Saposhnikovia divaricata mmanụ dị ọcha maka kandụl na ncha na-eme ka nnukwu diffuser mmanụ dị ọhụrụ maka ndị na-ere ọkụ ahịhịa amị.

nkowa nkenke:

 

2.1. Nkwadebe nke SDE

A zụrụ rhizomes nke SD dị ka ahịhịa a mịrị amị sitere na Hanherb Co. (Guri, Korea). Dokta Go-Ya Choi nke Korea Institute of Oriental Medicine (KIOM) kwadoro akụrụngwa osisi ahụ n'ụzọ taxonomically. Edebere akwụkwọ ikike (nọmba 2014 SDE-6) na Korea Herbarium nke Standard Herbal Resources. A na-ewepụta rhizomes akpọnwụ nke SD (320 g) ugboro abụọ na ethanol 70% (ya na 2 h reflux) wee tinyezie ihe ahụ n'okpuru nrụgide belatara. A na-enyocha decoction ahụ, lyophilized ma chekwaa ya na 4 Celsius. Mkpụrụ nke mịrị amị sitere na ihe mmalite crude bụ 48.13% (w/w).

 

2.2. Ntụle Chromatography Liquid Liquid High-Performance (HPLC).

Emere nyocha chromatographic site na sistemụ HPLC (Waters Co., Milford, MA, USA) yana ihe nchọta fotodiode array. Maka nyocha HPLC nke SDE, prim-OA zụrụ ọkọlọtọ glucosylcimifugin n'aka Korea Promotion Institute for Traditional Medicine Industry (Gyeongsan, Korea), nankeji-OGlucosylhamaudol na 4'-O-βD-glucosyl-5-O-methylvisamminol dịpụrụ adịpụ n'ime ụlọ nyocha anyị wee chọpụta ya site na nleba anya, nke bụ isi site na NMR na MS.

A na-agbaze ihe nlele SDE (0.1 mg) na 70% ethanol (10 ml). Ejiri kọlụm XSelect HSS T3 C18 mee nkewa nke chromatographic (4.6 × 250 mm, 5).μm, Waters Co., Milford, MA, USA). Usoro mkpanaka gụnyere acetonitrile (A) na 0.1% acetic acid n'ime mmiri (B) na ọsọ ọsọ nke 1.0 ml / min. A na-eji usoro gradient multistep mee ihe dị ka ndị a: 5% A (0 min), 5-20% A (0-10 min), 20% A (10-23 min), na 20-65% A (23-40 min). ). A na-enyocha ogologo ogologo oge nchọpụta ahụ na 210-400 nm wee dekọọ na 254 nm. Olu injection bụ 10.0μL. A kwadoro ngwọta ọkọlọtọ maka mkpebi nke chromone atọ na njedebe ikpeazụ nke 7.781 mg / ml (prim-OGlucosylcimifugin), 31.125 mg/mL (4 ′-O-βD-glucosyl-5-O-methylvisamminol), na 31.125 mg / ml (nkeji-O-glucosylhamaudol) na methanol ma debe ya na 4 Celsius.

2.3. Nyochaa Ọrụ Mgbochi ỌrịaNa Vitro

2.3.1. Omenala Cell na Nlereanya Ọgwụgwọ

E nwetara mkpụrụ ndụ RAW 264.7 site na mkpokọta Ụdị Omenala America (ATCC, Manassas, VA, USA) wee tolite na DMEM ọkara nwere 1% ọgwụ nje na 5.5% FBS. A na-etinye mkpụrụ ndụ n'ime ikuku iru mmiri nke 5% CO2 na 37 Celsius. Iji kpalie mkpụrụ ndụ ahụ, a na-eji DMEM dị ọhụrụ dochie usoro ahụ, na lipopolysaccharide (LPS, Sigma-Aldrich Chemical Co., St. Louis, MO, USA) na 1.μagbakwunyere g/mL na ọnụnọ ma ọ bụ enweghị SDE (200 ma ọ bụ 400μg/ml) maka awa 24 ọzọ.

2.3.2. Mkpebi nke nitric oxide (NO), Prostaglandin E2 (PGE2), Tumor Necrosis Factor-α(TNF-α), na Interleukin-6 (IL-6) Mmepụta

Ejiri SDE gwọọ sel ma kpalie ya na LPS maka awa 24. Ọ dịghị mmepụta e nyochara site n'ịtụ nitrite iji Griess reagent dị ka ọmụmụ gara aga [12]. Nzuzo nke cytokines mkpali PGE2, TNF-α, na IL-6 kpebisiri ike site na iji ngwa ELISA (Sistemụ R&D) dịka ntuziaka ndị nrụpụta si dị. Achọpụtara mmetụta nke SDE na NO na mmepụta cytokine na 540 nm ma ọ bụ 450 nm site na iji Wallac EnVision.™onye na-agụ microplate (PerkinElmer).

2.4. Nyocha nke Ọrụ AntiosteoarthritisNa Vivo

2.4.1. Ụmụ anụmanụ

A zụrụ ụmụ oke Sprague-Dawley (izu asaa) n'aka Samtako Inc. (Osan, Korea) ma nọrọ n'okpuru ọnọdụ a na-achịkwa ya na ọkụ 12-h / ọchịchịrị okirikiri.Celsius C na% iru mmiri. Enyere oke nri ụlọ nyocha na mmiriad libitum. Emere usoro nnwale niile na nrube isi nke National Institutes of Health (NIH) ma kwadoro site na Kọmitii Nlekọta Animal na Jiri nke mahadum Daejeon (Daejeon, Republic of Korea).

2.4.2. Ntinye OA na MIA na oke

A na-ekewa anụmanụ ndị ahụ ma kenye ya n'òtù ọgwụgwọ tupu mmalite nke ọmụmụ (kwa otu). MIA ngwọta (3 mg/50μL nke 0.9% saline) ka etinyere ya ozugbo n'ime oghere intra-articular nke ikpere aka nri n'okpuru anesthesia nke sitere na ngwakọta ketamine na xylazine. E kewara oke oke n'ime otu anọ: (1) otu saline na-enweghị mmịnye MIA, (2) otu MIA nwere ntụtụ MIA, (3) otu SDE na-agwọ (200 mg/kg) na ịgba ọgwụ MIA, na (4) ) ndị indomethacin- (IM-) a na-agwọ (2 mg/kg) nwere ntụtụ MIA. A na-eji ọnụ SDE na IM 1 nye oke oke tupu ịgba ọgwụ mgbochi MIA maka izu anọ. Usoro nke SDE na IM ejiri mee ihe n'ọmụmụ ihe a dabeere na ndị a na-arụ ọrụ na ọmụmụ ihe gara aga [10,13,14].

2.4.3. Nleta nke nkesa na-ebu ibu Hindpaw

Mgbe ntinye OA gasịrị, nguzozi izizi na ike ibu nke hindpaws kwụsịrị. A na-eji ihe nleba anya enweghị ike (Linton instrumentation, Norfolk, UK) iji nyochaa mgbanwe na nnabata ibu ibu. Ejiri nlezianya tinye oke n'ime ụlọ a na-atụ ihe. A na-agbakọ ike ibu arọ site n'akụkụ aka azụ n'ime ihe karịrị nkeji 3. A na-agbakọ oke nkesa arọ site na ngụkọ ndị a: [ịdị arọ n'akụkụ aka nri aka nri/( arọ na ụkwụ aka nri aka nri + arọ na ụkwụ aka ekpe)] × 100 [15].

2.4.4. Ntụle nke Serum Cytokine Ọkwa

A na-ekpuchi ihe nlele ọbara na 1,500 g maka 10 min na 4 ° C; Mgbe ahụ, a na-anakọta serum ma chekwaa ya na -70 Celsius C ruo mgbe ejiri ya. Ọnụ ego nke IL-1β, IL-6, TNF-α, na PGE2 na serum ka a tụrụ ya site na iji ngwa ELISA sitere na R&D Systems (Minneapolis, MN, USA) dịka ntuziaka ndị nrụpụta si dị.

2.4.5. Ọnụ ọgụgụ RT-PCR nke ezigbo oge

A na-ewepụta mkpokọta RNA site na anụ ahụ nkwonkwo ikpere site na iji TRI reagent® (Sigma-Aldrich, St. Louis, MO, USA), tụgharịa-sụgharịa ya na cDNA na PCR-amụbawanye site na iji ngwa TM One Step RT PCR nwere SYBR green (Applied Biosystems). , Grand Island, NY, USA). Emere PCR ezigbo oge site na iji sistemu Biosystems 7500 Real-Time PCR (Applied Biosystems, Grand Island, NY, USA). E gosipụtara usoro izizi na usoro nyocha na Tebụlụ1. Agbakwunyere Aliquots nke cDNA sample na nha nha nke GAPDH cDNA site na ngwakọta TaqMan® Universal PCR nwere DNA polymerase dịka ntuziaka ndị nrụpụta si dị (Applied Biosystems, Foster, CA, USA). Ọnọdụ PCR bụ 2 min na 50 Celsius C, 10 min na 94 Celsius C, 15 s na 95 Celsius C, yana 1 min na 60 Celsius C maka okirikiri 40. Achọpụtara mkpokọta mkpụrụ ndụ ihe nlebara anya site na iji ntụnyere Ct (ọnụọgụ okirikiri ụzọ n'ụzọ dị n'etiti atụmatụ nkwalite na ọnụ ụzọ), dịka ntuziaka ndị nrụpụta siri dị.

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