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tlhaloso e khutšoanyane:

 

2.1. Tokisetso ea SDE

Li-rhizome tsa SD li rekiloe e le setlama se omisitsoeng ho tsoa Hanherb Co. (Guri, Korea). Lisebelisoa tsa semela li tiisitsoe ka mokhoa oa taxonomic ke Dr. Go-Ya Choi oa Korea Institute of Oriental Medicine (KIOM). Sampole ea voucher (nomoro ea 2014 SDE-6) e ile ea kenngoa ho Korean Herbarium of Standard Herbal Resources. Li-rhizome tse omisitsoeng tsa SD (320 g) li ile tsa ntšoa habeli ka 70% ethanol (ka reflux ea lihora tse 2) 'me setlolo se ile sa kenngoa tlas'a khatello e fokotsehileng. Motsoako o ile oa sefshoa, oa etsoa lyophilized, 'me oa bolokoa ho 4°C. Pokello ea setlolo se omisitsoeng ho tsoa linthong tse qalang tse tala e ne e le 48.13% (w/w).

 

2.2. Tlhahlobo ea Liquid Chromatography (HPLC) ea Bongata bo Tshebetso e Phahameng

Tlhahlobo ea chromatographic e entsoe ka sistimi ea HPLC (Waters Co., Milford, MA, USA) le sesebelisoa sa ho lemoha li-photodiode array. Bakeng sa tlhahlobo ea HPLC ea SDE, prim-O-glucosylcimifugin standard e rekiloe ho Korea Promotion Institute for Traditional Medicine Industry (Gyeongsan, Korea), mmesec-O-glucosylhamaudol le 4′-O-β-D-glucosyl-5-O-methylvisamminol li ile tsa aroloa ka har'a laboratori ea rona 'me tsa khetholloa ka litlhahlobo tsa spectral, haholo-holo ke NMR le MS.

Disampole tsa SDE (0.1 mg) di qhibilihisitsoe ka ethanol ya 70% (10 mL). Karohano ya chromatographic e entswe ka kholomo ya XSelect HSS T3 C18 (4.6 × 250 mm, 5).μm, Waters Co., Milford, MA, USA). Mokhahlelo o tsamayang o ne o entswe ka acetonitrile (A) le 0.1% acetic acid metsing (B) ka sekgahla sa phallo sa 1.0 mL/min. Lenaneo la gradient la mehato e mengata le sebedisitswe ka tsela e latelang: 5% A (0 min), 5–20% A (0–10 min), 20% A (10–23 min), le 20–65% A (23–40 min). Bolelele ba leqhubu la ho lemoha bo skenetswe ho 210–400 nm mme ba rekotwa ho 254 nm. Bophahamo ba ente e ne e le 10.0μL. Litharollo tse tloaelehileng bakeng sa ho fumana li-chromone tse tharo li lokisitsoe ka mahloriso a ho qetela a 7.781 mg/mL (prim-O-glucosylcimifugin), 31.125 mg/mL (4′-O-β-D-glucosyl-5-O-methylvisamminol), le 31.125 mg/mL (sec-O-glucosylhamaudol) ka har'a methanol 'me e bolokiloe mochesong oa 4°C.

2.3. Tlhahlobo ea Mesebetsi ea ho Loantša ho RuruhaKa Vitro

2.3.1. Temo ea Lisele le Kalafo ea Sampole

Lisele tse RAW 264.7 li fumanoe ho tsoa ho American Type Culture Collection (ATCC, Manassas, VA, USA) 'me tsa holisoa ka har'a DMEM medium e nang le 1% ea lithibela-mafu le 5.5% FBS. Lisele li ile tsa beoa sebakeng se mongobo sa 5% CO2 ho 37°C. Ho hlasimolla lisele, medium e ile ea nkeloa sebaka ke DMEM medium e ncha, le lipopolysaccharide (LPS, Sigma-Aldrich Chemical Co., St. Louis, MO, USA) ho 1μg/mL e ekelitsoe ha SDE e le teng kapa e le sieo (200 kapa 400)μg/mL) bakeng sa lihora tse ling tse 24.

2.3.2. Ho fumana Nitric Oxide (NO), Prostaglandin E2 (PGE2), Tumor Necrosis Factor-α(TNF-α), le Tlhahiso ea Interleukin-6 (IL-6)

Lisele li ile tsa phekoloa ka SDE 'me tsa tsosolosoa ka LPS lihora tse 24. Ha ho tlhahiso e ileng ea hlahlojoa ka ho lekanya nitrite ho sebelisoa reagent ea Griess ho latela phuputso e fetileng [12]. Ho ntšoa ha li-cytokine tse ruruhang PGE2, TNF-α, 'me IL-6 e ile ea fumanoa ka ho sebelisa kit ea ELISA (litsamaiso tsa R&D) ho latela litaelo tsa moetsi. Litlamorao tsa SDE ho NO le tlhahiso ea cytokine li ile tsa fumanoa ho 540 nm kapa 450 nm ho sebelisoa Wallac EnVision.™sebali sa microplate (PerkinElmer).

2.4. Tlhahlobo ea Mosebetsi oa AntiosteoarthritisKa Vivo

2.4.1. Liphoofolo

Likhoto tse tona tsa Sprague-Dawley (tse libeke tse 7) li rekiloe ho Samtako Inc. (Osan, Korea) 'me li bolokiloe tlas'a maemo a laoloang ka potoloho ea khanya/lefifi ea lihora tse 12 ho°C le% mongobo. Likhoto li ile tsa fuoa lijo tsa laboratori le metsinako ea ho itokolla mosebetsingMekhoa eohle ea liteko e entsoe ho latela litataiso tsa National Institutes of Health (NIH) 'me ea amoheloa ke Komiti ea Tlhokomelo le Tšebeliso ea Liphoofolo ea univesithi ea Daejeon (Daejeon, rephaboliki ea Korea).

2.4.2. Ho kenngoa ha OA ka MIA ho Likhoto

Liphoofolo li ile tsa khethoa ka mokhoa o sa reroang 'me tsa abeloa lihlopha tsa kalafo pele ho qalo ea thuto (ka sehlopha). Tharollo ea MIA (3 mg/50μL ea saline ea 0.9% e ile ea kenngoa ka kotloloho sebakeng sa intra-articular sa lengole le letona tlas'a anesthesia e hlahisitsoeng ka motsoako oa ketamine le xylazine. Likhoto li ile tsa aroloa ka mokhoa o sa reroang ka lihlopha tse 'ne: (1) sehlopha sa saline se se nang ente ea MIA, (2) sehlopha sa MIA se nang le ente ea MIA, (3) sehlopha se phekoloang ke SDE (200 mg/kg) se nang le ente ea MIA, le (4) sehlopha se phekoloang ke indomethacin- (IM-) (2 mg/kg) se nang le ente ea MIA. Likhoto li ile tsa fuoa ka molomo le SDE le IM beke e le 'ngoe pele ho ente ea MIA libeke tse 4. Tekanyo ea SDE le IM e sebelisitsoeng thutong ena e ne e ipapisitse le tse sebelisitsoeng lithutong tse fetileng [10,13,14].

2.4.3. Litekanyo tsa Kabo ea Boima ba Hindpaw

Kamora ho kenngoa ha OA, tekanyo ea pele ea bokhoni ba ho jara boima ba maoto a ka morao e ile ea sitisoa. Sesebelisoa sa ho lekola ho se khone ho jara boima (Linton instrumentation, Norfolk, UK) se ile sa sebelisoa ho lekola liphetoho mamellong ea ho jara boima. Likhoto li ile tsa kenngoa ka hloko ka kamoreng ea ho lekanya. Matla a ho jara boima a sebelisoang ke leoto la ka morao a ile a lekanngoa ka nako ea metsotsoana e 3. Karolelano ea kabo ea boima e ile ea baloa ka equation e latelang: [boima leotong le ka morao le letona/(boima leotong le ka morao le letona + boima leotong le ka morao le letšehali)] × 100 [15].

2.4.4. Litekanyo tsa Maemo a Serum Cytokine

Disampole tsa madi di ile tsa tjhesetswa ka sentrifuge ho 1,500 g metsotso e 10 ho 4°C; ebe serum e bokellwa mme ya bolokwa ho −70°C ho fihlela e sebediswa.β, IL-6, TNF-α, le PGE2 ka hara serum di lekantswe ka ho sebedisa dikiti tsa ELISA tse tswang ho R&D Systems (Minneapolis, MN, USA) ho ya ka ditaelo tsa moetsi.

2.4.5. Tlhahlobo ea Bongata ba RT-PCR ea Nako ea Sebele

Kakaretso ea RNA e ntšoa liseleng tsa lengole ho sebelisoa reagent® ea TRI (Sigma-Aldrich, St. Louis, MO, USA), e fetiselitsoe morao ho cDNA 'me ea amplified ke PCR ho sebelisoa kit ea TM One Step RT PCR e nang le SYBR green (Applied Biosystems, Grand Island, NY, USA). Real-time quantitative PCR e entsoe ho sebelisoa sistimi ea Applied Biosystems 7500 Real-Time PCR (Applied Biosystems, Grand Island, NY, USA). Litatelano tsa primer le probe-sequence li bontšitsoe ho Tafole.1. Li-aliquot tsa li-cDNA tsa sampole le palo e lekanang ea GAPDH cDNA li ile tsa matlafatsoa ka motsoako o moholo oa TaqMan® Universal PCR o nang le DNA polymerase ho latela litaelo tsa moetsi (Applied Biosystems, Foster, CA, USA). Maemo a PCR e ne e le metsotso e 2 ho 50°C, metsotso e 10 ho 94°C, 15 s ho 95°C, le motsotso o 1 ho 60°C bakeng sa lipotoloho tse 40. Khokahano ea gene e reretsoeng e ile ea fumanoa ho sebelisoa mokhoa oa Ct o bapisang (nomoro ea potoloho ea moeli sebakeng se pakeng tsa amplification plot le moeli), ho latela litaelo tsa moetsi.

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