Name: preparation of herbal medicine: er-xian decoction and er-xian-containing serum for in vivo and in vitro experiments
Uploaded: 2017-05-31T15:00:00.000+00:00
Duration: 8 min 7 s
Description: Watch this Scientific Journal Video about Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments at JoVE.com

Bu çalışma Çin Ulusal Doğa Bilimleri Vakfı (81573992) tarafından desteklenmiştir. Dil düzenleme yardımı için Emily K. Lo ve Kathleen DiNapoli&#39;ye teşekkür ediyoruz.

Protokol, Şanghay Geleneksel Çin Tıbbı&#39;nın hayvan bakım rehberlerini takip eder ve hayvan deneyleri Şanghay Hayvan Etik Komitesi tarafından onaylanır. 1. Protokol I: EXD&#39;nin hazırlanması <ol><li> Hesaplama <ol><li> EXD&#39;yi 10 altı aylık bayan Baskı Kontrol Bölgesi (ICR) farelerine (0.02 kg / fare), haftada 5 gün 12 hafta süreyle uygulayan bir tedavi grubuna uygulayın. NOT: Toplam 1.702 EXD&#39;ye ihtiyaç vardır. EXD&#39;ler ayrı artışlarla ölçülür, çünkü gerçek uygulamada 2 EXD uygulayın. </li><li> Fare başına uygulanan EXD hacmini hesaplayın ( yani, V = (0.1418 EXD / kg • gün) * (0.02 kg / fare) * 50 mL / EXD = 0.14 mL / fare • gün. </li></ol></li><li> Islatma <ol><li> İki EXD&#39;nin ( örneğin Curculigo orchioides Gaertn (18 g), Herbaa Epimedi (18 g), Radix Morindae Officinalis&#39;in (18 g), RAdix Angelicae Sinensis (18 g), Cortex Phellodendri (12 g) ve Rhizoma Anemarrhenae (12 g), toplam 96 g) bir kapaklı bir kaba koydu. NOT: Seramik bir kap tavsiye edilir. </li><li> Çiğ otları 500 mL damıtılmış su ile 1 saat süreyle macerante edin; Su otları bir inç kadar kaplamalıdır. Tüm otları iyice ıslatın. </li></ol></li><li> İlk suda kaynatma <ol><li> Otlar, su kaynarcaya kadar yüksek sıcaklıkta bir gaz ocağı veya bir indüksiyon ocağı kullanarak ısıtın (yaklaşık 5 - 10 dakika, süre, ısıtma gücüne, kaba ve otların miktarı ve suya bağlıdır). Gücü 2 saat daha düşük bir kaynatma değere çevirin. </li></ol></li><li> İlk filtrasyon <ol><li> Normal filtre kağıdıyla veya gazlı bezle ve pamuklu, 500 mL&#39;lik cam bir behere koyun. Dikkatli bir şekilde, kaynatmayı filtre vasıtasıyla behergöre dökün. Şifalı bitkiler kabın içinde bırakın. </li><li> Geri kalan bitki kalıntılarını geri getirin oFiltre kağıdını kaba koyun. </li></ol></li><li> İkinci suda kaynatma <ol><li> Otlarla birlikte kaba damıtılmış su ekleyin; Otları bir inç kadar kapağı olsun. </li><li> Adım 1.3&#39;ü tekrarlayın. </li></ol></li><li> İkinci filtrasyon <ol><li> 1.4.1 ve 1.4.2 adımlarını tekrarlayın. </li><li> İkinci kaynatmayı aynı behere dökün. Birinci ve ikinci dekatikleri birlikte karıştırın. </li></ol></li><li> konsantrasyon <ol><li> Beferleri asbest içermeyen tel gazlı bezli bir gaz ocağının üzerine koyun, kaynatıp düşük bir kaynatmayla ısıtın ve yavaşça ve bir cam çubukla karıştırın. </li><li> Yoğuşma 200 mL&#39;ye düştüğünde, 500 mL&#39;lik bir behere aktarın. </li><li> Yıkama 100 mL&#39;ye düşene kadar adım 1.7.1&#39;i tekrarlayın. </li></ol></li><li> Depolama ve yönetim <ol><li> Konsantre kaynatmayı steril bir cam şişeye aktarın. Oda sıcaklığına kadar soğutunYeniden (RT). Bir hafta içinde kullanıyorsanız 4 ° C&#39;de veya uzun süre depolamada -70 ° C&#39;de saklayın. </li><li> Bir tenteli iğne kullanarak, ovarektomize (OVX) farelere günde bir kez, 12 hafta boyunca haftanın 5 günü, 0.14 mL / fare eksiltme uygulayın. </li></ol></li></ol> 2. Protokol II: EXD-içeren Serumun Hazırlanması <ol><li> Hesaplama <ol><li> Her sıçanın vücut ağırlığı (1 aylık) olarak 0.15 kg kullanarak sıçan eşdeğer dozunu (RED) hesaplayın: dB = (1/60) * (90/100) * (60 / 0.15) 1/3 = 0.111 EXD / kg / gün. NOT: 100 mL kültür ortamı (% 10 EXD içeren serum) hazırlamak için toplam 10 mL EXD içeren serum gereklidir. Her sıçandan 2 mL serum sağlaması bekleniyor. Böylece, 6 sıçan (% 20&#39;lik bir kayıp dikkate alınır), 3 gün boyunca günde bir kez EXD uygulaması için gereklidir. EXD sayısı: (0.111 EXD / kg • gün) * (0.15 kg / sıçan) * (6 sıçan) * (3 gün) = 0.300 EXD. Yine, EXD&#39;ler ayrı artışlarla ölçülür,1 EXD kullanın. </li><li> Sıçan başına uygulanan EXD hacmini hesaplayın ( yani, V = (0.111 EXD / kg • gün) * (0.15 kg / sıçan) * (50 mL / EXD) = 0.83 mL / sıçan • gün. </li></ol></li><li> Islatma <ol><li> 1 EXD&#39;ye ait hammaddeleri kapaklı bir kaba koyun. Çiğ otları damıtılmış suda 1 saat maceratlayın; Su otları bir inç kadar kaplamalıdır. Tüm otları iyice ıslatın. </li></ol></li><li> İlk suda kaynatma <ol><li> Otlar, yüksek kaynaklarda kaynar su gelene kadar indüksiyon ocağı kullanarak ısıtın (yaklaşık 5 - 10 dakika; süre, ısı gücüne, konteynere ve otlar ile suyun miktarına bağlıdır). Gücü 2 saat daha düşük bir kaynatma noktasına getirin. </li></ol></li><li> İlk filtrasyon <ol><li> Normal filtre kağıdıyla veya gazlı bezle ve pamuklu, 500 mL&#39;lik cam bir behere koyun. Dikkatli bir şekilde, kaynatmayı filtre vasıtasıyla behergöre dökün. Şifalı bitkiler kabın içinde bırakın. </li><li&gt; Filtre kağıdındaki bitki kalıntılarını kaba geri koyun. </li></ol></li><li> İkinci suda kaynatma <ol><li> Otlarla birlikte kaba damıtılmış su ekleyin; Otları bir inç kadar kapağı olsun. </li><li> Adım 2.4&#39;ü tekrarlayın. </li></ol></li><li> İkinci filtrasyon <ol><li> 2.5.1 ve 2.5.2 adımlarını tekrarlayın. </li><li> İkinci sıkma işlemini, adım 2.4.1&#39;deki gibi aynı behça boşaltın ve birinci ve ikinci dekatikleri birlikte karıştırın. </li></ol></li><li> konsantrasyon <ol><li> Asfalt içermeyen tel gazlı bezli bir gaz ocağı üzerine beheri koyun, kaynatmayı düşük bir kaynatmayla ısıtın ve yavaşça ve bir cam çubukla karıştırın. </li><li> Yoğuşma 100 mL&#39;ye düştüğünde, 100 mL&#39;lik bir behere aktarın. </li><li> Yıkama 50 mL&#39;ye düşene kadar adım 2.7.1&#39;i tekrarlayın. </li></ol></li><li> Depolama ve yönetim <ol><li> Konsantre kaynatmayı sterilizeIle cam şişe. Oda sıcaklığına soğumasını bekleyin. Bir hafta içinde kullanıyorsanız 4 ° C&#39;de veya uzun süre depolamada -70 ° C&#39;de saklayın. </li><li> Intragastional olarak 0.83 mL / sıçan günde bir kez 3 gün boyunca uygulayın. </li></ol></li><li> EXD içeren serum preparatı <ol><li> Farelerin son uygulanmasından 1 saat sonra intraperitonal olarak 300 mL / 100 g 80 mg / kg ketamin ve 10 mg / kg xylazin enjekte ederek anestezi uygulayın. Çimdikleme ile doğru anestezi doğrulayın. </li><li> Sıçanın cildi ve peritonunu, karın bölgesinden göğüs dibine bir bistül (veya düz ameliyat makası) kullanarak incise edin. Sırasıyla yaklaşık 5 cm ve 0.5 cm&#39;lik bir uzunluk ve derinlik kesiği oluşturun. Doku kağıdı kullanarak karın iç organını sola doğru hareket ettirin. </li><li> Doku kağıdı kullanarak, damarı açıkça ortaya çıkarmak için karın aortunun bağ dokusunu çıkarın. </li><li> Karbon aortundan, 10 mL&#39;lik, 22 ölçüslü bir şırınga kullanarak kanı yavaşça çekin. Şuraya aktar:İğneyi çıkardıktan sonra 15 mL&#39;lik steril bir tüp; Bir fare 8-10 mL kan üretebilir. NOT: Kan çizimine başlanırken ve çizim tamamlandıktan sonra ölü olan sıçan canlı olmalıdır ( yani, abdominal aort titreşirken). </li><li> Oda RT&#39;da 30 - 60 dakika boyunca dik bir konumda kan pıhtılaştırın. 500-600 xg&#39;de 20 dakika boyunca santrifüjleyin. Tüm süpernatanı (serum) dikkatli bir şekilde bir 50 mL steril tüp içine yerleştirin ve tüm serumu (farklı hayvanlardan) karıştırın. </li><li> Serumu 56 ° C&#39;lik bir su banyosunda 30 dakika inkübe ederek ısı inaktivasyonu yapın. Serumu, 0.22 um gözenek boyutlu bir hidrofil polietersülfon membranlı bir şırınga filtresi kullanarak filtreleyin. Serumu taze veya -20 ° C&#39;de saklayın. </li></ol></li><li> Kontrollü ilaç içeren serum preparatı (kontrol grubunda kullanılır) <ol><li> Her bir fare günde bir kez aynı miktarda tuzlu su (0.83 mL) ile 3 gün süre ile idare edilir. Diğer adımlar adım 2.9&#39;daki ile aynıdır.</li></ol></li><li> Uygulama <ol><li> Her 100 ml&#39;lik orta ve artı% 1 penisilin-streptomisin (PS) 23&#39;e 10 mL EXD içeren serum veya ilaç içeren serum ekleyin. </li></ol></li></ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+major+chemical+constituents+and+their+metabolites+in+rat+plasma+and+various+organs+after+oral+administration+of+effective+Erxian+Decoction+(EXD)+fraction+by+liquid+chromatography-mass+spectrometry.">Identification of the major chemical constituents and their metabolites in rat plasma and various organs after oral administration of effective Erxian Decoction (EXD) fraction by liquid chromatography-mass spectrometry.</a> Biomed Chromatogr. 24 (5), 479-489 (2010).</li><li>Xue, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+and+interaction+of+icariin,+curculigoside,+and+berberine+in+er-xian+decoction,+a+traditional+chinese+medicinal+formula,+on+osteoclastic+bone+resorption.">Effects and interaction of icariin, curculigoside, and berberine in er-xian decoction, a traditional chinese medicinal formula, on osteoclastic bone resorption.</a> Evid Based Complement Alternat Med. , (2012).</li><li>Nair, A. B., Jacob, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+practice+guide+for+dose+conversion+between+animals+and+human.">A simple practice guide for dose conversion between animals and human.</a> J Basic Clin Pharm. 7 (2), 27-31 (2016).</li><li>Xu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effect+of+the+traditional+Chinese+medicine+xuesaitong+on+intestinal+ischemia-reperfusion+injury+in+rats.">Protective effect of the traditional Chinese medicine xuesaitong on intestinal ischemia-reperfusion injury in rats.</a> Int J Clin Exp Med. 8 (2), 1768-1779 (2015).</li><li>Li, Z., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+methods+for+Chinese+herbs+serum+pharmcology.">On the methods for Chinese herbs serum pharmcology.</a> Zhong Guo Zhong Yi Yao Xing Xi Za Zhi. 9 (2), 5-6 (2002).</li><li>Jiang, Y. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+chinese+herbal+drug-containing+serum+for+activating-blood+and+dispelling-toxin+on+ox-LDL-induced+inflammatory+factors'+expression+in+endothelial+cells.">Effect of chinese herbal drug-containing serum for activating-blood and dispelling-toxin on ox-LDL-induced inflammatory factors' expression in endothelial cells.</a> Chin J Integr Med. 18 (1), 30-33 (2012).</li><li>Guo, C. Y., Ma, X. J., Liu, Q., Yin, H. J., Shi, D. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Effect+of+Chinese+herbal+drug-containing+serum+for+activating+blood,+activating+blood+and+dispelling+toxin+on+TNF-alpha-induced+adherence+between+endothelial+cells+and+neutrophils+and+the+expression+of+MAPK+pathway].">[Effect of Chinese herbal drug-containing serum for activating blood, activating blood and dispelling toxin on TNF-alpha-induced adherence between endothelial cells and neutrophils and the expression of MAPK pathway].</a> Zhongguo Zhong Xi Yi Jie He Za Zhi. 35 (2), 204-209 (2015).</li><li>Li, Y., Xia, J. Y., Chen, W., Deng, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Ling+Qi+Juan+Gan+capsule+drug-containing+serum+on+PDGF-induced+proliferation+and+JAK/STAT+signaling+of+HSC-T6+cells].">Effects of Ling Qi Juan Gan capsule drug-containing serum on PDGF-induced proliferation and JAK/STAT signaling of HSC-T6 cells].</a> Zhonghua Gan Zang Bing Za Zhi. 21 (9), 663-667 (2013).</li><li>Li, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Some+issues+in+methology+of+Chinese+herbs+serum+pharmcology].">[Some issues in methology of Chinese herbs serum pharmcology].</a> Zhong Yao Xin Yao Yu Lin Chuang Yao Li. 10 (5), 263 (1999).</li><li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[A+review+of+Chinese+herbs+serum+pharmcology+methodological+study].">[A review of Chinese herbs serum pharmcology methodological study].</a> Nan Jing Zhong Yi Yao Da Xue Xue Bao. 18 (4), 254 (2002).</li><li>Zhang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Issues+and+strategies+for+study+of+serum+pharmcology+in+oncology].">[Issues and strategies for study of serum pharmcology in oncology].</a> Zhong Yi Yan Jiu. 17 (5), 13-14 (2004).</li></ol>

Yazarlar, rekabet eden mali çıkarlarının olmadığını beyan ettiler.

Son yıllarda binlerce yıldır Doğu dünyasında klinik ortamda uygulanan alternatif tıp türlerinden biri olan bitkisel ilaçlara daha fazla önem verilmektedir. Modern tıbbın &quot;tezgahdan tezgah&quot; deseninden farklı olarak, geleneksel bitkisel tıbbın ilk önce kendi mekanizmalarını açıklamak için &quot;tezgah yatağı&quot; örneğini gerektirir. Bunu, tezgah aşamasında yapılan doğrulama ve yeni optimize edilmiş ilaçların geliştirilmesi için işleme takip edilebilir. Şu ana kadar, bütün ...

Çalışmaya ve geleneksel bitkisel tıbbın uygulanmasına olan ilgi günümüzde artmaktadır. Kimyasal maddelerin kesin olduğu modern ilaçların aksine bitkisel formüllerin bilinmeyen bazı bileşenleri vardır ve aktif bileşiklerinin verilmesini sağlamak için ekstraksiyon işlemleri gerektirir. Çoğu araştırma, bütün ot veya bitkisel formülün temsilcisi olarak iyi bilinen bir yapıya sahip küçük bir bileşik seçmeye çalışsa da, ne farmakolojik etkinlikler ne de mekanizmalar eşdeğer 1 , 2 olarak kabul edilemez. Parmak izi kompleks bitkisel formüllerin bileşenlerini analiz etmeyi sağlarken, bazı bileşenler hala net bir şekilde analiz edilmediğinden, çalışma 3 için tüm özütleri birleştirirken bir meydan okumaya neden olur. Çok sayıda bileşenin / ekstraktların etkileşimleri, bitkisel ilaçların terapötik etkilerine aracılık eder. Bu avantajı korumak için, geleneksel ekstrakt form-dekoctİyon hala klinikte yaygın olarak kullanılmaktadır. Ekstrakt prosedürü, terapötik etkinlik üzerinde büyük bir etkiye sahip olduğu için, özellikle in vivo çalışmalar için 4 , 5 için geleneksel su sıkılmasının standart bir protokolü gereklidir. Öte yandan, farmakolojik mekanizmaları araştırırken, in vitro veya ex vivo çalışmalar sırasında bitkisel açma maddelerinin uygulanması da bir mücadeledir. İlaç içeren serumun konsepti ilk kez Tashino tarafından 1988&#39;de 6 önerildi. O zamandan beri, artan sayıda araştırmacı onu bitkisel ilaç 7 , 8 , 9&#39;a uyguladı. İlaç içeren serumun metodu, serumun belli bileşenlerinin etkisi gibi bazı kısıtlamalara sahip olmakla birlikte, fizyolojik koşulları yakından taklit eden bir yöntem olarak kabul edilmektedir. 14 , menopoz osteoporozu 15 , 16 , 17 , prematür over yetmezliği 18 , meme kanseri 19 , yumurtalık kanseri 20 ve ertelenmiş ergenlik 21&#39;in tedavisinde de uygulanmıştır. Burada, er-xian ekstraksiyonunun (EXD) ve ilacı içeren serumun hazırlanması için ayrıntılı protokoller sunuyoruz. Buna ek olarak, fare menopoz osteoporotik modellerine EXD ve EXD içeren serumun uygulanmasını açıklıyoruz. Bir EXD, 9 gr Curculigo orchioides Gaertn , Herbaa Epimedii&#39;den oluşur </eM&gt;, Radix Morindae Officinalis ve Radix Angelicae Sinensis ve her biri yetişkin hasta başına günde 6 g Cortex Phellodendri ve Rhizoma Anemarrhenae . Bir fare için eşdeğer doz, aşağıdaki denklem 22&#39;ye dayanılarak 0.1418 EXD / kg / gün&#39;dür: dB = dA * RB / RA * (WA / WB) 1/3 . DA ve dB, sırasıyla insan ve fare vücut ağırlığı başına doz (mg / kg) anlamına gelir. Dozun mg / kg olarak, EXD / kg sayısı ile değiştirilir. RA ve RB sırasıyla vücut yüzey alanı (m 2 ) / vücut ağırlığı (kg) ile orantılı insan vücut faktörünü ve fare gövde faktörünü temsil eder) 2/3 (Bkz. Tablo 1 ). WA ve WB sırasıyla insan ve fare vücut ağırlığını (kg) gösterir.

<table><tbody><tr><td>Curculigo orchioides Gaertn (9&amp;thinsp;g), Herbaa Epimedii (9&amp;thinsp;g), Radix Morindae Officinalis (9&amp;thinsp;g), Radix Angelicae Sinensis (9&amp;thinsp;g), Cortex Phellodendri (6&amp;thinsp;g), and Rhizoma Anemarrhenae (6&amp;thinsp;g)</td><td>Kang-qiao Chinese Medicine Yinpian Co. Ltd (Shanghai, CN)</td><td>160922</td><td>EXD components</td></tr><tr><td>Filter paper</td><td>GElifesciences</td><td>99-103-952</td><td>Filter EXD decoction before concentration</td></tr><tr><td>Imprinting Control Region (ICR) mice</td><td>Shanghai Laboratory Animal Center</td><td>SCXK 2007-0005</td><td>In vivo study</td></tr><tr><td>Sprague Dawley rats</td><td>Shanghai Laboratory Animal Center</td><td>SCXK 2007-0005</td><td>EXD-containing serum preparation</td></tr><tr><td>Syringe filter</td><td>Millipore</td><td>SLGP033RB</td><td>0.22 &amp;micro;m</td></tr><tr><td>gavage needles (10 ml)</td><td>Shanghai BO Ge trade sales department</td><td>59104274</td><td>Adminstration of EXD</td></tr><tr><td>Ketamine (80 mg/kg)&amp;nbsp;</td><td>Fujian Gutian Pharma Co. Ltd</td><td>H35020148</td><td>Anesthesia</td></tr><tr><td>Xylazine (10 mg/kg)</td><td>Sunway Pharma Co. Ltd</td><td>CB07591</td><td>Anesthesia</td></tr><tr><td>Dulbecco&amp;rsquo;s modification of Eagle&amp;rsquo;s medium Dulbecco (DMEM) culture medium</td><td>Gibco</td><td>12800-116</td><td>DMEM with 2 mM L-glutamine and without ribonucleosides and ribonucleotides</td></tr><tr><td>Streptomycin</td><td>Sigma</td><td>1277</td><td>100 &amp;micro;g / ml</td></tr><tr><td>Penicillin</td><td>Sigma</td><td>4687</td><td>100 &amp;micro;g / ml</td></tr></tbody></table>

preparation of herbal medicine: er-xian decoction and er-xian-containing serum for in vivo and in vitro experiments

Klinik ortamda alternatif bir ilaç olan geleneksel bitki tıbbı, son yıllarda artan bir ilgi görmüştür. Vücutta teslim edilmeden önce, aktif çiğneme maddelerini çiğ otlardan ayırmak için ek bir ekstraksiyon prosedürü yaygın olarak gereklidir. Su dekompozisyonu, klinik ortamda halen yaygın olarak kullanılan klasik bir ekstraksiyon prosedürüdür. Burada, deneysel araştırmalara bitki özütleri uygulamak için er-xian kaynatma işlemi (EXD) için ayrıntılı bir protokol önermekteyiz. Hayvana uygun doz hesaplamanın yanı sıra EXD&#39;nin dört ana aşamasını açıklar: ıslatma, su dekompozisyonu, filtrasyon ve konsantrasyon. Buna ek olarak, serum içeren EXD, in vitro doğrulama aracı olarak farelere verilir. Burada sıçanlar oral yoldan üç gün süreyle EXD ile tedavi edildi. Daha sonra kan numuneleri toplandı, inaktive edildi, santrifüj edildi ve filtrelendi. Kültür ortamı ile seyreltilmiş serum, v&#39;de hücreleri veya dokuları tedavi etmek için kullanılabilirItro. Örneğin, EXD, hem in vivo hem de in vitro çalışmalarda uygulanmış ve EXD&#39;nin osteojenezi arttırdığını göstermiştir. Bu protokol bitkisel ilaçların hazırlanması ve uygulanması için referans olarak kullanılabilir.

EXD&#39;nin OVX farelerinin kemik yoğunluğuna etkisi Omurga vertebrası bölümündeki hematoksilin ve eozin boyama, OVX grubundaki kişilerle ( Şekil 1A , sol panel) karşılaştırıldığında in vivo EXD tedavisinden sonra artmış kemik trabekülleri gösterir ( Şekil 1A , sağ panel). Şekil 1B , 12 haftalık EXD tedavisine sahip OVX farelerindeki ( Şe...

Watch this Scientific Journal Video about Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments at JoVE.com

Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

Burada, erüe, ekstraksiyon, filtrasyon ve konsantrasyon olmak üzere dört basamakta bir er-xian ekstraksiyonunun (EXD) hazırlanması ve hazırlanmış bir EXD içeren serumun sıçanlara uygulanmasını gösteriyoruz. Bu yöntemler, geleneksel Çin ilaçları gibi bitkisel dekompozisyonların in vivo ve in vitro çalışması için geçerlidir.

Bitkisel Tıbbın Hazırlanması: Er-Xian Kaynatma ve Er-Xian içeren Serum<emIn vivo</emVe<em&gt; In vitro</em&gt; Deneyler

Traditional herbal medicine, an alternative medicine in the clinical setting, has received increased attention in recent years. Before delivery to the ...

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Medicine

Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

10.9K Görüntüleme.  Shanghai University of Traditional Chinese Medicine.  Burada, erüe, ekstraksiyon, filtrasyon ve konsantrasyon olmak üzere dört basamakta bir er-xian ekstraksiyonunun (EXD) hazırlanması ve hazırlanmış bir EXD içeren serumun sıçanlara uygulanmasını gösteriyoruz. Bu yöntemler, geleneksel Çin ilaçları gibi bitkisel dekompozisyonların in vivo ve in vitro çalışması için geçerlidir. 

Video: Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro

Epidemiological studies indicate that increased flavonoid intake correlates with decreased mortality due to cardiovascular diseases (CVD) in the United States (US) and Europe. Berries are widely consumed in the US and have a high polyphenolic content. Polyphenols have been shown to interact with many molecular targets and to exert numerous positive biological functions, including antioxidant, anti-inflammatory, and cardioprotective effects. Polyphenols isolated from blackberry (BL), raspberry (RB), and black raspberry (BRB) reduce oxidative stress and cellular senescence in response to angiotensin II (Ang II). This work provides a detailed description of the protocol used to prepare the polyphenol extracts from freeze-dried berries. Polyphenol extractions from freeze-dried berry powder were performed using 80% aqueous ethanol and an ultrasonic-assisted extraction method. The crude extract was further purified and fractionated using chloroform and ethyl acetate, respectively. The effects of both crude and purified extracts were tested on Vascular Smooth Muscle Cells (VSMCs) in culture.

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro

This work details a step-by-step method to prepare polyphenol-rich extracts from freeze-dried berry powder. In addition, it provides a thorough description of how to use these polyphenol-rich extracts in cell culture in the presence of the peptide hormone angiotensin II (Ang II) using Vascular Smooth Muscle Cells (VSMCs).

Dondurularak Kurutulmuş Berry Tozundan Polifenollerin Vasküler Düz Kas Hücrelerinin Tedavisinde Ekstraksiyonu ve Saflaştırılması<em&gt; In vitro</em

Epidemiological studies indicate that increased flavonoid intake correlates with decreased mortality due to cardiovascular diseases (CVD) in the United ...

Kimya

Ultra High-Performance Liquid Chromatography-Mass Spectrometry and Self-established Database Analysis of Chinese Herbal Medicine Components

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) is the most widely used method in qualitative analysis of compounds. The method includes standardized and programmed protocols for sample pretreatment, MS tune, MS acquisition, and data processing. The sample pretreatments include collection, pulverization, solvent extraction, ultrasound, centrifugation, and filtration. Data post-processing was described in detail and includes data importing, self-established database construction, method establishment, data processing, and other manual operations. The above-ground part of the alpine yarrow herb, Achillea millefolium L., is used to treat inflammation, gastrointestinal disturbances, and pain and its 3-oxa-guaianolides could be useful leads for anti-inflammatory drug development. Three representative compounds in AML were identified, combining TOF-MS with a self-established database. Moreover, the differences from existing literature, liquid-phase parameter optimization, scan mode selection, ion source suitability, collision energy adjustment, isomer screening, method limitation, and possible solutions were discussed. This standardized analysis method is universal and can be applied to identify complex compounds in Chinese herbal medicine.

We describe a general protocol and systematic design that could be applied to separate and recognize complex components in alpine yarrow herb, Achillea millefolium L., a Chinese herbal medicine.

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

In vivo ve In vitro deneyler için Gushukang (GSK) granül hazırlanması

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...

Tıp

Preparation of Naringenin Solution for In Vivo Application

The preparation of a compound (phytochemical) solution is an overlooked but critical step prior to its application in studies such as drug screening. The complete solubilization of the compound is necessary for its safe use and relatively stable results. Here, a protocol for preparing naringenin solution and its intraperitoneal administration in a high-fat diet and streptozotocin (STZ)-induced diabetic model is demonstrated as an example. A small amount of naringenin (3.52-6.69 mg) was used to test its solubilization in solvents, including ethanol, dimethylsulfoxide (DMSO), and DMSO plus Tween 80 reconstituted in physiological saline (PS), respectively. Complete solubilization of the compound is determined by observing the color of the solution, the presence of precipitates after centrifugation (2000 x g for 30 s), or allowing the solution to stand for 2 h at room temperature (RT). After obtaining a stable compound/phytochemical solution, the final concentration/amount of the compound required for in vivo studies can be prepared in a solvent-only (no PS) stock solution, and then diluted/mixed with PS as desired. The antidiabetic osteoporotic effects of naringenin in mice (intraperitoneal administration at 20 mg/kg b.w., 2 mg/mL) were assessed by measuring blood glucose, bone mass (micro-CT), and bone resorption rate (TRAP staining and ELISA). Researchers looking for detailed organic/phytochemical solution preparations will benefit from this technique.

Preparation of Naringenin Solution for In Vivo Application

Here, the protocol presents the preparation of naringenin solution for in vivo intraperitoneal administration. Naringenin is fully dissolved in a mixture of dimethylsulfoxide, Tween 80, and saline. The antidiabetic osteoporotic effects of naringenin were assessed by blood glucose testing, tartrate-resistant acid phosphatase staining, and enzyme-linked immunosorbent assay.

In Vivo Uygulama için Naringenin Solüsyonunun Hazırlanması

The preparation of a compound (phytochemical) solution is an overlooked but critical step prior to its application in studies such as drug screening. The ...

An In Vitro Dissolution Determination of Multi-Index Components in Tibetan Medicine Rhodiola Granules

The composition of the Tibetan medicine Rhodiola granules (RG) is complex, and the overall quality of RG is difficult to determine. Therefore, establishing a method to determine the multi-component in vitro dissolution of RG is of great significance for quality control. This study uses the second paddle method of the fourth general rule 0931 from the Chinese Pharmacopoeia (2020 edition), compliant with apparatus 2 of the United States Pharmacopeia (USP). The dissolution apparatus was set to a rotation speed of 100 rpm with ultrapure water as the dissolution medium. A sample volume of 1 mL was collected at each timepoint. Furthermore, the cumulative dissolution of gallic acid, salidroside, and ethyl gallic acid in RG at different time points was determined by high-performance liquid chromatography (HPLC). Finally, the dissolution curves were drawn, and the curves were fitted to the GompertzMod, the Gompertz, the Logistic, and the Weibull equations. The results showed that the cumulative dissolution of gallic acid in RG was over 80% at 1 min, the cumulative dissolution of salidroside and ethyl gallic acid was over 65% at 5 min, and the cumulative dissolution of each index component decreased after 30 min. The curve fitting demonstrated that the GompertzMod equation was the best-fitting model for each index component of RG. In conclusion, the dissolution test method described in this protocol is simple, accurate, and reliable. It can characterize the dissolution behavior of the index components in RG in vitro, which provides a methodological reference for quality control of RG and quality evaluation of other ethnic compounds.

An In Vitro Dissolution Determination of Multi-Index Components in Tibetan Medicine Rhodiola Granules

Here, we test the dissolution of Rhodiola granules (RG) in vitro, draw dissolution curves of salidroside, gallic acid, and ethyl gallate in ultrapure water, and fit the curves to different mathematical models. This protocol provides information and guidance for in vivo bioequivalence and in vivo-in vitro correlation studies of RG.

Tibet Tıbbında Rhodiola Granüllerinde Çok İndeksli Bileşenlerin In Vitro Çözünme Tayini

The composition of the Tibetan medicine Rhodiola granules (RG) is complex, and the overall quality of RG is difficult to determine. Therefore, ...

Exploring the Two Herb Combination Strategy to Treat Injured PC12 Cells

In view of the advantages of the combination of traditional Chinese medicine (TCM) in the treatment of cerebral ischemia, we studied the differences in the efficacy and mechanism between the preparation combination and the component combination in order to explore the two herb combination strategy to treat injured PC12 cells. Cobalt chloride (CoCl2) combined with a glucose-free medium was employed to induce oxidative damage of PC12 cells. Then, the optimal combination of Astragalus mongholicus (Ast) and Erigeron breviscapus (Eri) injection was selected and combined following uniform design methods after screening their safe and effective concentration on PC12 cells. Further, the component combination screened comprises 10 &#181;M astragaloside A, 40 &#181;M scutellarin, and 75 &#181;M chlorogenic acid in two herbs. Then, MTT, Annexin V-FITC/PI, immunofluorescence, and Western blot analysis were used to evaluate the efficacy and mechanism of the preparation combination and the component combination on injured PC12 cells. The results showed that the optimal preparation combination for cell pro-survival was Ast injection and Eri capsule with a concentration of 6:1.8 (&#181;M). The component combination (10 &#181;M astragaloside A, 40 &#181;M scutellarin, and 75 &#181;M chlorogenic acid) was more effective than the preparation combination. Both combinations remarkably reduced apoptotic rate, the fluorescence intensity of caspase-3, and intracellular reactive oxygen species (ROS) level; meanwhile, they upregulated the expression levels of p-Akt/Akt, Bcl-2/Bax, and Nrf2. These effects were more evident in the component combination. In conclusion, both combinations can inhibit the injury induced by CoCl2 combined with a glucose-free medium on PC12 cells, thus promoting cell survival. However, the efficiency of the component combination over the preparation combination may be due to its stronger regulation of the PI3K/Akt/Nrf2 signaling pathway related to oxidative stress and apoptosis.

This protocol provides a combination strategy of two herbs to treat injured PC12 cells. The protocol provides a reference for optimizing the best application mode of traditional Chinese medicine (TCM).

Yaralı PC12 Hücrelerini Tedavi Etmek için İki Bitki Kombinasyonu Stratejisini Keşfetmek

In view of the advantages of the combination of traditional Chinese medicine (TCM) in the treatment of cerebral ischemia, we studied the differences in ...

Optimization of the Epimedii Folium Mutton-Oil Processing Technology and Testing Its Effect on Zebrafish Embryonic Development

As a traditional Chinese medicine (TCM), Epimedii folium (EF) has a history in medicine and food that is &#62; 2,000 years old. Clinically, EF processed with mutton oil is often used as a medicine. In recent years, reports of safety risks and adverse reactions of products that use EF as a raw material have gradually increased. Processing can effectively improve the safety of TCM. According to TCM theory, mutton-oil processing can reduce the toxicity of EF and enhance its tonifying effect on the kidneys. However, there is a lack of systematic research and evaluation of EF mutton-oil processing technology. In this study, we used the Box-Behnken experimental design-response surface methodology to optimize the key parameters of the processing technology by assessing the contents of multiple components. The results showed that the optimal mutton-oil processing technology of EF was as follows: heating the mutton oil at 120 &#176;C &#177; 10 &#176;C, adding the crude EF, stir-frying it gently to 189 &#176;C &#177; 10 &#176;C until it is evenly shiny, and then removing it and cool. For every 100 kg of EF, 15 kg of mutton oil should be used. The toxicities and teratogenicities of an aqueous extract of crude and mutton-oil processed EF were compared in a zebrafish embryo developmental model. The results showed that the crude herb group was more likely to cause zebrafish deformities, and its half-maximal lethal EF concentration was lower. In conclusion, the optimized mutton-oil processing technology was stable and reliable, with good repeatability. At a certain dose, the aqueous extract of EF was toxic to the development of zebrafish embryos, and the toxicity was stronger for the crude drug than for the processed drug. The results showed that mutton-oil processing reduced the toxicity of crude EF. These findings can be used to improve the quality, uniformity, and clinical safety of mutton oil-processed EF.

In this protocol, the mutton-oil processing technology of Epimedii folium (EF) was optimized by applying a Box-Behnken experimental design-response surface methodology, and the effect of crude and optimized water-extracted EF on zebrafish embryonic development was preliminarily investigated.

Epimedii Folyum koyun eti yağı işleme teknolojisinin optimizasyonu ve zebra balığı embriyonik gelişimi üzerindeki etkisinin test edilmesi

As a traditional Chinese medicine (TCM), Epimedii folium (EF) has a history in medicine and food that is &#62; 2,000 years old. Clinically, EF processed ...

Impact of Sanleng Jiashen Formula on Orthotopic Hepatocellular Carcinoma in Mice

Establishment of Hepatocarcinoma in BALB/c-nu Mice and Investigation of the Therapeutic Effect of the Sanleng Jiashen Formula

The lethality of liver cancer and the resistance to chemical drugs have forced the search for effective prescriptions of traditional herbs for liver cancer. Animal models that are repeatable, easy to manipulate, and highly mimic the pathophysiological processes of liver cancer are the prerequisite for the successful screening of effective drug candidates. Meanwhile, reliable drug efficacy evaluation indicators and means are also the guarantee of anti-liver cancer drug research and development.
Sanleng Jiashen formula, a representative prescription of traditional Chinese medicine containing Sparganium stoloni erum, Buch. -Ham. (Sanleng), Panax ginseng C. A. Mey. (ginseng), Rheum officinale Baill. (rhubarb), and Ligusticum chuanxiong Hort. (Chuanxiong), is prescribed to nourish the liver and clear heat, remove toxins, and promote blood circulation to treat liver cancer. This experimental protocol describes the preparation of lyophilized Sanleng Jiashen formula and the establishment process of in-situ liver cancer in BALB/c-nu mice. Histopathological staining, immunohistochemical detection of cancer markers, in vivo imaging of mice, and chick embryo chorioallantoic membrane test were used to explore the inhibition and anti-angiogenesis effect of Sanleng Jiashen formula on malignant proliferation of liver cancer tissue. The data show that the Sanleng Jiashen formula can effectively resist the malignant proliferation of liver cancer tissue, which is manifested by reduced tumor mass volume, improved pathological damage, and lower levels of the cancer marker ki67.
The superior inhibition of angiogenesis also suggests that the Sanleng Jiashen formula may have the potential to treat and prevent the progression and deterioration of liver cancer. The whole experimental scheme shows a comprehensive process of traditional Chinese medicine components in the treatment of mouse liver cancer, which provides a reference for the establishment and optimization of a liver cancer model, as well as the research and development of drugs to prevent and treat liver cancer.

Here, we present the step-by-step protocol to investigate the therapeutic and vascular inhibitory effects of the Sanleng Jiashen formula on BALB/c-nu mice with orthotopic hepatocellular carcinoma.

The lethality of liver cancer and the resistance to chemical drugs have forced the search for effective prescriptions of traditional herbs for liver ...

Osteosarkom Farelerinde Bitki Kaynatma Formülasyonu ve Gavaj Standardizasyonu

Gavage Strategy for Decoction Formula of Traditional Chinese Medicine in Osteosarcoma Model Mice

Decoction formula is the most commonly used dosage form in traditional Chinese medicine and applied in clinical practice for thousands of years by trans-oral administration, which is characterized by quick effect, easy absorption, and individualized treatment based on the specific syndromes of patients. The quality of the decoction formula is directly responsible for the clinical efficacy of traditional Chinese medicine; therefore, the standardization process of the decoction formula is important to avoid differences in decoction quality caused by subjective factors. Meanwhile, due to the limitations of performing clinical experiments, small animals bearing human diseases, such as mice, are often used in medical research to explore the therapeutic efficacy and comprehensive mechanisms of different interventions, including the decoction formula for traditional Chinese medicine.&#160;Consequently, as an important trans-oral administration method, the skilled gavage technique is particularly important to avoid potential esophagus damage and drug spillage, which will ensure an equal amount of medicine being administered to each model animal, leading to accurate experimental results. Furthermore, the standardized method of decoction formula preparation and skilled gavage strategy are necessary to protect animal welfare and minimize the number of animals used. Here, we reported a detailed standardization process of the decoction formula and gavage technique with Yiqi Jiedu decoction in osteosarcoma mouse model as an example. The efficacy was evaluated by the tumor volume. This protocol will maximize animal protection and improve the reliability of research data, therefore providing effective strategies for future investigating therapeutic efficacy and molecular mechanisms of decoction formula for traditional Chinese medicine in vivo.

Yazar Spotlight: Gelişmiş Hayvan Çalışmaları için Standartlaştırılmış Bitkisel Kaynatma Protokolü

The protocol provides a detailed standardization process of decoction formula and gavage technique with Yiqi Jiedu decoction in the osteosarcoma mouse model as an example. It describes animal protection and improves reliability of research data, providing effective strategies for investigating therapeutic efficacy and molecular mechanisms of decoction formulas in vivo.

Bitkisel Tıbbın Hazırlanması: Er-Xian Kaynatma ve Er-Xian içeren Serum

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