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
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...

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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 border="0" cellpadding="0" cellspacing="0" width="651">
	
	<tbody>
		<tr height="165">
			<td height="165" style="height:165px;width:216px;">Curculigo orchioides Gaertn (9&#8201;g), Herbaa Epimedii (9&#8201;g), Radix Morindae Officinalis (9&#8201;g), Radix Angelicae Sinensis (9&#8201;g), Cortex Phellodendri (6&#8201;g), and Rhizoma Anemarrhenae (6&#8201;g)</td>
			<td style="width:149px;">Kang-qiao Chinese Medicine Yinpian Co. Ltd (Shanghai, CN)</td>
			<td style="width:119px;">160922</td>
			<td style="width:167px;">EXD components</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Filter paper</td>
			<td style="width:149px;">GElifesciences</td>
			<td style="width:119px;">99-103-952</td>
			<td style="width:167px;">Filter EXD decoction before concentration</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Imprinting Control Region (ICR) mice</td>
			<td style="width:149px;">Shanghai Laboratory Animal Center</td>
			<td style="width:119px;">SCXK 2007-0005</td>
			<td style="width:167px;">In vivo study</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Sprague Dawley rats</td>
			<td style="width:149px;">Shanghai Laboratory Animal Center</td>
			<td style="width:119px;">SCXK 2007-0005</td>
			<td style="width:167px;">EXD-containing serum preparation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Syringe filter</td>
			<td style="width:149px;">Millipore</td>
			<td style="width:119px;">SLGP033RB</td>
			<td style="width:167px;">0.22 &#181;m</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">gavage needles (10 ml)</td>
			<td style="width:149px;">Shanghai BO Ge trade sales department</td>
			<td align="right" style="width:119px;">59104274</td>
			<td style="width:167px;">Adminstration of EXD</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Ketamine (80 mg/kg)&#160;</td>
			<td style="width:149px;">Fujian Gutian Pharma Co. Ltd</td>
			<td style="width:119px;">H35020148</td>
			<td>Anesthesia</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Xylazine (10 mg/kg)</td>
			<td style="width:149px;">Sunway Pharma Co. Ltd</td>
			<td style="width:119px;">CB07591</td>
			<td>Anesthesia</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">Dulbecco&#8217;s modification of Eagle&#8217;s medium Dulbecco (DMEM) culture medium</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:119px;">12800-116</td>
			<td style="width:167px;">DMEM with 2 mM L-glutamine and without ribonucleosides and ribonucleotides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Streptomycin</td>
			<td style="width:149px;">Sigma</td>
			<td align="right" style="width:119px;">1277</td>
			<td style="width:167px;">100 &#181;g / ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin</td>
			<td style="width:149px;">Sigma</td>
			<td align="right" style="width:119px;">4687</td>
			<td style="width:167px;">100 &#181;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 ...

The overall goal of this procedure is to prepare an er-xian decoction, a traditional Chinese herbal medicine for its in vivo and in vitro application. This method can help answer key questions in herbal medicine field about preparation and application of a traditional Chinese medicine. The main advantage of this technique is that the water extraction steps are similar to those used in a clinical setting.Place the raw materials for a single er-xian decoction into a container with a lid, and add enough distilled water that the herbs are covered by about an inch. Put the lid on the container, and macerate the raw herbs for one hour. Then use a gas cooker on high power to heat the herbs until the water boils.Turn the power down to a low simmer, and decoct the herbs for two hours. At the end of the first water decoction, cover a 500-milliliter glass beaker with gauze and cotton, and carefully pour the decoction liquid only into the beaker through the filter. Return any herbal residue on the filter paper to the container, and add enough fresh distilled water to cover the herbs with another inch of water.After a second water decoction as just demonstrated, filter the decoction into the 1, 000-milliliter beaker, and place the beaker onto a gas cooker covered with asbestos-free wire gauze gas. Heat the decoction with a low simmer using a glass rod to slowly and continuously stir the solution. When the decoction reduces to 200 milliliters, transfer the solution to a 500-milliliter beaker, and reduce the volume to 100 milliliters as just demonstrated.After confirming the appropriate level of sedation by toe pinch, use straight operating scissors to make a five-centimeter long, 0.5-centimeter deep incision in the skin and peritoneum of an er-xian decoction-treated animal one hour after the last decoction treatment from the abdomen to the bottom of the thorax. Use tissue paper to move the abdominal viscera, and remove the connective tissue of the abdominal aorta to expose the vessel. Next, use a 10-milliliter syringe equipped with a 22-gauge needle to slowly draw eight to 10 milliliters of blood from the abdominal aorta, and transfer the blood to a 15-milliliter sterile tube.Clot the blood for 30 to 60 minutes at room temperature, then collect the cells by centrifugation. Carefully pool the serum from each animal into a single sterile 50-milliliter tube, and heat and activate the serum in a 56 degrees Celsius water bath for 30 minutes. Finally, use a syringe filter with a 0.22-micrometer pore size, and hydrophilic polyethersulfone membrane to filter the serum into a 50-milliliter conical tube for immediate use.Hematoxylin and eosin staining of the lumbar vertebral section of an ovariectomized or OVX mouse reveals an increase in bone trabeculae after er-xian decoction treatment in vivo. Compare to control OVX animals. Representative micro-CT images of the fourth lumbar of a control OVX mouse and an OVX mouse treated with the er-xian decoction for two weeks reveals that more trabecular bones are observed on the inside of the lumbar vertebra of the er-xian decoction-treated animals than those of the control OVX mice.Indeed, quantitative analysis of these micro-CT data indicates an increase in bone to tissue volume, trabecular number and thickness, and a decrease in the trabecular spacing of the fourth lumbar of the er-xian decoction-treated animals. Fat droplets automatically form when bone mesenchymal stem cells from OVX mice are cultured for seven days. In cultures from er-xian decoction-treated animals, bone nodules form instead.Further, more alkaline phosphatase-positive cells can be detected in er-xian decoction-treated bone mesenchymal stem cells than in stem cells from control OVX animals. In addition, hierarchical clustering demonstrates that the expression of 389 genes in bone mesenchymal cells as reveal from microarray is full change greater than 1.5 times between OVX and er-xian decoction-treated mice in vivo. Once mastered, the er-xian decoction can be prepared in eight hours, and the contained herb serum can be prepared in two hours.For internal gastric demonstration, first confirm that the IG tube is properly positioned within the esophagus, then gently delivers the decoction to the animal. According to tradition, preparation method of a Chinese herbal medicine, the medicine or materials should be extracted with water rather than external. Rotary evaporators are commonly used for concentrating the decoction but gas can be used as necessary.Traditional Chinese medicine requires the standardized preparation procedure and general medicinal materials for in this procedure, the consistent quality will be controlled. After watching this video, you should have a good understanding of how to standardize medicinal materials extraction, and to prepare contained herbal serum. Please keep in mind that the boiling decoction is very hot.Wear waterproof heat-resistant gloves as necessary.

Research

JoVE Journal

Medicine

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

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