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

Ce travail a été soutenu par la National Natural Science Foundation of China (81573992). Nous remercions Emily K. Lo et Kathleen DiNapoli pour leur aide à l&#39;édition de langue.

Le protocole suit les directives sur les soins des animaux de l&#39;Université de médecine traditionnelle chinoise de Shanghai et est approuvé par l&#39;expérimentation animale du Comité d&#39;éthique animale de Shanghai. 1. Protocole I: Préparation de l&#39;EXD <ol><li> Calcul <ol><li> Administrer l&#39;EXD à un groupe de traitement de 10 souris de six mois de la région de contrôle d&#39;impression féminine (ICR) (0,02 kg / souris), 5 jours par semaine pendant ...

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S., 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=An+in+vitro+and+in+vivo+investigation+of+the+antimetastatic+effects+of+a+Chinese+medicinal+decoction,+erxian+decoction,+on+human+ovarian+cancer+models.">An in vitro and in vivo investigation of the antimetastatic effects of a Chinese medicinal decoction, erxian decoction, on human ovarian cancer models.</a> Integr Cancer Ther. 12 (4), 336-346 (2013).</li><li>Zhu, Z., Li, L., Jin, X., Fang, J., 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=Er-Xian+Decoction,+a+traditional+Chinese+herbal+formula,+intervening+early+in+hypothalamic-pituitary+axis+of+male+rats+with+delayed+puberty.">Er-Xian Decoction, a traditional Chinese herbal formula, intervening early in hypothalamic-pituitary axis of male rats with delayed puberty.</a> Pharmacogn Mag. 10 (40), 517-521 (2014).</li><li>Miao, M. 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Au cours des dernières années, on a accordé plus d&#39;attention aux médicaments à base de plantes, un type de médicament alternatif appliqué dans le milieu clinique dans le monde oriental depuis des milliers d&#39;années. Différente du modèle de la médecine moderne &quot;banc à lit&quot;, la médecine traditionnelle à base de plantes nécessite d&#39;abord le modèle &quot;lit à banc&quot; pour expliquer leurs mécanismes. Cela peut être suivi d&#39;une validation effectuée au niveau du banc et du trait...

L&#39;intérêt pour l&#39;étude et l&#39;application de la phytothérapie traditionnelle augmente actuellement. Par opposition aux médicaments modernes, dans lesquels les ingrédients chimiques sont définis, les formules à base de plantes contiennent des ingrédients inconnus et nécessitent des procédés d&#39;extraction pour permettre la distribution de leurs composés actifs. Bien que de nombreuses études tentent de sélectionner un petit composé avec une structure bien connue comme représentant de l&#39;herbe entière ou de la formule à base de plantes, ni les efficacités pharmacologiques ni les mécanismes ne peuvent être considérés comme équivalents 1 , 2 . Bien que les empreintes digitales permettent d&#39;analyser les constituants des formules à base de plantes complexes, certains constituants ne sont toujours pas clairement analysés, ce qui entraîne un défi lors de la combinaison de tous les extraits d&#39;étude 3 . Les interactions de nombreux constituants / extraits mesurent les effets thérapeutiques des plantes médicinales. Pour conserver cet avantage, la forme-décoct traditionnelle d&#39;extraitIon - est encore largement utilisé dans la clinique. Comme la procédure d&#39;extraction a un impact important sur l&#39;efficacité thérapeutique, un protocole standard de décoction traditionnelle d&#39;eau est nécessaire, en particulier pour les études in vivo 4 , 5 . D&#39;autre part, lors de l&#39;exploration de mécanismes pharmacologiques, l&#39;administration de décoctions à base de plantes lors d&#39;études in vitro ou ex vivo est également un défi. La notion de sérum contenant des médicaments a d&#39;abord été proposée par Tashino en 1988 6 . Depuis lors, un nombre croissant de chercheurs l&#39;ont appliquée à la phytothérapie 7 , 8 , 9 . Bien que la méthode du sérum contenant du médicament présente certaines limites, telles que l&#39;influence de certains composants du sérum, elle est encore considérée comme une méthode qui imite de près les conditions physiologiques. 10 , 11 , 12 , 13 . Il a également été appliqué au traitement de l&#39;anémie aplasique 14 , l&#39;ostéoporose ménopausique 15 , 16 , 17 , l&#39;insuffisance ovarienne prématurée 18 , le cancer du sein 19 , le cancer de l&#39;ovaire 20 et la puberté retardée 21 . Ici, nous présentons des protocoles détaillés pour la préparation à la fois de la décoction er-xian (EXD) et de son sérum contenant des médicaments. De plus, nous décrivons l&#39;application de sérum contenant EXD et EXD aux modèles d&#39;ostéoporose ménopausique murine. Un EXD est composé de 9 g chacun de Curculigo orchioides Gaertn , Herbaa Epimedii </eM&gt;, Radix Morindae Officinalis et Radix Angelicae Sinensis et 6 g de Cortex Phellodendri et Rhizoma Anemarrhenae par patient adulte par jour. La dose équivalente pour une souris est de 0,1418 EXD / kg / jour selon l&#39;équation suivante 22 : dB = dA * RB / RA * (WA / WB) 1/3 . DA et dB font référence à la dose par poids corporel (mg / kg) de l&#39;homme et de la souris, respectivement. La dose, en mg / kg, est remplacée par le nombre d&#39;EXD / kg. La RA et la RB représentent respectivement le facteur corporel humain et le facteur corporel de la souris, qui sont proportionnels à la (surface corporelle (m 2 ) / poids corporel (kg)) 2/3 (voir tableau 1 ). WA et WB indiquent le poids corporel (kg) de l&#39;homme et de la souris, respectivement.

<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

La médecine traditionnelle à base de plantes, une médecine alternative dans le milieu clinique, a reçu une attention accrue ces dernières années. Avant la livraison au corps, une procédure d&#39;extraction supplémentaire est communément requise pour libérer les constituants actifs des herbes brutes. La décoction de l&#39;eau est une procédure d&#39;extraction classique qui est encore largement utilisée dans les milieux cliniques. Ici, nous proposons un protocole détaillé pour la décoction er-xian (EXD) afin d&#39;appliquer des décoctions à base de plantes à des études expérimentales. On décrit le calcul d&#39;une dose adaptée aux animaux, ainsi que les quatre étapes principales de l&#39;EXD: trempage, décoction de l&#39;eau, filtration et concentration. De plus, l&#39;EXD contenant du sérum est introduit chez les rats comme moyen de validation in vitro . Ici, les rats ont été administrés par voie orale EXD pendant trois jours. Les échantillons de sang ont ensuite été recueillis, inactivés, centrifugés et filtrés. Le sérum, dilué avec le milieu de culture, peut être utilisé pour traiter les cellules ou les tissus dans vItro. Par exemple, EXD a été appliqué à la fois à des études in vivo et in vitro et a démontré que EXD améliore l&#39;ostéogenèse. Ce protocole peut être utilisé comme référence pour la préparation et l&#39;application de plantes médicinales.

L&#39;effet d&#39;EXD sur la densité osseuse des souris OVX La coloration de l&#39;hématoxyline et de l&#39;éosine (H &amp; E) de la colonne vertébrale lombaire montre des trabécules osseuses accrues après traitement EXD in vivo ( figure 1A , panneau droit) par rapport à celles du groupe OVX ( figure 1A , panneau de gauche). La figure 1B montre les images μCT rep...

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

Ici, nous présentons la préparation d&#39;une décoction er-xian (EXD) en quatre étapes, trempage, décoction, filtration et concentration, et démontrons l&#39;administration d&#39;un sérum contenant EXD préparé à des rats. Ces méthodes s&#39;appliquent à l&#39;étude in vivo et in vitro des décoctions à base de plantes telles que les médicaments traditionnels chinois.

Préparation de la phytothérapie: la décoction Er-Xian et le sérum contenant de l&#39;er-Xian pour<em&gt; In vivo</em&gt; Et<em&gt; In Vitro</em&gt; Expériences

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.

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

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