Mafuta oyera a Saposhnikovia divaricata a makandulo ndi sopo kupanga mafuta ophatikizira mafuta ofunikira atsopano kwa zoyatsira bango

Mafuta oyera a Saposhnikovia divaricata a makandulo ndi sopo opanga mafuta ophatikizira mafuta ofunikira atsopano kwa zoyatsira bango

Kufotokozera mwachidule:

2.1. Kukonzekera kwa SDE

Ma rhizomes a SD adagulidwa ngati zitsamba zouma kuchokera ku Hanherb Co. (Guri, Korea). Zomerazo zidatsimikiziridwa mwaukadaulo ndi Dr. Go-Ya Choi wa Korea Institute of Oriental Medicine (KIOM). Chitsanzo cha voucher (nambala 2014 SDE-6) chinayikidwa mu Korea Herbarium of Standard Herbal Resources. Ma rhizomes owuma a SD (320 g) adatengedwa kawiri ndi 70% ethanol (yokhala ndi 2 h reflux) ndipo zotulutsazo zidayikidwa movutikira. Decoction imasefedwa, lyophilized, ndikusungidwa pa 4 ° C. Zokolola za zouma zouma kuchokera kuzinthu zoyambira zopanda pake zinali 48.13% (w/w).

2.2. Quantitative High-Performance Liquid Chromatography (HPLC) Analysis

Kusanthula kwa Chromatographic kunachitika ndi makina a HPLC (Waters Co., Milford, MA, USA) ndi chowunikira cha photodiode. Pakuwunika kwa HPLC kwa SDE, prim-O-glucosylcimifugin standard idagulidwa ku Korea Promotion Institute for Traditional Medicine Viwanda (Gyeongsan, Korea), ndimphindi-O-glucosylhamaudol ndi 4'-O-βD-glucosyl-5-O-methylvisamminol inapatulidwa mkati mwa labotale yathu ndipo idazindikirika ndi kusanthula kwa spectral, makamaka ndi NMR ndi MS.

Zitsanzo za SDE (0.1 mg) zinasungunuka mu 70% ethanol (10 mL). Kulekanitsa chromatographic kunachitika ndi XSelect HSS T3 C18 column (4.6 × 250 mm, 5μm, Waters Co., Milford, MA, USA). Gawo la mafoni linali la acetonitrile (A) ndi 0.1% acetic acid m'madzi (B) pamtunda wa 1.0 mL / min. Pulogalamu ya multistep gradient idagwiritsidwa ntchito motere: 5% A (0 min), 5–20% A (0–10 min), 20% A (10–23 min), ndi 20–65% A (23–40 min. ). Kutalika kwa mawonekedwewo kudasinthidwa pa 210-400 nm ndikujambulidwa pa 254 nm. Kuchuluka kwa jekeseni kunali 10.0μL. Mayankho anthawi zonse otsimikizira ma chromone atatu adakonzedwa pomaliza 7.781 mg/mL (prim-O-glucosylcimifugin), 31.125 mg/mL (4′-O-βD-glucosyl-5-O-methylvisamminol), ndi 31.125 mg/mL (mphindi-O-glucosylhamaudol) mu methanol ndikusungidwa pa 4 ° C.

2.3. Kuunika kwa Ntchito Yotsutsana ndi KutupaMu Vitro

2.3.1. Chikhalidwe cha Maselo ndi Zitsanzo za Chithandizo

Maselo a RAW 264.7 adapezedwa kuchokera ku American Type Culture Collection (ATCC, Manassas, VA, USA) ndipo amakula mu DMEM sing'anga yomwe ili ndi 1% maantibayotiki ndi 5.5% FBS. Maselo anakulungidwa mu mpweya wa 5% CO2 pa 37°C. Pofuna kulimbikitsa maselo, sing'angayo inasinthidwa ndi DMEM yatsopano, ndi lipopolysaccharide (LPS, Sigma-Aldrich Chemical Co., St. Louis, MO, USA) pa 1μg/mL idawonjezedwa pamaso kapena kusapezeka kwa SDE (200 kapena 400μg/mL) kwa maola 24 owonjezera.

2.3.2. Kutsimikiza kwa Nitric Oxide (NO), Prostaglandin E2 (PGE2), Tumor Necrosis Factor-α(TNF-α), ndi Interleukin-6 (IL-6) Kupanga

Maselo amathandizidwa ndi SDE ndikulimbikitsidwa ndi LPS kwa 24 h. Palibe kupanga komwe kunawunikidwa poyesa nitrite pogwiritsa ntchito Griess reagent malinga ndi kafukufuku wakale [12]. Kutulutsa kwa ma cytokines otupa PGE2, TNF-α, ndipo IL-6 idatsimikiziridwa pogwiritsa ntchito zida za ELISA (R&D machitidwe) molingana ndi malangizo opanga. Zotsatira za SDE pa NO ndi kupanga cytokine zinatsimikiziridwa pa 540 nm kapena 450 nm pogwiritsa ntchito Wallac EnVision.™owerenga microplate (PerkinElmer).

2.4. Kuwunika kwa Antiosteoarthritis ActivityMu Vivo

2.4.1. Zinyama

Makoswe aamuna a Sprague-Dawley (masabata 7) adagulidwa kuchokera ku Samtako Inc. (Osan, Korea) ndipo amasungidwa pansi pazikhalidwe zoyendetsedwa ndi 12-h kuwala / mdima kuzungulira.°C ndi% chinyezi. Makoswe anapatsidwa chakudya cha labotale ndi madzindi libitum. Njira zonse zoyesera zidachitika motsatira malangizo a National Institutes of Health (NIH) ndikuvomerezedwa ndi Komiti Yoyang'anira Zanyama ndi Ntchito ya yunivesite ya Daejeon (Daejeon, Republic of Korea).

2.4.2. Kulowetsedwa kwa OA ndi MIA mu Makoswe

Zinyamazo zinasinthidwa mwachisawawa ndikupatsidwa magulu achipatala musanayambe phunzirolo (pa gulu). MIA yankho (3 mg/50μL ya 0.9% saline) analowetsedwa mwachindunji mu intra-articular danga la bondo lamanja pansi pa anesthesia yopangidwa ndi ketamine ndi xylazine. Makoswe adagawidwa mwachisawawa m'magulu anayi: (1) gulu la saline lopanda jekeseni wa MIA, (2) gulu la MIA ndi jekeseni wa MIA, (3) gulu la SDE (200 mg / kg) ndi jekeseni wa MIA, ndi (4) ) gulu la mankhwala a indomethacin- (IM-) (2 mg/kg) ndi jekeseni wa MIA. Makoswe ankaperekedwa pamlomo ndi SDE ndi IM 1 sabata pamaso pa jekeseni wa MIA kwa masabata a 4. Mlingo wa SDE ndi IM wogwiritsidwa ntchito mu phunziroli unachokera pa omwe anagwiritsidwa ntchito m'maphunziro apitalo [10,13,14].

2.4.3. Miyezo ya Hindpaw Weight-Bearing Distribution

Pambuyo pa kulowetsedwa kwa OA, mphamvu yolemetsa yolemetsa ya ma hindpaws inasokonekera. Woyesa wolephera (Linton instrumentation, Norfolk, UK) adagwiritsidwa ntchito poyesa kusintha kwa kulekerera kulemera. Makoswe ankawaika mosamala m’chipinda choyezeramo. Mphamvu yonyamula zolemetsa yoperekedwa ndi mwendo wakumbuyo udachitika pafupifupi 3 s. Chiŵerengero cha kagawidwe ka kulemera chinaŵerengedwa ndi chiŵerengero chotsatirachi: [kulemera pa mwendo wakumbuyo wakumanja/(kulemera ku nsonga yakumanja + kulemera kudzanja lakumanzere)] × 100 [15].

2.4.4. Kuyeza kwa Serum Cytokine Levels

Zitsanzo za magazi zinali centrifuged pa 1,500 g kwa 10 min pa 4 ° C; ndiye seramuyo idasonkhanitsidwa ndikusungidwa pa −70 ° C mpaka itagwiritsidwa ntchito. Miyezo ya IL-1β, IL-6, TNF-α, ndi PGE2 mu seramu adayesedwa pogwiritsa ntchito zida za ELISA kuchokera ku R & D Systems (Minneapolis, MN, USA) malinga ndi malangizo a wopanga.

2.4.5. Real-Time Quantitative RT-PCR Analysis

RNA yonse inachotsedwa ku minofu ya mawondo pogwiritsa ntchito TRI reagent® (Sigma-Aldrich, St. Louis, MO, USA), yolembedwanso mu cDNA ndi PCR-amplified pogwiritsa ntchito TM One Step RT PCR kit ndi SYBR green (Applied Biosystems). , Grand Island, NY, USA). PCR yochulukira nthawi yeniyeni idachitidwa pogwiritsa ntchito pulogalamu ya Applied Biosystems 7500 Real-Time PCR (Applied Biosystems, Grand Island, NY, USA). Kutsatizana koyambirira ndi kutsatizana kwa probe kukuwonetsedwa mu Table1. Ma Aliquots a zitsanzo za cDNAs ndi kuchuluka kofanana kwa GAPDH cDNA adakulitsidwa ndi TaqMan® Universal PCR master mix yomwe ili ndi DNA polymerase malinga ndi malangizo a wopanga (Applied Biosystems, Foster, CA, USA). Mikhalidwe ya PCR inali 2 min pa 50 ° C, 10 min pa 94 ° C, 15 s pa 95 ° C, ndi 1 min pa 60 ° C kwa 40 mizungu. Kuchulukira kwa jini yomwe chandamale idatsimikiziridwa pogwiritsa ntchito njira yofananizira ya Ct (nambala yozungulira podutsa pakati pa chiwembu chokulitsa ndi pachimake), molingana ndi malangizo a wopanga.

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Titumizireni imelo

Tsatanetsatane wa Zamalonda

Zolemba Zamalonda

Nyamakazi ya Osteoarthritis (OA) ndi matenda ofala kwambiri a minofu ndi mafupa omwe amapezeka mwa okalamba.1]. OA ndi vuto lomwe limadza chifukwa cha kuvulala, kuwonongeka kwa cartilage ndi magwiridwe antchito, komanso kusokoneza njira zoyambitsa kutupa ndi zotsutsana ndi kutupa [2,3]. Zimakhudza kwambiri cartilage ndi subchondral bone ya synovial joints ndipo zimabweretsa kulephera kwa mgwirizano, zomwe zimayambitsa kupweteka pa kulemera kuphatikizapo kuyenda ndi kuyimirira [4].

OA alibe mankhwala, chifukwa n'kovuta kwambiri kubwezeretsa chichereŵechereŵe chikawonongeka [5]. Zolinga za chithandizo ndi kuchepetsa ululu, kusunga kapena kupititsa patsogolo kuyenda kwamagulu, kuwonjezera mphamvu zamagulu, ndi kuchepetsa zotsatira zolepheretsa za matendawa. Thandizo la pharmacological la OA limayesetsa kuchepetsa ululu kuti awonjezere kugwira ntchito limodzi kwa wodwalayo komanso moyo wabwino. Ngakhale kuwonongeka kwa cartilage ndiye chochitika chachikulu mu OA, kuwonongeka kwa collagen ndiye chochitika chofunikira chomwe chimatsimikizira kupita patsogolo kosasinthika kwa OA mogwirizana ndi kutupa [6,7]. Kuchiza ndi anti-yotupa ndi chondroprotective ntchito akuyembekezeredwa kuthetsa ululu ndi kusunga umphumphu wa matrix mwa odwala OA.

Chifukwa chake, kuchepetsa kutupa kungakhale kopindulitsa pakuwongolera OA. Kafukufuku waposachedwa akuwonetsa kuti chitetezo chazitsamba chikupitilira kukula kwa OA, pochepetsa kutupa kwa chondrocyte komanso kuwononga chichereŵechereŵe, chifukwa cha kuthekera kwawo kuyanjana ndi minyewa yolumikizana ndi olowa, zomwe zimapangitsa kuchepetsa kupweteka kwa mafupa [8].

Muzu waSaposhnikovia divaricataSchischkin (Umbelliferae) wakhala akugwiritsidwa ntchito kwambiri mu mankhwala achikhalidwe pochiza mutu, ululu, kutupa, ndi nyamakazi ku Korea ndi China [9,10]. The zosiyanasiyana pharmacological zotsatira zaSaposhnikovia divaricata(SD) imaphatikizanso anti-yotupa, analgesic, antipyretic, ndi antiarthritic properties [9,11]. Kafukufuku waposachedwa wasonyeza kuti SD chromone extract ili ndi zotsatira za antirheumatoid nyamakazi mu mtundu wa mbewa wa nyamakazi yopangidwa ndi collagen [10]; komabe, kafukufuku wochepa wachitika kuti athandizire odana ndi yotupa ndi odana ndi nyamakazi ntchito yaSaposhnikovia divaricatakuchotsa (SDE).

Chifukwa chake, kafukufuku wapano adafufuza za anti-yotupa ndi antiosteoarthritis za 70% ya ethanol yochokera ku SD. Choyamba, anti-inflammatory effect ya SDE inayesedwamu vitrom'maselo a RAW 264.7 opangidwa ndi LPS. Kenaka, mphamvu ya antiosteoarthritis ya SDE inayesedwa poyesa kugawa kulemera, kuwonongeka kwa cartilage ya articular, ndi mayankho otupa mu rat model of monosodium iodoacetate- (MIA-) induced OA.