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

この研究は、中国国立自然科学財団（81573992）によって支持された。私たちはEmily K. LoとKathleen DiNapoliの言語編集支援に感謝します。

このプロトコルは上海漢方医学の動物飼育指針に従っており、動物実験上海動物倫理委員会の承認を受けています。 1.プロトコルI：EXDの調製<ol><li> 計算 <ol><li> 6ヵ月齢の女性刷本管理領域（ICR）マウス（0.02kg /マウス）10匹の治療群にEXDを投与し、1週間あたり5日間12週間投与する。 注：合計1.702 EXDが必要です。 EXDは離散的な増分で測定されるため、実際には2つのEXDを管理します。 </li><li>マウス1匹につき適用されるEXDの量を計算する（ すなわち、 V =（0.1418 EXD / kg・日）*（0.02kg /マウス）* 50mL / EXD = 0.14mL /マウス・日）。 </li></ol></li><li> 浸漬 <ol><li> 2つのEXDのすべての原材料（ すなわち、Curculigo orchioides Gaertn （18g）、 Herba Epimedii （18g）、 Radix Morindae Officinalis （18g）、 R（18g）、 Cortex Phellodendri （12g）、 Rhizoma Anemarrhenae （12g）、合計96g）を蓋付き容器に入れた。 注：セラミック容器を推奨します。 </li><li>生ハーブを500mLの蒸留水で1時間洗浄します。水はハーブを約1インチ覆うべきです。すべてのハーブを徹底的に浸しましょう。 </li></ol></li><li> 最初の水の煎じ薬 <ol><li>水が沸騰するまで（約5〜10分;加熱時間、容器、およびハーブと水の量に依存します）、ガス炊飯器または誘導加熱調理器を使用してハーブを高熱で加熱します。低煮沸の電源を2時間倒します。 </li></ol></li><li> 最初のろ過 <ol><li>定期的なろ紙またはガーゼと綿で500 mLガラスビーカーを覆う。フィルターを通して、煎じ薬を慎重にビーカーに注ぎます。容器にハーブを残す。 </li><li>残った薬草残渣を返す容器にろ紙を移す。 </li></ol></li><li> 2回目の水煎じ <ol><li>ハーブで蒸留水を容器に加えます。ハーブを約1インチ覆うようにしましょう。 </li><li>手順1.3を繰り返します。 </li></ol></li><li> 二次ろ過 <ol><li>ステップ1.4.1と1.4.2を繰り返します。 </li><li> 2回目の煎じ薬を同じビーカーに注ぎます。 1回目と2回目の煎じ薬を混ぜる。 </li></ol></li><li> 濃度 <ol><li>ビーカーをアスベストフリーのワイヤーガーゼを挟んでガス炊飯器の上に置き、低煮沸器で加熱し、ガラス棒でゆっくりと連続的に攪拌する。 </li><li>煎剤が200 mLになったら、500 mLのビーカーに移す。 </li><li>煎じ薬が100mLになるまでステップ1.7.1を繰り返します。 </li></ol></li><li> 保管と管理 <ol><li>濃縮した煎じ薬を滅菌ガラス瓶に移す。それは室温に冷やしてみましょう再（RT）。 1週間以内に使用する場合は4℃で、長期間保存する場合は-70℃で保管してください。 </li><li>強制栄養針を用いて、1日1回、1週間に5日、12週間、卵巣摘出（OVX）マウスに煎じ薬0.14mL /マウスを投与する。 </li></ol></li></ol> 2.プロトコルII：EXD含有血清の調製<ol><li> 計算 <ol><li>各ラット（1ヶ月齢）の体重として0.15kgを用いて、ラット相当用量（RED）を計算する：dB =（1/60）*（90/100）*（60 / 0.15） 1/3 = 0.111 EXD / kg /日。 注：100mLの培地（10％EXD含有血清）を調製するためには、合計10mLのEXD含有血清が必要です。各ラットは2mLの血清を提供すると予想される。したがって、EXD投与には1日1回3日間、6匹のラット（20％の損失を考慮する）が必要である。 EXDの数は、（0.111 EXD / kg・日）*（0.15kg /ラット）*（6匹のラット）*（3日）= 0.300 EXD。再び、EXDは離散的な増分で測定されるので、1 EXDを使用します。 </li><li>ラット（ すなわち、 V =（0.111 EXD / kg・日）*（0.15kg /ラット）*（50mL / EXD）= 0.83mL /ラット・日）に適用されるEXDの容量を計算する。 </li></ol></li><li> 浸漬 <ol><li> 1 EXD用の原料を蓋付き容器に入れます。生ハーブを蒸留水に1時間浸します。水はハーブを約1インチ覆うべきです。すべてのハーブを徹底的に浸しましょう。 </li></ol></li><li> 最初の水の煎じ薬 <ol><li>水が沸騰するまで（約5〜10分;時間は熱出力、容器、およびハーブと水の量に依存します）、誘導加熱器を高出力で使用してハーブを加熱します。電源を2時間低煮沸にします。 </li></ol></li><li> 最初のろ過 <ol><li>定期的なろ紙またはガーゼと綿で500 mLガラスビーカーを覆う。フィルターを通して、煎じ薬を慎重にビーカーに注ぎます。容器にハーブを残す。 </li><li&gt;ろ紙上のハーブ残渣を容器に戻します。 </li></ol></li><li> 2回目の水煎じ <ol><li>ハーブで蒸留水を容器に加えます。ハーブを約1インチ覆うようにしましょう。 </li><li>ステップ2.4を繰り返します。 </li></ol></li><li> 二次ろ過 <ol><li>手順2.5.1と2.5.2を繰り返します。 </li><li>ステップ2.4.1と同じビーカーに2回目の煎じ薬を注ぎ、1回目と2回目の煎じ薬を混ぜます。 </li></ol></li><li> 濃度 <ol><li>ビーカーをアスベストフリーのワイヤーガーゼを挟んだガス炊飯器に入れ、低煮沸器で加熱し、ガラス棒でゆっくりと連続的に攪拌する。 </li><li>煎じ薬が100mLになったら、100mLビーカーに移す。 </li><li>煎じ薬が50mLになるまで2.7.1の手順を繰り返します。 </li></ol></li><li> 保管と管理 <ol><li>濃縮煎じ薬をステーに移すガラス瓶。それをRTに冷やす。 1週間以内に使用する場合は4℃で、長期間保存する場合は-70℃で保管してください。 </li><li> 1日1回、0.83mL /ラットを3日間胃内投与する。 </li></ol></li><li> EXD含有血清調製物 <ol><li>最後のEXD投与の1時間後に300mg / 100gの80mg / kgケタミンおよび10mg / kgキシラジンを腹腔内注射することによりラットを麻酔する。つま先で適切な麻酔を確認してください。 </li><li>メスを使用して腹部から胸郭の底にラットの皮膚と腹膜を切開する（またはまっすぐに操作するはさみ）。それぞれ約5cmと0.5cmの長さと深さの切開を作成します。ティッシュペーパーを使用して腹部内臓を左に動かします。 </li><li>ティシュペーパーを使用して、腹部大動脈の結合組織を除去して、血管をはっきりと露出させる。 </li><li> 10mL、22ゲージのシリンジを使用して、腹部大動脈からゆっくりと血液を採取する。それを転送する針を除去した後の15mLの滅菌チューブ; 1匹のラットは8〜10mLの血液を産生することができる。 注：採血が始まるとラットは生きている（ すなわち、腹部大動脈が拍動する）。 </li><li>室温RTで30〜60分間、直立姿勢で血液を凝固させる。 500-600×gで20分間遠心分離する。すべての上清（血清）を注意深く1つの50mLの滅菌チューブに入れ、すべての血清（異なる動物由来）を一緒に混合する。 </li><li>血清を56℃の水浴中で30分間インキュベートすることにより熱不活性化を行う。 0.22μmの孔径の親水性ポリエーテルスルホン膜を有するシリンジフィルターを用いて血清を濾過する。新鮮な血清を使用するか、-20℃で保存してください。 </li></ol></li><li> 対照薬物含有血清調製物（対照群において使用される） <ol><li> 1日1回3日間同じ容量の生理食塩水（0.83mL）で各ラットを投与する。他のステップはステップ2.9と同じです。</li></ol></li><li> 応用 <ol><li> EXD含有血清または薬物含有血清10mLを各100mLの培地に加え、1％ペニシリン - ストレプトマイシン（PS） 23に加える。 </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>

著者らは、競合する金銭的利益がないと宣言している。

近年、東アジアの臨床現場で何千年も使用されてきた代替薬の一種であるハーブ薬が注目されています。伝統的な漢方薬は、現代医学の「ベンチ・ベッド」のパターンとは異なり、まずそのメカニズムを説明するために「ベッド・ツー・ベンチ」のパターンが必要です。これに続いて、ベンチ段階で行われた検証と、新たに最適化された薬物の開発のための処理が続く。これまで、数式また?...

伝統的な漢方薬の研究と応用に関心が高まっています。化学成分が明確な現代薬とは対照的に、ハーブ処方には未知の成分があり、活性成分の送達を可能にするために抽出プロセスが必要です。多くの研究は、ハーブまたはハーブの処方全体の代表として、よく知られている構造を持つ1つの小さな化合物を選択しようとしていますが、薬理学的有効性やメカニズムは同等ではありません1,2 。フィンガープリンティングは複雑な漢方式の構成成分の分析を可能にするが、一部の成分は依然として明確に分析されていないため、研究3のすべての抽出物を組み合わせる際に問題が生じる。多数の成分/抽出物の相互作用は、漢方薬の治療効果を媒介する。この利点を保持するために、伝統的な抽出物の形態イオンは、依然として診療所で広く使用されています。抽出手順は治療効果に大きな影響を与えるため、特にインビボ研究では、伝統的な水煎出の標準プロトコールが必要である4,5。 他方、薬理学的機構を調べる場合、 インビトロまたはエクスビボの研究中の薬草煎じ薬の投与も課題である。薬物含有血清の概念は、1988年にTashinoによって最初に提案された6 。それ以来、数多くの研究者が漢方薬に適用しています7,8,9。薬物含有血清の方法は、血清の特定の成分の影響などのいくつかの制限があるが、依然として生理学的条件を厳密に模倣する方法であると考えられている。 14 、閉経骨粗鬆症15,16,17、早期卵巣不全18 、乳癌19 、卵巣癌20 、思春期遅発21の治療にも適用されています。ここでは、エラシアン煎剤（EXD）とその薬物含有血清の両方の調製のための詳細なプロトコールを提示する。加えて、我々は、ネズミ更年期骨粗鬆症モデルに対するEXDおよびEXD含有血清の適用を記載する。 EXDは、 9gのCurculigo orchioides Gaertn 、 Herba Epimedii </em&gt;、 Radix Morindae Officinalis 、 Radix Angelicae Sinensis 、 Cortex PhellodendriおよびRhizoma Anemarrhenaeそれぞれ6gを 1日当たりに投与する。マウスの等価線量は、以下の式22に基づいて0.1418EXD / kg /日である。dB = dA * RB / RA *（WA / WB） 1/3 。 dAおよびdBは、それぞれ、ヒトおよびマウスの体重当たりの用量（mg / kg）を示す。用量は、mg / kgで、EXD / kgの数で置き換えられる。 RAおよびRBは、（体表面積（m 2 ）/体重（kg）） 2/3 （ 表1参照）に比例するヒト身体因子およびマウス体係数をそれぞれ表す。 WAおよびWBは、それぞれヒトおよびマウスの体重（kg）を示す。

<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

近年、臨床現場での代替薬である伝統的な漢方薬が注目されています。身体に送達する前に、生薬から活性成分を放出するためには、一般的に追加の抽出手順が必要である。水煎出は、臨床の場で広く使用されている古典的な抽出手順です。ここでは、薬草煎じ薬を実験研究に適用するために、エキソ煎じ薬（EXD）の詳細なプロトコルを提案する。 EXDの主要な4つのステップ（浸漬、水煎出、濾過、濃縮）と同様に、動物に適した投与量の計算が記載されています。さらに、血清含有EXDをインビトロでの検証の手段としてラットに導入する。ここでは、ラットにEXDを3日間経口投与した。次いで、血液サンプルを収集し、不活性化し、遠心分離し、濾過した。培養培地で希釈した血清を用いて、細胞または組織をvitro。例えば、EXDはインビボおよびインビトロ研究の両方に適用され、EXDが骨形成を増強することが実証された。このプロトコールは、漢方薬の調製および応用の参考として用いることができる。

OVXマウスの骨密度に対するEXDの効果腰椎切片のヘマトキシリンおよびエオシン（H＆E）染色は、OVX群（ 図1A 、左パネル）と比較して、 インビボでの EXD処置後の骨梁の増加を示す（ 図1A 、右パネル）。 図1Bは、OVXマウス（ 図1B 、左パネル）および12週間のEXD処置を行...

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

ここでは、浸漬、煎出、ろ過、および濃縮の4段階でerxian decoction（EXD）の準備を行い、準備したEXD含有血清のラットへの投与を実証する。これらの方法は、伝統的な漢方薬などの薬草煎じ薬のin vivoおよびin vitro研究に適用可能である。

漢方薬の準備：Er-Xian煎じ薬とEr-Xian含有血清<em&gt;インビボ</em&gt;と<em&gt;インビトロ</em&gt;実験

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

preparation-herbal-medicine-er-xian-decoction-er-xian-containing

Research

JoVE Journal

Medicine

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

10.9K ビュー.  Shanghai University of Traditional Chinese Medicine. ここでは、浸漬、煎出、ろ過、および濃縮の4段階でerxian decoction（EXD）の準備を行い、準備したEXD含有血清のラットへの投与を実証する。これらの方法は、伝統的な漢方薬などの薬草煎じ薬のin vivoおよびin vitro研究に適用可能である。 

ビデオ: 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).

血管平滑筋細胞の処理のための凍結乾燥ベリー粉末からのポリフェノールの抽出および精製<em&gt;インビトロ</em

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

化学

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.

インビボおよびインビトロ実験用グシュカン(GSK)顆粒の調製

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

医学

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アプリケーションのためのナリンゲニン溶液の調製

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.

チベット医学ロディオラ顆粒におけるマルチインデックス成分の in vitro 溶解測定

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).

損傷したPC12細胞を治療するための2つのハーブの組み合わせ戦略の探索

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フォリウムマトン油加工技術の最適化とゼブラフィッシュ胚発生への影響の検証

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 ...

骨肉腫マウスにおけるハーブ煎じ薬製剤と強制経口投与の標準化

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.

著者スポットライト:強化された動物研究のための標準化されたハーブ煎じ薬プロトコル

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.

漢方薬の準備：Er-Xian煎じ薬とEr-Xian含有血清

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血管平滑筋細胞の処理のための凍結乾燥ベリー粉末からのポリフェノールの抽出および精製