Este trabalho foi apoiado pela Fundação Nacional de Ciências Naturais da China (81973569, 82274207 e 82104533), pelo Xinglin Scholar Research Promotion Project da Universidade de Chengdu da TCM (XKTD2022013) e pelo Programa de Pesquisa e Desenvolvimento Chave de Ningxia (2023BEG02012).

Rhodiola crenulata é coletada da Montanha de Neve de Zhuoda, Condado de Ganzi, Prefeitura Autônoma Tibetana de Ganzi, Província de Sichuan, China (N 31.44570°, E 99.96086°, 4892 m). As plantas são autenticadas como genuínas pelo professor Yi Zhang na Escola de Medicina Étnica da Universidade de Medicina Tradicional Chinesa de Chengdu.
1. Coleção de Rhodiola crenulata
<ol><li>Fotografe o mapa de habitat de Rhodiola crenulata.</li>
	<li>Fotografe toda a planta, folhas, cálice e rizoma de Rhodiola crenulata.</li>
	<li>Use uma pá para limpar as ervas daninhas e pedras quebradas dentro de 1 m da Rhodiola crenulata para garantir a mineração suave subsequente.</li>
	<li>Desenterre o solo com uma enxada até que toda a rizosfera seja vista e colete a raiz da torneira. NOTA: As raízes e rizomas de Rhodiola crenulata usados em partes medicinais devem ser coletados no outono, quando os caules da flor murcham.</li>
</ol>2. Identificação das características
<ol><li>Observe os traços de aparência de Rhodiola crenulata a olho nu: raízes e rizomas cilíndricos e curtos, superfície marrom, epiderme amarela membranosa com padrão rosa e fatias vermelho-alaranjadas ou bordô.</li>
	<li>Identifique-o pelo cheiro: Dá um cheiro perfumado quando perto do nariz.</li>
	<li>Identifique-o pelo gosto: Leve um pequeno pedaço de raiz para a boca, primeiro beba e depois mastigue, gosto ligeiramente amargo, depois doce.</li>
</ol>3. Identificação microscópica de grânulos de amido em pó medicinal
<ol><li>Retire o solo na superfície da Rhodiola crenulata com um pincel, coloque-o no forno a 40 °C e vire as ervas a cada 24 h. OBS: A facilidade de quebra de materiais medicinais é considerada o padrão para secagem de umidade.</li>
	<li>Pulverizar os materiais medicinais secos usando uma máquina de pó e filtrar o pó medicinal usando a peneira medicamentosa nº 3 (ver Tabela de Materiais).</li>
	<li>Pegue uma lâmina limpa (consulte Tabela de Materiais), cave o pó com uma agulha dissecante (consulte Tabela de Materiais) e coloque-o uniformemente em um terço da lâmina dentro de 2 mm.</li>
	<li>Use um conta-gotas de vidro (consulte Tabela de Materiais) para adicionar uma gota de água deionizada ao pó. Use uma pinça (consulte Tabela de Materiais) para segurar uma extremidade do vidro da tampa (consulte Tabela de Materiais) para tocar o nível de líquido rapidamente e cobrir o pó. NOTA: Use uma agulha anatômica para misturar suavemente água e pó medicinal para garantir uma mistura uniforme da amostra. Não deve haver bolhas de ar entre a lâmina, o pó e o vidro da tampa.</li>
	<li>Abra o microscópio (ver Tabela de Materiais) e coloque a lâmina na etapa 3.4 na plataforma para prendê-la. Ajuste a fonte de luz e a espiral de foco grosso para ver o pó. Ajuste a espiral parafocal fina até que os tecidos sejam vistos claramente. Mude para uma objetiva de 40x e observe os grânulos de amido. NOTA: Amido os grãos que se apresentam como grãos únicos ou múltiplos, e o ponto umbilical aparece em forma de espinha de arenque ou rachadura.</li>
</ol>4. Identificação microscópica de cateteres, células de cortiça, fibras, células do parênquima da madeira e massas pigmentares em pó medicinal
<ol><li>Pegue uma lâmina limpa (consulte Tabela de Materiais), cave o pó com uma agulha dissecante (consulte Tabela de Materiais) e coloque-a em um terço da lâmina.</li>
	<li>Use um conta-gotas de vidro (consulte a Tabela de Materiais) para adicionar uma gota de hidrato de cloral (consulte a Tabela de Materiais) ao pó. Pegue o slide com uma pinça (veja Tabela de Materiais) e aqueça-o na lâmpada de álcool três vezes, cada vez por 1 s. NOTA: As bolhas devem ser evitadas durante o aquecimento. O líquido permanece sem fluir, indicando que a penetração está completa.</li>
	<li>Use um conta-gotas de vidro para adicionar uma gota de glicerina (consulte a Tabela de Materiais). Use uma pinça para segurar uma extremidade do vidro da tampa para tocar o nível do líquido rapidamente.</li>
	<li>Abra o microscópio e coloque a lâmina na plataforma para prendê-la. Ajuste a fonte de luz e a espiral de foco grosso para ver o pó. Ajuste a espiral parafocal fina até que os tecidos sejam vistos claramente. Observe o cateter, as células de cortiça, as fibras, as células do parênquima da madeira e o bloqueio de pigmentos mudando para uma lente objetiva de 40x. NOTA: Poligonal ou poligonal longa de células de cortiça, vaso espiral com estrutura helicoidal óbvia, parênquima de xilema contendo cristais de areia de oxalato de cálcio e bloco de pigmento vermelho ou vermelho-acastanhado.</li>
</ol>5. Preparação de amostras cromatográficas em camada delgada (CCD) de Rhodiola crenulata e sua referência
<ol><li>Coloque o papel de pesagem na balança (ver Tabela de Materiais) e pese 3 g de pó de Rhodiola crenulata.</li>
	<li>Leve o pó para um frasco cônico de 100 mL (consulte a Tabela de Materiais) e adicione 25 mL de metanol com uma pipeta de barriga grande (consulte a Tabela de Materiais). Coloque o frasco cônico no instrumento ultrassônico. Defina a potência para 250 W, frequência para 40 kHz e tempo para 30 min (consulte Tabela de Materiais) e ligue o instrumento. NOTA: O objetivo do instrumento de ultrassom é garantir que o pó de Rhodiola crenulata seja completamente dissolvido sem afetar os resultados de experimentos cromatográficos de camada fina subsequentes.</li>
	<li>Retire o frasco cónico e lave o frasco exterior com água corrente até à temperatura ambiente (RT).</li>
	<li>Aspirar 800 μL de líquido preparado na etapa 5.3 com uma seringa de 1 mL. Filtrar com membrana filtrante microporosa de 0,22 μm (ver Tabela de Materiais) e coletar 400 μL de solução de amostra midstream de Rhodiola crenulata em um frasco de amostra cromatográfica.</li>
	<li>Pesar e adicionar 2 mg de salidrósido, tirosol e ácido gálico (ver Tabela de Materiais) em 3 frascos cónicos separados de 100 ml, respetivamente. Adicione 25 mL de metanol com uma pipeta de barriga grande em cada frasco cônico.</li>
	<li>Coloque o frasco cônico no instrumento ultrassônico, ajuste a potência para 250 W, a frequência para 40 kHz e o tempo para 30 minutos e repita a etapa 5.3 (consulte a Tabela de Materiais).</li>
	<li>Aspirar 800 μL de líquido preparado na etapa 5.6 com uma seringa de 1 mL. Filtrar com membrana filtrante microporosa de 0,22 μm (ver Tabela de Materiais) e recolher 400 μL de solução padrão de salidrósido, tirosol e ácido gálico nos frascos de amostra cromatográficos correspondentes.</li>
</ol>6. Identificação do CPT
<ol><li>Pipeta triclorometano (5 mL), acetato de etila (4 mL), metanol (2 mL) e ácido fórmico (0,5 mL) (ver Tabela de Materiais). Adicione a um lado do cilindro de cromatografia de tanque duplo (consulte a Tabela de Materiais), agite e misture uniformemente. Cubra o cabeçote superior.</li>
	<li>Coloque o ácido gálico, o salidrósido, a solução padrão de tirosol e a solução de Rhodiola crenulata no rack de amostras nas posições A1-A4.</li>
	<li>Coloque a folha de silicone de 5 cm x 10 cm (ver Tabela de Materiais) na tabela de amostragem. Ligue a máquina de amostragem automática (consulte Tabela de Materiais) e abra a válvula de controle de ar.</li>
	<li>Abra o software visionCATS (consulte a Tabela de Materiais). Clique em Nova > Nova pasta (chamada Rhodiola crenulata teste de exemplo) > OK. Clique em Novo método (nome Rhodiola crenulata sample test) > OK > ATS 4.</li>
	<li>Clique em Concluir Definição da Etapa. Clique em Rastrear atribuição para editar a descrição (ácido gálico, salidrósido, tirosol e Rhodiola crenulata amostra).</li>
	<li>Clique em Etapas HPTLC. Defina os parâmetros da camada fina (5 cm x 10 cm). Selecione Tipo de aplicativo (banda), defina os parâmetros (Tabela 1) e clique no botão OK .</li>
	<li>Qualidade de enxágue/enxágue aberto. Marque Preencher somente Volume Programado. Defina o nível inferior do frasco para injetáveis (mm) como 0,5 e clique no botão OK.</li>
	<li>Clique em Executar método.</li>
	<li>Clique no botão Controlar Atribuição , marque Central e defina os parâmetros (Tabela 2).</li>
	<li>Clique no botão Continuar para obter amostras automáticas.</li>
	<li>Desligue a máquina de amostragem automática e a válvula de ar. Remova a folha de silicone da máquina de amostragem automática.</li>
	<li>Coloque a folha de silicone no outro lado do cilindro de cromatografia de tanque duplo na etapa 6.1, cubra o cabeçote superior e pré-saturar por 20 min.</li>
	<li>Aperte suavemente a extremidade superior da placa de camada fina com um tweezer, coloque rapidamente a folha de silicone no agente revelador e cubra a cabeça superior do cilindro. NOTA: Retire a folha de silicone quando a borda frontal de desdobramento estiver a 0,5-1,0 cm de distância da extremidade superior da placa de camada fina.</li>
	<li>Depois que o solvente orgânico evaporar, pulverize a solução cromogênica na superfície da folha de silicone à temperatura ambiente para obter resultados cromogênicos. NOTA: A solução cromogênica é uma solução aquosa contendo 2% de FeCl3 e 1% de K3[Fe(CN)6].</li>
</ol>

Caracterização microscópica e identificação baseada em TLC de crenulato de Rhodiola

<ol>
	<li>Zhang, J., 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=Traditional+herbal+medicine+and+nanomedicine:+Converging+disciplines+to+improve+therapeutic+efficacy+and+human+health.">Traditional herbal medicine and nanomedicine: Converging disciplines to improve therapeutic efficacy and human health.</a> Advanced Drug Delivery Reviews. 178, 113964 (2021).</li><li>Yin, 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=The+positive+role+and+mechanism+of+herbal+medicine+in+Parkinson's+Disease.">The positive role and mechanism of herbal medicine in Parkinson's Disease.</a> Oxidative Medicine and Cellular Longevity. 2021, 9923331 (2021).</li><li>Li, F. S., Weng, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demystifying+traditional+herbal+medicine+with+modern+approach.">Demystifying traditional herbal medicine with modern approach.</a> Nature Plants. 3, 17109 (2017).</li><li>da Fonseca, L. 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=Herbal+medicinal+products+from+Passiflora+for+anxiety:+An+unexploited+potential.">Herbal medicinal products from Passiflora for anxiety: An unexploited potential.</a> The Scientific World Journal. 2020, 6598434 (2020).</li><li>Aziato, L., Antwi, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facilitators+and+barriers+of+herbal+medicine+use+in+Accra,+Ghana:+an+inductive+exploratory+study.">Facilitators and barriers of herbal medicine use in Accra, Ghana: an inductive exploratory study.</a> BMC Complementary and Alternative Medicine. 16, 142 (2016).</li><li>Tao, H., 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=Rhodiola+species:+A+comprehensive+review+of+traditional+use,+phytochemistry,+pharmacology,+toxicity,+and+clinical+study.">Rhodiola species: A comprehensive review of traditional use, phytochemistry, pharmacology, toxicity, and clinical study.</a> Medicinal Research Reviews. 39 (5), 1779-1850 (2019).</li><li>Wang, Y., 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=The+dietary+supplement+Rhodiola+crenulata+extract+alleviates+dextran+sulfate+sodium-induced+colitis+in+mice+through+anti-inflammation,+mediating+gut+barrier+integrity+and+reshaping+the+gut+microbiome.">The dietary supplement Rhodiola crenulata extract alleviates dextran sulfate sodium-induced colitis in mice through anti-inflammation, mediating gut barrier integrity and reshaping the gut microbiome.</a> Food &amp; Function. 12 (7), 3142-3158 (2021).</li><li>Xie, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodiola+crenulate+alleviates+hypobaric+hypoxia-induced+brain+injury+via+adjusting+NF-&#954;B/NLRP3-mediated+inflammation.">Rhodiola crenulate alleviates hypobaric hypoxia-induced brain injury via adjusting NF-&#954;B/NLRP3-mediated inflammation.</a> Phytomedicine. 103, 154240 (2022).</li><li>Wang, 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=Salidroside,+a+phenyl+ethanol+glycoside+from+Rhodiola+crenulata,+orchestrates+hypoxic+mitochondrial+dynamics+homeostasis+by+stimulating+Sirt1/p53/Drp1+signaling.">Salidroside, a phenyl ethanol glycoside from Rhodiola crenulata, orchestrates hypoxic mitochondrial dynamics homeostasis by stimulating Sirt1/p53/Drp1 signaling.</a> Journal of Ethnopharmacology. 293, 115278 (2022).</li><li>Ma, D., 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=Chemical+characteristics+of+Rhodiola+Crenulata+and+its+mechanism+in+acute+mountain+sickness+using+UHPLC-Q-TOF-MS/MS+combined+with+network+pharmacology+analysis.">Chemical characteristics of Rhodiola Crenulata and its mechanism in acute mountain sickness using UHPLC-Q-TOF-MS/MS combined with network pharmacology analysis.</a> Journal of Ethnopharmacology. 294, 115345 (2022).</li><li>Wang, 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=Rhodiola+crenulata+attenuates+apoptosis+and+mitochondrial+energy+metabolism+disorder+in+rats+with+hypobaric+hypoxia-induced+brain+injury+by+regulating+the+HIF-1&#945;/microRNA+210/ISCU1/2(COX10)+signaling+pathway.">Rhodiola crenulata attenuates apoptosis and mitochondrial energy metabolism disorder in rats with hypobaric hypoxia-induced brain injury by regulating the HIF-1&#945;/microRNA 210/ISCU1/2(COX10) signaling pathway.</a> Journal of Ethnopharmacology. 241, 111801 (2019).</li><li>d'Avigdor, E., Wohlmuth, H., Asfaw, Z., Awas, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+current+status+of+knowledge+of+herbal+medicine+and+medicinal+plants+in+Fiche,+Ethiopia.">The current status of knowledge of herbal medicine and medicinal plants in Fiche, Ethiopia.</a> Journal of Ethnobiology and Ethnomedicine. 10, 38 (2014).</li><li>S&#225;nchez, M., Gonz&#225;lez-Burgos, E., Divakar, P. K., G&#243;mez-Serranillos, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-based+authentication+and+metabolomics+analysis+of+medicinal+plants+samples+by+DNA+barcoding+and+ultra-high-performance+liquid+chromatography/triple+quadrupole+mass+spectrometry+(UHPLC-MS).">DNA-based authentication and metabolomics analysis of medicinal plants samples by DNA barcoding and ultra-high-performance liquid chromatography/triple quadrupole mass spectrometry (UHPLC-MS).</a> Plants (Basel, Switzerland). 9 (11), 1601 (2020).</li><li>. Rhodiola renulata (Hook.fil &amp; Thomson) H.Ohba Available from: <a target="_blank" href="https://www.gbif.org/species/4200529">https://www.gbif.org/species/4200529</a> (2023)</li><li>. Plant + data center Available from: <a target="_blank" href="https://www.iplant.cn/">https://www.iplant.cn/</a> (2023)</li><li>. Chinese virtual herbarium Available from: <a target="_blank" href="https://www.cvh.ac.cn/">https://www.cvh.ac.cn/</a> (2023)</li><li>Cunningham, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=There+&amp;#34;ain't+no+mountain+high+enough&amp;#34;?:+The+drivers,+diversity+and+sustainability+of+China's+Rhodiola+trade.">There &amp;#34;ain't no mountain high enough&amp;#34;?: The drivers, diversity and sustainability of China's Rhodiola trade.</a> Journal of Ethnopharmacology. 252, 112379 (2020).</li><li>Wang, Q., Ruan, X., Jin, Z. H., Yan, Q. C., Tu, S. <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+Rhodiola+species+by+using+RP-HPLC.">Identification of Rhodiola species by using RP-HPLC.</a> Journal of Zhejiang University. Science. B. 6 (6), 477-482 (2005).</li><li>Dong, X., Guo, Y., Xiong, C., Sun, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+two+major+Rhodiola+species+and+the+systemic+changing+characteristics+of+metabolites+of+Rhodiola+crenulata+in+different+altitudes+by+chemical+methods+combined+with+UPLC-QqQ-MS-based+metabolomics.">Evaluation of two major Rhodiola species and the systemic changing characteristics of metabolites of Rhodiola crenulata in different altitudes by chemical methods combined with UPLC-QqQ-MS-based metabolomics.</a> Molecules. 25 (18), 4062 (2020).</li><li>Zhao, W., 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=HPLC+fingerprint+differentiation+between+natural+and+ex+situ+populations+of+Rhodiola+sachalinensis+from+Changbai+Mountain,+China.">HPLC fingerprint differentiation between natural and ex situ populations of Rhodiola sachalinensis from Changbai Mountain, China.</a> PloS One. 9 (11), 112869 (2014).</li><li>Yu, Y. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+evaluation+of+Tibetan+medicine+Rhodiolae+crenulatae+radix+et+rhizome+based+on+sensory+recognition+and+near+infrared+spectrum+analysis.">Quality evaluation of Tibetan medicine Rhodiolae crenulatae radix et rhizome based on sensory recognition and near infrared spectrum analysis.</a> Chengdu University of Traditional Chinese Medicine. , (2020).</li><li>Lv, X. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+determination+of+5+chemical+components+in+3+kinds+of+Rhodiola+rosea+by+HPLC.">Simultaneous determination of 5 chemical components in 3 kinds of Rhodiola rosea by HPLC.</a> China Pharmacy. 29 (18), 2515-2519 (2018).</li><li>Dong, T., Sha, Y., Liu, H., Sun, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altitudinal+variation+of+metabolites,+mineral+elements+and+antioxidant+activities+of+Rhodiola+crenulata+(Hook.f.+&amp;+Thomson)+H.Ohba.">Altitudinal variation of metabolites, mineral elements and antioxidant activities of Rhodiola crenulata (Hook.f. &amp; Thomson) H.Ohba.</a> Molecules. 26 (23), 7383 (2021).</li><li>Ma, D., 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=Application+of+UHPLC+fingerprints+combined+with+chemical+pattern+recognition+analysis+in+the+differentiation+of+six+Rhodiola+species.">Application of UHPLC fingerprints combined with chemical pattern recognition analysis in the differentiation of six Rhodiola species.</a> Molecules. 26 (22), 6855 (2021).</li><li>Li, X. H., 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=Metabolic+discrimination+of+different+Rhodiola+species+using+1H-NMR+and+GEP+combinational+chemometrics.">Metabolic discrimination of different Rhodiola species using 1H-NMR and GEP combinational chemometrics.</a> Chemical &amp; Pharmaceutical Bulletin. 67 (2), 81-87 (2019).</li><li>Liu, Y., 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+Hippophae+species+(Shaji)+through+DNA+barcodes.">Identification of Hippophae species (Shaji) through DNA barcodes.</a> Chinese Medicine. 10, 28 (2015).</li></ol>

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Existem mais de 90 espécies de plantas Rhodiola no mundo, e mais de 60% de todas as espécies são encontradas na China, incluindo Rhodiola crenulata, Rhodiola rosea, Rhodiolas achalinensis, e Rhodiola algida17. Rhodiola crenulata, registrada na primeira parte da Farmacopeia Chinesa (2020), é um medicamento tradicional tibetano cultivado em grandes altitudes. A demanda do mercado para Rhodiola crenulata está aumentando anualmente, portanto, garantir...

A fitoterapia tem uma longa história e rica experiência de aplicação na China, e foi o primeiro registro sistemático no clássico herbal de Shennong1. A descoberta da artemisinina aplicada à malária promoveu o desenvolvimento da fitoterapia em uma nova etapa1. O uso de tecnologia científica moderna para desvendar o mecanismo exato da fitoterapia aumenta a taxa de utilização e a demanda por fitoterápicos, abrindo um novo mercado internacional para amesma2,3,4. No entanto, isso levou a uma série de efeitos negativos. Os não-profissionais têm uma compreensão vaga das características da fitoterapia, o que faz com que o uso de fitoterápicos enfrente um enorme risco de segurança5.
Rhodiola crenulata, uma das plantas da espécie Rhodiola, distribui-se principalmente no Tibete, noroeste de Yunnan e oeste de Sichuan da China (Figura 1)6,7. Rhodiola crenulata compreende salidrósido, tirosol, ácido gálico e outros compostos para o tratamento de doenças relacionadas à hipóxia através da função de "revigorar o qi e promover a circulação sanguínea, limpar o pulso e acalmar a asma"8,9,10,11. A investigação de campo mostra que Rhodiola crenulata pode ser encontrada nas zonas de talus alpino, encostas de voçorocas e fendas rochosas a uma altitude de 4.000-5.600 m. Seu ambiente de crescimento é frio, cheio de sol e intensa radiação, e pertence ao ecossistema de prados alpinos. Rhodiola crenulata pode ser distribuída em populações lamelares e pontiagudas de acordo com o terreno de crescimento, e o fluxo gênico pode ser realizado através da polinização cruzada.
O aborto polínico do gênero Rhodiola, a escavação ilegal e o ambiente ecológico degenerado fazem da Rhodiola crenulata uma espécie ameaçada de extinção 6,12. Tendo em vista o alto valor medicinal da Rhodiola crenulata, espera-se que os produtos falsificados fluam para o mercado. Este artigo apresenta o habitat de Rhodiola crenulata e alguns métodos convenientes de identificação laboratorial. Primeiramente, observamos o ambiente de crescimento da Rhodiola crenulata e suas propriedades medicinais. Em segundo lugar, a microestrutura do pó medicinal foi observada ao microscópio. O último passo é o ponto-chave. Os componentes representativos de Rhodiola crenulata foram separados e identificados de acordo com as diferentes propriedades de adsorção ou dissolução desses componentes em uma determinada substância. Métodos de autenticação baseados em DNA ou análise metabolômica de plantas medicinais são complicados e caros13. Estes métodos básicos, convenientes, e econômicos podem identificar rapidamente Rhodiola crenulata.

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			<td>ATS 4</td>
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			<td>Chloral hydrate</td>
			<td>Fuzhou Brunei Technology Co., Ltd</td>
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			<td>Zhejiang ALWSCI Technology Co., Ltd</td>
			<td>C0000008</td>
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			<td>Conical flask</td>
			<td>Sichuan Shubo Co., Ltd</td>
			<td>1121</td>
			<td></td>
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			<td>Cover glass</td>
			<td>Citotest Labware Manufacturing Co., Ltd</td>
			<td>10211818c</td>
			<td></td>
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			<td>Dissecting needle</td>
			<td>Shanghai Bingyu Fluid Technology Co., Ltd</td>
			<td>BY-5026</td>
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			<td>Electronic balance</td>
			<td>SHIMADZU</td>
			<td>ATX124</td>
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			<td>Chengdu Kelong Chemical Co., Ltd</td>
			<td>2022120901</td>
			<td></td>
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			<td>Formic acid</td>
			<td>Chengdu Kelong Chemical Co., Ltd</td>
			<td>2021110801</td>
			<td></td>
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			<td>Gallic acid</td>
			<td>Chengdu Herbpurify Co., Ltd</td>
			<td>M-017</td>
			<td></td>
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			<td>Glycerol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10010618</td>
			<td></td>
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			<td>High speed&#160; crusher</td>
			<td>Beijing Zhongxingweiye Instrument Co., Ltd</td>
			<td>FW-100</td>
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			<td>Methanol</td>
			<td>Chengdu Kelong Chemical Co., Ltd</td>
			<td>20230108</td>
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			<td>Microscope</td>
			<td>Chongqing Oprec Nistrument Co.,&#160; Ltd</td>
			<td>B203</td>
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			<td>Microscope slide</td>
			<td>Citotest Labware Manufacturing Co., Ltd</td>
			<td>7105P-G</td>
			<td></td>
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			<td>Oven</td>
			<td>Shanghai Yuejin Medical Equipment Co., Ltd</td>
			<td>DHG-8145</td>
			<td></td>
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			<td>Pharmacopoeia sieve</td>
			<td>Hangzhou Xingrun sieve factory</td>
			<td>572423281330</td>
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			<td>Pipette</td>
			<td>Changde BKMAM Biotechnology Co., Ltd</td>
			<td>120302008</td>
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			<td>Salidroside</td>
			<td>Chengdu Herbpurify Co., Ltd</td>
			<td>H-040</td>
			<td></td>
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			<td>Saturate tank&#160;</td>
			<td>Yancheng Liegu Technology Co., Ltd</td>
			<td>10*20 P-1</td>
			<td></td>
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			<td>Silica gel plate</td>
			<td>Yantai Jiangyou Silica Gel Development&#160; Co., Ltd</td>
			<td>HSG20211227</td>
			<td></td>
		</tr>
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			<td>Trichloromethane</td>
			<td>Chengdu Kelong Chemical Co., Ltd</td>
			<td>20221013-1</td>
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			<td>Tweezer&#160;</td>
			<td>Changde BKMAM Biotechnology Co., Ltd</td>
			<td>130302027</td>
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			<td>Tyrosol</td>
			<td>Chengdu Herbpurify Co., Ltd</td>
			<td>L-042</td>
			<td></td>
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			<td>Ultrasound equipment</td>
			<td>Ningbo Xinyi Ultrasonic Equipment Co., Ltd</td>
			<td>SB-8200DTS</td>
			<td></td>
		</tr>
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			<td>Volumetric pipet</td>
			<td>Changde BKMAM Biotechnology Co., Ltd</td>
			<td>120301006</td>
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field collection and laboratory routine identification of rhodiola crenulata

A identificação de materiais medicinais é premissa e garantia da segurança medicamentosa. A maioria dos pesquisadores científicos é obrigada a favorecer o processo de identificação simples, rápido, eficaz e barato de ervas. Rhodiola crenulata é um medicamento tradicional tibetano cultivado em grandes altitudes, distribuído principalmente no Tibete, Yunnan, e Sichuan regiões da China. Crenulato de Rhodiola possui múltiplas bioatividades, tais como anti-inflamatório, anti-hipóxia, e propriedades antioxidantes, e tem grande potencial para o desenvolvimento. Com a crescente demanda do mercado e uma rápida diminuição no conteúdo de recursos, um grande número de produtos confusos de Rhodiola crenulata têm incomodado as pessoas. Portanto, este protocolo introduz um processo padrão para a identificação de Rhodiola crenulata no campo combinado com testes laboratoriais de rotina. A combinação de habitat, características microscópicas e cromatografia em camada fina sem dúvida identificará Rhodiola crenulata de forma rápida, eficiente e econômica, contribuindo para o desenvolvimento contínuo da medicina tibetana e o controle de qualidade de materiais medicinais.

Este protocolo experimental descreve a identificação e coleta de crenulato de Rhodiola no campo. Rhodiola crenulate tende a viver nas zonas de talus alpino, encostas de voçorocas, e fendas rochosas em grandes altitudes. O habitat, planta inteira, flor e folhas de Rhodiola crenulate podem ser mostrados na Figura 2. O crenulado de Rhodiola tem um rizoma marrom-avermelhado (Figura 3A). Uma imagem representativa do pó medicina...

Watch this Scientific Journal Video about Field Collection and Laboratory Routine Identification of Rhodiola crenulata at JoVE.com

Autor em destaque: Desenvolvimento de métodos de identificação para Rhodiola crenulata e investigação de sua distribuição de recursos e efeitos farmacológicos

Aqui, descrevemos a identificação de Rhodiola Crenulata a partir de habitat, morfologia vegetal, propriedades medicinais, características microscópicas e cromatografia em camada delgada.

Coleta de campo e identificação de rotina laboratorial de Rhodiola crenulata

The identification of medicinal materials is the premise and guarantee of drug safety. The majority of scientific researchers are bound to favor the ...

O escopo de nossa pesquisa está centrado na realização de um levantamento de recursos da medicina étnica. Especificamente, este estudo visa desenvolver métodos rápidos e convenientes para a identificação de Rhodiola crenulata. O protocolo que oferecemos apresenta um método econômico, direto e rápido para identificar vários materiais fitoterápicos medicinais.Nosso laboratório concentrará seus futuros esforços de pesquisa na investigação de duas áreas principais, a distribuição da pesquisa associada com Rhodiola crenulata e os impactos farmacológicos desta espécie.

field-collection-laboratory-routine-identification-rhodiola

Research

JoVE Journal

Medicine

Field Collection and Laboratory Routine Identification of Rhodiola crenulata

482 visualizações.  Chengdu University of Traditional Chinese Medicine. O escopo de nossa pesquisa está centrado na realização de um levantamento de recursos da medicina étnica. Especificamente, este estudo visa desenvolver métodos rápidos e convenientes para a identificação de Rhodiola crenulata. O protocolo que oferecemos apresenta um método econômico, direto e rápido para identificar vários materiais fitoterápicos medicinais.Nosso laboratório concentrará seus futuros esforços de pesquisa na investigação de duas áreas principais, a dis...

Vídeo: Autor em destaque: Desenvolvimento de métodos de identificação para Rhodiola crenulata e investigação de sua distribuição de recursos e efeitos farmacológicos

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

Protocolo de Coleta para Pâncreas Humano

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Medicina

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

A utilização de recursos primários fibroblastos humanos de Monitoramento Fenótipos mitocondriais no Campo da Doença de Parkinson

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Localization, Identification, and Excision of Murine Adipose Depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

Localização, identificação e excisão de Depots Murino adiposo

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

Methodology for Sputum Induction and Laboratory Processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which is interesting in various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis. This technique is well tolerated, safe and non-invasive, but is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff. The success rate of sputum induction and analysis is about 80%.
Here, we describe the induction and laboratory processing of sputum samples. Sputum is induced by inhalation of hypertonic or isotonic saline with salbutamol. For the processing, we use the whole sputum technique. Dithiothreitol (DTT) is used to allow mucolysis of sputum samples. The primary aim of sputum processing is to obtain a differential cell count to study the cell types present in the airway lumen. Additional analyses may also be performed on sputum supernatant and sputum cells, which may allow further investigation into inflammatory processes and immune mechanisms. Examples include studying mediators in sputum supernatant and performing a large spectrum of analysis on sputum cells such as flow cytometry, genomics, or proteomics.
Finally, representative results of sputum analysis in healthy controls, asthmatics, and COPD patients are presented.

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

Metodologia para a indução de escarro e laboratório de processamento

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...

Routine Screening Method for Microparticles in Platelet Transfusions

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood banks. Cells fragment off microparticles (MP) when they are stressed. Blood and blood components may contain cellular fragments from a variety of cells, most notably from activated platelets. When performing their roles as innate immune cells and major players in coagulation and hemostasis, platelets change shape and generate microparticles. With dynamic light scattering (DLS)-based microparticle detection, it is possible to differentiate activated (high microparticle) from non-activated (low microparticle) platelets in transfusions, and optimize the use of this scarce blood product. Previous research suggests that providing non-activated platelets for prophylactic use in hematology-oncology patients could reduce their risk of becoming refractory and improve patient care. The goal of this screening method is to routinely differentiate activated from non-activated platelets. The method described here outlines the steps to be performed for routine platelet inventory management in a hospital blood bank: obtaining a sample from a platelet transfusion, loading the sample into the capillary for DLS measurement, performing the DLS test to identify microparticles, and using the reported microparticle content to identify activated platelets.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative in hospital blood banks. The goal is to differentiate activated from non-activated platelets to optimize the use of platelets. Providing non-activated platelets to hematology-oncology patients might reduce their high risk to become refractory.

Método de triagem para micropartículas em transfusões de plaquetas de rotina

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood ...

Remote Laboratory Management: Respiratory Virus Diagnostics

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a multitude of issues ranging from transportation, access, facilities, equipment, and communication to provider training. To address this need, we have developed an innovative, scalable, logistics-enhanced, mobile, laboratory facility for emergencies and epidemics in resource-constrained global settings. Utilizing a background in clinical operations as an academic medical center, we designed a rapidly-deployable, modular BSL-2 and BSL-3 facility with user-friendly software for tracking and management of drugs and supplies in remote regions during epidemics and outbreaks. Here, we present our intermodal, mobile, expandable shipping-container laboratory units. The design of the laboratory facilitates off-grid usage by minimizing power consumption and allowing alternate water sources. The unit's information communication technology (ICT) platform provides (i) user-friendly tablet-based documentation, (ii) enhanced tracking of patients and supplies, and (iii) integrated communication onsite with built-in telehealth capabilities. To ensure quality in remote environments, we have developed a checklist for a basic laboratory workflow and a protocol for respiratory viral diagnosis using reverse-transcription polymerase chain reaction (RT-PCR). As described, this innovative and comprehensive approach allows for the provision of laboratory capability in resource-limited global environments.

A rapidly-deployable, off-grid laboratory has been designed and built for remote, resource-constrained global settings. The features and critical aspects of the logistically-enhanced, expandable, multifunctional laboratory modules are explored. A checklist for a basic laboratory workflow and a protocol for a respiratory viral diagnostic test are developed and presented.

Gerenciamento remoto do laboratório: Diagnósticos de vírus respiratório

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a ...

Learning Modern Laryngeal Surgery in a Dissection Laboratory

Surgery for laryngeal malignancies requires millimetric accuracy from the different endoscopic and open techniques available. Practice of this surgery is almost completely reserved to a few referral centers that deal with a large proportion of this pathology. Practice on human specimens is not always possible for ethical, economic, or availability reasons. The aim of this study is to provide a reproducible method for the organization of a laryngeal laboratory on ex vivo animal models where it is possible to approach, learn, and refine laryngeal techniques. Porcine and ovine larynges are ideal, affordable, models to simulate laryngeal surgery given their similarity to the human larynx in their anatomical layout and tissue composition. Herein, the surgical steps of transoral laser surgery, open partial horizontal laryngectomy, and total laryngectomy are reported. The merging of endoscopic and exoscopic views guarantees an inside-out perspective, which is vital for the comprehension of the complex laryngeal anatomy. The method was successfully adopted during three sessions of a dissection course &#34;Lary-Gym&#34;. Further perspectives on robotic surgical training are described.

The purpose of this paper is to illustrate how to organize a reproducible laboratory for laryngeal surgery on affordable and closely similar animal laryngeal models in order to improve anatomical and surgical knowledge and skills.

Aprendendo cirurgia laríngea moderna em um laboratório de dissecção

Surgery for laryngeal malignancies requires millimetric accuracy from the different endoscopic and open techniques available. Practice of this surgery is ...

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.

Uma determinação de dissolução in vitro de componentes multiíndices em grânulos de Rhodiola da medicina tibetana

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

Ferric Chloride-Induced Arterial Thrombosis and Sample Collection for 3D Electron Microscopy Analysis

Cardiovascular diseases are a leading cause of mortality and morbidity worldwide. Aberrant thrombosis is a common feature of systemic conditions like diabetes and obesity, and chronic inflammatory diseases like atherosclerosis, cancer, and autoimmune diseases. Upon vascular injury, usually the coagulation system, platelets, and endothelium act in an orchestrated manner to prevent bleeding by forming a clot at the site of the injury. Abnormalities in this process lead to either excessive bleeding or uncontrolled thrombosis/insufficient antithrombotic activity, which translates into vessel occlusion and its sequelae. The FeCl3-induced carotid injury model is a valuable tool in probing how thrombosis initiates and progresses in vivo. This model involves endothelial damage/denudation and subsequent clot formation at the injured site. It provides a highly sensitive, quantitative assay to monitor vascular damage and clot formation in response to different degrees of vascular damage. Once optimized, this standard technique can be used to study the molecular mechanisms underlying thrombosis, as well as the ultrastructural changes in platelets in a growing thrombus. This assay is also useful to study the efficacy of antithrombotic and antiplatelet agents. This article explains how to initiate and monitor FeCl3-induced arterial thrombosis and how to collect samples for analysis by electron microscopy.

The present protocol describes how to use a FeCl3-mediated injury to induce arterial thrombosis, and how to collect and prepare arterial injury samples at various stages of thrombosis for electron microscopy analysis.

Trombose Arterial Induzida por Cloreto Férrico e Coleta de Amostras para Análise de Microscopia Eletrônica 3D

Cardiovascular diseases are a leading cause of mortality and morbidity worldwide. Aberrant thrombosis is a common feature of systemic conditions like ...

Um protocolo para isolar, cultivar e identificar células de nicho límbico humano

Isolation and Identification of Limbal Niche Cells

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used for LNCs isolation. The tissue was divided into 12 pieces under aseptic conditions and digested for 18 h at 37 &#176;C in the cell culture incubator using collagenase A to obtain cell clusters with LNCs and limbal&#160;epithelial progenitor cells. The cell clusters were further digested for 15 min at 37 &#176;C using 0.25% trypsin-EDTA to obtain single cells and then cultured in modified embryonic stem cell medium (MESCM) on a plastic surface coated with 5% Matrigel. Cells were passaged upon 70% confluence, and LNCs were identified using immunofluorescence, real-time quantitative PCR (qPCR), and flow cytometry. Primary LNCs were isolated and passaged more than 12 times. The proliferation activity of LNCs from P4 to P6 was the highest. LNCs expressed higher stem cell markers than BMMSCs (SCF, Nestin, Rex1, SSEA4, CD73, CD90, MSX1, P75NTR, and PDGFR&#946;). Furthermore, results showed that P4 LNCs uniformly expressed VIM, CD90, CD105, and PDGFR&#946;, but not Pan-CK, which could be used as a marker for the identification of LNCs. Flow cytometric analysis showed that approximately 95%, 97%, 92%, and 11% of LNCs expressed CD73, CD90, CD105, and SCF respectively, while they were&#160;68%, 99%, 20%, and 3% in&#160;BMMSCs. The standard process for LNC&#160;isolation and identification could provide&#160;a reliable laboratory basis for the widespread use of LNCs.

Autor em destaque: Padronizando o isolamento e a caracterização de células de nicho límbico (LNC) para apoiar a pesquisa generalizada de LNC 

Here, we present a protocol to isolate and identify the human limbal niche cells.

Isolamento e Identificação de Células de Nicho Limbal

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used ...