhaleni sangaphakathi kwedolo lesokudla ngaphansi kwe-anesthesia ebangelwe yingxube ye-ketamine ne-xylazine. Amagundane ahlukaniswe ngokungahleliwe abe amaqembu amane: (1) iqembu le-saline elingenayo i-MIA injection, (2) iqembu le-MIA elinomjovo we-MIA, (3) iqembu elaphathwa yi-SDE (200 mg/kg) elinomjovo we-MIA, kanye (4) iqembu elaphathwa yi-indomethacin- (IM-) (2 mg/kg) elinomjovo we-MIA. Amagundane anikezwa ngomlomo nge-SDE kanye ne-IM isonto eli-1 ngaphambi komjovo we-MIA amasonto ama-4. Umthamo we-SDE kanye ne-IM osetshenziswe kulolu cwaningo wawusekelwe kulawo asetshenziswe ezifundweni zangaphambilini [10,13,14].

2.4.3. Ukulinganiswa Kokusabalalisa Isisindo Se-Hindpaw

Ngemva kokungeniswa kwe-OA, ibhalansi yokuqala emandleni okuthwala isisindo sama-hindpaws yaphazamiseka. Isihloli sokungakwazi ukusebenza (i-Linton instrumentation, eNorfolk, e-UK) sasetshenziswa ukuhlola izinguquko ekubekezeleni kokuthwala isisindo. Amagundane afakwa ngokucophelela ekamelweni lokulinganisa. Amandla okuthwala isisindo asetshenziswa yilungu langemuva alinganiswa phakathi nenkathi yemizuzwana emi-3. Isilinganiso sokusatshalaliswa kwesisindo sabalwa ngesibalo esilandelayo: [isisindo esithweni sangemuva sokudla/(isisindo esithweni sangemuva sokudla + isisindo esithweni sangemuva kwesobunxele)] × 100 [15].

2.4.4. Ukulinganiswa Kwamazinga E-Serum Cytokine

Amasampula egazi afakwa ku-centrifuge ku-1,500 g imizuzu eyi-10 ku-4°C; bese i-serum iqoqwa futhi igcinwe ku-−70°C kuze kube yilapho isetshenziswa.β, IL-6, TNF-α, kanye ne-PGE2 ku-serum kwalinganiswa kusetshenziswa ama-ELISA kit avela ku-R&D Systems (eMinneapolis, MN, e-USA) ngokwemiyalelo yomkhiqizi.

2.4.5. Ukuhlaziywa Kwe-RT-PCR Yesikhathi Sangempela

I-RNA iyonke ikhishwe ezicutshini zamadolo kusetshenziswa i-TRI reagent® (Sigma-Aldrich, St. Louis, MO, USA), yahunyushwa kabusha yaba yi-cDNA futhi yakhuliswa yi-PCR kusetshenziswa ikhithi ye-TM One Step RT PCR ene-SYBR green (Applied Biosystems, Grand Island, NY, USA). I-Real-time quantitative PCR yenziwe kusetshenziswa uhlelo lwe-Applied Biosystems 7500 Real-Time PCR (Applied Biosystems, Grand Island, NY, USA). Ukulandelana kwe-primer kanye nokulandelana kwe-probe kuboniswe kuThebula.1. Ama-aliquot amasampula e-cDNA kanye nenani elilinganayo le-GAPDH cDNA akhuliswe ngengxube ye-TaqMan® Universal PCR master equkethe i-DNA polymerase ngokwemiyalelo yomkhiqizi (i-Applied Biosystems, Foster, CA, USA). Izimo ze-PCR zaziyimizuzu emi-2 ku-50°C, imizuzu eyi-10 ku-94°C, imizuzwana eyi-15 ku-95°C, kanye nomzuzu o-1 ku-60°C imijikelezo engama-40. Ukuhlushwa kwe-target gene kunqunywe kusetshenziswa indlela yokuqhathanisa i-Ct (inombolo yomjikelezo womngcele endaweni ewela phakathi kwesakhiwo sokukhulisa kanye nomkhawulo), ngokwemiyalelo yomkhiqizi.

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