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

Este trabalho foi apoiado pela National Natural Science Foundation of China (81573992). Agradecemos a Emily K. Lo e Kathleen DiNapoli pela assistência de edição de idiomas.

O protocolo segue as diretrizes de cuidados com os animais da Universidade de Medicina Tradicional Chinesa de Xangai e é aprovado pelo experimento animal Comitê de Ética Animal de Xangai. 1. Protocolo I: Preparação do EXD <ol><li> Cálculo <ol><li> Administrar o EXD a um grupo de tratamento de 10 camundongos de região de controle de Imprinting Control (ICR) de seis meses de idade (0,02 kg / mouse), 5 dias por semana durante 12 semanas. NOTA: Um total de 1,702 ...

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Nos últimos anos, tem sido dada mais atenção às ervas medicinais, um tipo de medicina alternativa que foi aplicada no cenário clínico no mundo oriental há milhares de anos. Diferente do padrão de &quot;banco para cama&quot; da medicina moderna, a medicina herbal tradicional exige primeiro o padrão de &quot;cama a bancada&quot; para explicar seus mecanismos. Isso pode ser seguido de validação realizada no estágio do banco e processamento para o desenvolvimento de novos medicamentos otimizados. Até agora, exi...

O interesse no estudo e na aplicação de ervas medicinais tradicionais está crescendo atualmente. Ao contrário das drogas modernas, nas quais os ingredientes químicos são definitivos, as fórmulas à base de plantas possuem alguns ingredientes desconhecidos e requerem processos de extração para permitir a entrega de seus compostos ativos. Embora muitos estudos tentam selecionar um composto pequeno com uma estrutura bem conhecida como representante da erva inteira ou fórmula herbal, nem as eficiências farmacológicas nem os mecanismos podem ser considerados equivalentes 1 , 2 . Embora as impressões digitais permitam a análise dos constituintes de fórmulas ervas complexas, alguns constituintes ainda não são claramente analisados, levando a um desafio ao combinar todos os extratos para o estudo 3 . As interações de vários constituintes / extratos medeiam os efeitos terapêuticos das ervas medicinais. Para manter essa vantagem, o extrato tradicional decoctaIon - ainda é amplamente utilizado na clínica. Uma vez que o procedimento de extração tem um grande impacto sobre a eficácia terapêutica, é necessário um protocolo padrão de decocção de água tradicional, especialmente para estudos in vivo 4 , 5 . Por outro lado, ao explorar mecanismos farmacológicos, a administração de decocções de ervas durante estudos in vitro ou ex vivo também é um desafio. O conceito de soro contendo drogas foi proposto pela primeira vez por Tashino em 1988 6 . Desde então, um número crescente de pesquisadores aplicou-o à medicina herbal 7 , 8 , 9 . Embora o método do soro contendo fármaco tenha algumas limitações, como a influência de certos componentes do soro, ainda é considerado um método que imita de perto as condições fisiológicas. 10 , 11 , 12 , 13 . Também foi aplicado ao tratamento de anemia aplástica 14 , osteoporose menopausa 15 , 16 , 17 , insuficiência ovariana prematura 18 , câncer de mama 19 , câncer de ovário 20 e puberdade retardada 21 . Aqui, apresentamos protocolos detalhados para a preparação da decocção er-xian (EXD) e do seu soro contendo fármaco. Além disso, descrevemos a aplicação de EXD e EXD contendo soro para modelos osteoporóticos da menopausa murina. Um EXD é composto por 9 g de cada um de Curculigo Orchioides Gaertn , Herbaa Epimedii </eM&gt;, Radix Morindae Officinalis e Radix Angelicae Sinensis e 6 g de Cortex Phellodendri e Rhizoma Anemarrhenae por paciente adulto por dia. A dose equivalente para um mouse é 0.1418 EXD / kg / dia com base na seguinte equação 22 : dB = dA * RB / RA * (WA / WB) 1/3 . DA e dB referem-se à dose por peso corporal (mg / kg) do ser humano e do mouse, respectivamente. A dose, em mg / kg, é substituída pelo número de EXD / kg. RA e RB representam o fator do corpo humano e o fator do corpo do mouse, respectivamente, que são proporcionais à (área de superfície do corpo (m 2 ) / peso corporal (kg)) 2/3 (ver Tabela 1 ). WA e WB indicam o peso corporal (kg) do humano e do mouse, respectivamente.

<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

A medicina tradicional à base de plantas, uma medicina alternativa no cenário clínico, recebeu atenção crescente nos últimos anos. Antes da entrega ao corpo, um procedimento de extração adicional é comumente requerido para liberar os constituintes ativos das ervas cruas. A decocção de água é um procedimento de extração clássico que ainda é amplamente utilizado nas configurações clínicas. Aqui, propomos um protocolo detalhado para a decocção er-xian (EXD), a fim de aplicar decocções à base de plantas para estudos experimentais. O cálculo de uma dose apropriada para animais é descrito, bem como os quatro passos principais de EXD: imersão, decocção de água, filtração e concentração. Além disso, o EXD contendo soro é introduzido em ratos como meio de validação in vitro . Aqui, os ratos foram administrados oralmente EXD por três dias. As amostras de sangue foram então recolhidas, inactivadas, centrifugadas e filtradas. O soro, diluído com o meio de cultura, pode ser utilizado para tratar células ou tecidos em vItro. Por exemplo, EXD foi aplicado tanto em estudos in vivo como in vitro e demonstrou que o EXD melhora a osteogênese. Este protocolo pode ser usado como referência para a preparação e aplicação de medicamentos à base de plantas.

O efeito de EXD sobre a densidade óssea de camundongos OVX A coloração com hematoxilina e eosina (H &amp; E) da vértebra lombar mostra aumento da trabécula óssea após o tratamento com EXD in vivo ( Figura 1A , painel direito) em comparação com os do grupo OVX ( Figura 1A , painel esquerdo). A Figura 1B mostra as imagens μCT representativas do 4º lom...

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

Aqui, apresentamos a preparação de uma decocção er-xian (EXD) em quatro etapas - imersão, decocção, filtração e concentração - e demonstram a administração de um soro contendo EXD preparado para ratos. Estes métodos são aplicáveis ​​ao estudo in vivo e in vitro de decocções à base de plantas, como medicamentos tradicionais chineses.

Preparação de Fitoterapia: Decocção Er-Xian e Soro contendo Er-Xian para<em&gt; In Vivo</em&gt; E<em&gt; In Vitro</em&gt; Experimentos

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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