Science topic

Plant Extracts - Science topic

Concentrated pharmaceutical preparations of plants obtained by removing active constituents with a suitable solvent, which is evaporated away, and adjusting the residue to a prescribed standard.
Questions related to Plant Extracts
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Which is the standard cell lines used for checking cytotoxicity activity of medicinal plant extract ?
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there are no such standardization for cytotoxicity assay by any diffrent assay like MTT,MMT or XXT nd it depends on ur aim of experiment nd its objective like if working with breast cancer thn use std. Hela or SiHa nd ny one could help u more if u make clear wid ur wrking objective or for general checking use fibroblast or CHO line....best of luk
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To check the chemical behaviour of nickel with plant extracts.
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Trigonelline has been shown to form a few coordination complexes with Cu2+, and chloride, and it's possible that a similar reaction could occur with Ni2+.
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I have a plant extract which needs to be concentrated and I do not have lyophilizer. Can anyone please suggest me any conventional method.
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With a reference link.
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The method by Kooy et al. (1994) have been used by several researchers already. So I think it is an effective method.
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I would like to investigate brassinosteroid activity of crude plant extracts but cannot reach the original paper of Wada et al 1981. Does somebody have experience with this assay? Is there some other assay for detection of brassinosteroid activity that does not require dwarf mutants? I have an experience with extraction and physiological activity detection of other plant hormones, but have never worked with brassinosteroids. What solvent is the best for brassinosteroid extraction from plant tissues (grown in vitro)?
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you will find you answer in the paper that has the following link
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Can anyone explain the exact procedure of fluorescent analysis of a plant extract and its requirements?
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I think that you need to be more specific of what you are trying to do. There are a lot of different fluorescent pigment containing complexes in plants.
What are you trying to do exactly?
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I research biofuels from different plants, and I am looking for the best method for oil extraction from plants (fresh and dry). Any suggestions?
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Fatty oil of biofuel interest can be extracted by using non-polar organic solvents like n-Hexane, petroleum ether & toluene. Cost involved for extractant and final recovery of fatty oil should also be taken into account.. Considering the two issues n-hexane appears to be the most appropriate.
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There are many solvents like water, acetone, ethanol, methanol. In which is the best one for the clinical applications and nanoparticles synthesis?
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Water is the best solvent as it is also very compatible and non-reactive.
Check the following book chapter where a clear differentiation of the role of various solvents based on polarity in extraction for NP synthesis is explained.
"M. Sathishkumar, A. Mahadevan, S. Pavagadhi, R. Kaushik, V. Sharma, R.
Balasubramanian, 2013. Biological synthesis of silver nanoparticles and
assessment of their bactericidal activity, In book: Green Sustainable
Nanotechnology and the Environment, American Chemical Society, USA."
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I'm looking for accredited lab/company providing PT for bioactive compounds in plant extracts and/or herbal volatile oil. I'm interested in Geraniol, Vincristin, Allicin
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The Central Institute of Medicinal and Aromatic Plants, Lucknow, India is a Government funded National laboratory working on all aspects of medicinal and aromatic plants. The Institute has well-equipped laboratories to analyse plant samples, volatile oils for their composition. You may visit the Institute's website: www.cimap.res.in for full details about the activities of the Institute. For sample analysis, contact the Institute's director: director@cimap.res.in.
I hope this information is useful to you.
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I know there are obstacles in antimicrobial peptides but can't find anything about Fabatins specifically.
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Ok, Is that a probability or sure . I noticed that the article published on 1997 .
Just I want to be sure it isn't refused as I want to work on Fabatins .
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I have prepared TLC plates using Silica gel G (Merk). I could not even get the surface. Can anyone help me?
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you have the applicator to make plates? cause after making the plates you need to put them @ 100 degree Celsius for two hours to activate them.
you need something like this.. more info can be found in this link.
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Can anyone tell me about the crystal growth procedure from plant extracts?
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Prof. Ruben D. Torrenegra G. rtorrenegra@udca.edu.co
PURIFICACIÓN, ETAPAS
Introducción.
Mucho se ha hecho para llegar a la purificación de una sustancia a partir de mezclas complejas( extractos) y esto requiere de hacer un proceso de varias pasos o etapas que incluyen : fraccionamiento por polaridades, aislamiento por extracciones sólido – líquido o líquido-líquido, según el caso; separación en mezclas de pocos compuestos por cromatografías preparativas y por último sublimación o cristalización.
La purificación de una sustancia, que se podría considerar un arte, requiere de la habilidad práctica y conceptual del químico o el analista. Cada caso puede necesitar una estrategia diferente que depende de los constituyentes de la mezcla, si hay mezclas de sustancias orgánicas e inorgánicas o son sustancias de una misma naturaleza, son compuestos de una serie homóloga, de qué función química, etc; lo anterior necesita que se conozca sobre la composición y complejidad de la mezcla y para ello se puede iniciar con una cromatografía en capa delgada (CCD) en varias fases y con varios solventes.
QUE ES PUREZA?
Se considera pura una sustancia que genere los mismos datos al determinársele sus propiedades físicas, químicas y espectroscópicas, dentro de los rangos establecidos; por ejemplo punto de fusión dentro de un rango menor de dos grados, CCD o HPTLC de una sola mancha en varios sistemas de solventes, un solo pico en cromatografía de gases (CG) o cromatografía líquida de alta resolución (HPLC) en condiciones óptimas de separación.
El grado de pureza, porcentaje de la sustancia en la muestra, que se necesita que tenga una sustancia depende del experimento que con ella se requiera hacer, lo ideal sería 100%, pero en algunos casos un 80% de pureza puede generar el dato que se necesita en un experimento. Para la determinación de las constantes físicas y la actividad biológica es mejor una alta pureza, mayor del 98%. La definición de “puro” es más o menos arbitraria, decir que el proceso de purificación es “completo” no significa que la sustancia se encuentre totalmente libre de impurezas de otros compuestos, sino que ellos se encuentran en un nivel aceptable y que no producirán interferencias en los resultados del experimento que se deba hacer.
ELIMINACION DE SALES
Los metabolitos secundarios que se requieren purificar tienen comúnmente varios cientos de masa molecular y pueden ser separados de sales inorgánicas y macromoléculas por cromatografía de permeación en gel, con geles adecuados de tamaño de poro ( 10, 20, por ej.) los componentes de masa molecular mas grandes eluyen primero y los componentes inorgánicos, muy pequeños, son eluídos mucho mas tarde y podrían ser retenidos en la columna, de tal manera que las sustancias a purificar saldrían en las fracciones intermedias.
Las sustancias iónicas pueden ser retenidas en resinas de intercambio iónicas aniónicas y catiónicas. Sustancias de muy baja polaridad relativa también pueden ser eliminadas por una cromatografía con RP-18 ya que ésta fase estacionaria retiene fuertemente las sustancias apolares. Como es de entender se deben seleccionar las fases móviles adecuadas.
EXTRACCIÓN LÍQUIDO LÍQUIDO
Los extractos de productos naturales orgánicos, la mayoría de las veces, se hacen con solventes polares que extraen casi todos los componentes del material que se estudia. La concentración de estos extractos podrían generar extractos no totalmente secos que deben ser redisueltos en mezclas hidro-etanólicas (p.ej 50% v/v etanol o metanol: agua) para luego fraccionarlo por extracción líquido-líquido con solventes de baja y mediana polaridad inmiscibles con agua.
Estas extracciones permiten separar compuestos de acuerdo a su coeficiente de reparto Kd; en la fase hidrofílica o la fase lipofílica dependiendo de su afinidad por esas fases. Los compuestos de menor polaridad quedarán disueltos en la fase lipofílica y los de mayor polaridad en la fase hidrofílica. Los solventes lipofílicos recomendados son Petrol, éter etílico, acetato de etilo que son menos densos que el agua y cloroformo o diclorometano que son mas densos que el agua. Un fraccionamiento secuencial podría iniciarse con petrol, continuar con cloroformo y
será rica en los compuestos de alta polaridad. La extracción se puede hacer en un sistema continuo l-l o con embudos de decantación, en este último caso se deben usar volúmenes de extracción pequeños en varias etapas que un volumen grande en una sola etapa; por ejemplo extraer cuatro veces con 25ml que una sola vez con 100ml.
EXTRACCION SOLIDO LIQUIDO.
Si un extracto se ha podido llevar a sequedad es posible someterlo extracción sólido liquido con solventes de polaridad creciente, desde petrol o hexano hasta etanol. Los compuestos se separarán dependiendo de su solubilidad, aunque la polaridad del solvente varia con los solutos que se extraen y la especificidad varia no solo con la polaridad del solvente puro. La extracción sólido líquido se puede llevar a cabo con el extracto adsorbido sobre una fase estacionaria, como silica gel (60-230mm) o RP 18, en cada caso los solventes se deben seleccionar según la fase escogida. Para RP18 se debe iniciar con mezclas hidrofílcas y terminar con diclorometano. Este último proceso se denomina fraccionamiento por percolación y se desarrolla en una columna.
SECADO
De las extracciones o purificaciones las sustancias se obtienen en solución por lo que es necesario secarlas eliminando el solvente. Esto que puede ser obvio se justifica por:
-Estabilidad física y química, el compuesto o compuestos son mas estables secos que en solución.
-Rendimiento, para poder determinarla en necesario obtener el peso del compuesto.
-Espectros, algunas veces el proceso de purificación es seguido o monitoreado por alguna técnica espectroscópica y el agua u otro solvente interfieren en la toma del espectro.
Varios son los métodos de secado que se pueden aplicar al compuesto a nivel de laboratorio y su escogencia depende de lo que se pretenda hacer con la muestra sólida: a) con gas inerte (N2), el cual se aplica en frio o en caliente sobre la superficie o el interior de la solución desplazándose el equilibrio liq-vap hacia la evaporación. El sólido queda como una película adherida a la superficie del recipiente que contenía la solución que es difícil de manipular. b) Evaporación en un rota evaporador, se puede recuperar el solvente por destilación pero persiste la desventaja de la formación de una película de sólido. c) Secado al vacío, este proceso es similar al anterior sólo que se aplica vacio reduciéndose la temperatura de ebullición y protegiéndose así los compuestos termolábiles. d) centrifugación a vacío, permite situar el sólido obtenido en el fondo del recipiente y trabajar a bajas temperaturas, es útil para secar relativamente grandes cantidades de sustancia. d) Secado en congelación o liofilización, como se lleva a alto vacio y en congelación, involucra procesos de sublimación del agua, el hielo se evapora y condensa a parte en un tubo a temperaturas muy bajas, -70ºC. Debido a las bajas temperaturas las sustancias lábiles no sufren daños en su estrutura y terminan como sólidos porosos listos para ser redisueltos o suspendidos en cualquier líquido.
CRISTALIZACION
Los procesos de cristalización pretenden obtener sólidos puros con alguna estructura cristalina definida y ordenada. Los cristales se formarán a partir de soluciones saturadas en las que las moléculas del compuesto menos soluble se organizan como un sólido según su estructura y propiedades moleculares.
La cristalinidad es sinónimo de orden y es éste el que permite reconocer el material cristalino por sus propiedades tales como el punto de fusión, la difracción de Rx y la presencia de superficies planas ordenadas (caras) con lados rectos. El término policristalino se refiere a agregados cristalinos muy pequeños difíciles de observar a ojo. Se deben obtener cristales porque una unidad cristalina permite ver la disposición de los átomos en un espacio tridimensional usando la difracción de Rx, lo que es mas directo que la RMN para la obtención de la configuración molecular. La difracción de Rx permite determinar, confirmar o completar estructuras moleculares de sustancias que presenten dudas y poder establecer la conformación molecular y su estereoquímica.
OBTENCION DE CRISTALES
Los cristales de productos naturales se obtiene a partir de soluciones y los procesos pueden ser descritos como aquellos en los cuales:
1- Una solución saturada de uno o mas compuestos de interés llega a ser sobresaturada.
2- Ocurre una nucleación y el cristal comienza a crecer.
La cristalización es un proceso de colisiones, las moléculas chocan formando un cluster llamado núcleo a partir del cual se desarrolla el cristal con una estructura interna característica y una forma externa definida. Las colisiones o choques moleculares son afectadas por factores como agitación y grado de sobresaturación, la velocidad de sobresaturación se ve afectada por la evaporación del solvente y por ende la temperatura.
Un primer paso para llevar a cabo la cristalización de un compuesto es la escogencia del solvente en el que no debe ser ni muy soluble o insoluble. El solvente puede ser cualquiera de la serie eluotrópica desde el hexano o petrol hasta el metanol o etanol, pero siempre de alta pureza. Debe ser volátil y miscible con otros solventes que permitan cambiar su polaridad de tal manera que en la mezcla, la solubilidad del compuesto disminuya para llegar a la sobresaturación.
La solución sobresaturada se prepara dependiendo de la cantidad de sustancia con que se cuente, si se tiene cantidad suficiente del compuesto se toma un volumen del solvente seleccionado y se le agrega el compuesto hasta que no se disuelva mas. Si se cuenta con poca cantidad de soluto, esta se disuelve en el solvente y luego se le adiciona gota a gota otro solvente en el que no es soluble el soluto, hasta que la solución se torne turbia, momento en el cual la solución está sobresaturada. Una solución saturada llega a sobre saturarse por enfriamiento, puesto que se disminuye la solubilidad o por evaporación del solvente ya que se aumenta la concentración del soluto.
La evaporación se produce dejando la solución abierta a la atmosfera a temperatura ambiente. Se reduce la velocidad de evaporación cubriendo la solución con papel de aluminio o se aumenta pasando una corriente de nitrógeno sobre ella.
La temperatura juega un papel importante en la cristalización, la energía cinética de las moléculas aumenta con ella y el empaquetamiento para formar la red cristalina también. La solubilidad de moléculas pequeñas de metabolitos secundarios se ve afectada por la temperatura, comúnmente disminuye al decrecer la temperatura , es así que controlando la rata de enfriamiento en la solución de la sustancia que se quiere cristalizar, se controla la velocidad con que se alcanza la sobresaturación, la rata de nucleación y la rata de crecimiento del cristal. La ratas de enfriamiento demasiado rápidas permiten la formación de muchos microcristales debido al aumento de núcleos de cristalización. La rata de enfriamiento se controla con b año de agua fría. Si el control del enfriamiento no es exitoso para la obtención de” buenos” cristales se puede controlar la sobresaturación por difusión de vapor del solvente, para ello se introduce el recipiente con la solución en otro recipiente que contenga un solvente altamente miscible con el de la solución a cristalizar, por ejemplo si la solución está en acetona el solvente externo puede ser agua. Como es lógico el solvente externo debe ser menos volátil que el de la solución, como se muestra en el caso 1 (acetona –agua), en el caso 2 es al contrario y en el solvente externo el compuesto a cristalizar no debe ser soluble ( acetona- petrol), como se ve en la figura siguiente. En diagrama 2 se ejemplariza la cristalización en capilar, para ello se estira un capilar de vidrio y se deja evaporar la gota de solución dentro de un recipiente adecuado. En la figura 3 se usa un cristal madre que se introduce en la solución saturada y este cristal semilla se deja crecer.
1.Cristalización por difusión
2.Cristalización por evaporación en la gota capilar
3. Cristalización por crecimiento con un cristal semilla.
CRISTALIZACIÓN PARA PURIFICACIÓN DE COMPUESTOS.
El proceso de purificación por cristalización se conoce como cristalización fraccionada, en el que las distintas sustancias que componen las mezcla tienen diferentes solubilidades y por ello alcanzan la sobre saturación en distintos momentos y cristalizan en tiempos diferentes. Si los componentes son solubles, unos en frio y otros en caliente, se utiliza esta particularidad para separarlos por filtración. La escogencia del solvente es un paso crítico en este proceso. Cada vez que se obtienen cristales se deben ir separando por decantación y los líquidos madres deben dejarse cristalizar nuevamente y repetirse este proceso hasta cuando todos los componentes se hayan cristalizado. Por lo general, en la cristalización fraccionada se obtienen cristales impurificados con los componentes de solubilidades cercanas. En este caso ellos se recristalizarían en el mismo u otro solvente mas adecuado.
Por ejemplo en una mezcla de A+B+C se debe escoger un solvente que disuelva a B y C a cualquier temperatura y no a A; el enfriamiento producirá cristales de A con poca impureza de B y C. Se repite el proceso con A impuro y con solvente puro hasta obtener A “libre” de B y C. Como se debe entender esta cristalización no garantiza que obtenga un compuesto puro en un solo paso.
En este proceso se suele perder algo del compuesto ya que este sólo cristaliza en sobresaturación. Si se cuenta con cantidades mayores de 80 o 100mg es mejor usar métodos cromatográficos.
La pericia y conocimiento del analista son clave para diseñar y seguir estrategias exitosas, la selección del solvente, el calentamiento y enfriamiento, la decantación o filtración en el momento justo, son etapas que se cumplen con atención y control del proceso.
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Methanol quality and extraction process (cold and hot extraction).
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I think that cold extraction is better than hot one because the thermal degradation of some compounds. The methanol grade depends on the type of analyses but the more pure the best, at least reagent grade is correct. If you are going to perform MS or HPLC analyses the grade must to be MS or HPLC.
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I am working on isolation, purification and characterization of biomolecules from leaf extracts. I have methanolic extract of leaf and I want to remove the chlorophyll from it first.
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Extract with carbon tetrachloride. It is the best solvent for removing chlorophyll. Also, being highly non-polar, it will not remove secondary metabolites from an extract which is methanolic.
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I am following protocols using spectrophotometer.
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You do not get a colour since the conversion of xanthine to uric acid can be followed using UV/VIS - try phosphate buffer pH 7.4 and measure at 298 nm - check p. 68 from http://ufdcimages.uflib.ufl.edu/UF/E0/02/17/81/00001/affum_a.pdf
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Could someone please suggest a method for metabolites profiling of plant extract through GCMS (aglient 5975c series)? Also, which part of the column would be suitable for plant extract?
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Can i inject crude extract for plant sample directly for GC?
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I'm looking for an anti-fungal agent for a macerate in which water is the solvent. After several days initiating maceration I start to get hyphae covering the surface.
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Few drops of toluene may be used to check the fungal spores, provided your experiment is not for edible purposes. 
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I weighed 0.1 g of plant extract in 2ml methanol then I performed the total antioxidant test by the phosphomolebdenum method, I have plotted a st curve using ascorbic acid now I want to know how to convert the read I get to an ascorbic acid equivalent?
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You cannot get the molecular weight of the extract, unless you know the identity or concentration of its active component. You can report the conc in terms of mg/ml instead of molar conc.
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I conducted water extraction on my plant and deduced that saponins are present. As far as I know, foam test and blood haemolysis test are generally used to detect saponins. Are these the only reliable tests? There are papers stating that saponins are biosurfactants, but are all saponins also biosurfactants? If this is true, tests such are drop collapse and emulsification stability would also indirectly prove the presence of saponins. Please advise.
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Dear Delhousie,
your approach is questionable because saponins are not the only foaming and interfacial tension lowering substances.
If you are after using the extract for a special purpose then best to use a related test.
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I am planning to add either a qualitative detection or quantification of cyanogenic glycosides on my research. Please help me find protocols and methods in doing such analyses. Depending on the availability of reagents on our lab, I will decide later on if I'll have to do quali or quanti. For qualitative analysis, I only find methods that use picrate paper. Is there another alternative? Thanks in advance.
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Perhaps (if you haven't tried yet) you could fractionate them by Flash chromatography and analyze by TLC or perhaps even better: VLC+TLC? I don't, however, know which other types of phytochemicals you have in your material: separation might be somewhat tricky to achieve. I wish you good luck, though!
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I tried Lowry, I am not getting consistent answer. I am trying Bradford now.
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I feels that both methods have its own demerits. So, it is better to try for Micro-Kjeldahl method. Any suggestions?
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I am working on three different types of tea(black tea,green tea and Rooibos tea). I want to check their anti-inflmamatory property...So,please suggest some assays for that.
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SRBC (Sheep Red Blood Cells) membrane stabilization method is a simple method to check anti-inflammatory activity.
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I am doing research in antidiabetic activity of plant extracts. I want to find the secondary metabolites by HPTLC analysis.
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This paper should help you for now, I guess. Will try to come up with a detailed answer shortly. :-)
Regards,
Subramaniyam
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I extracted plant material with ethanol for anticonvulsant activity. DMSO is used to dissolve the ethanolic extract. But ethanol itself has CNS stimulant activity. So please suggest a method by which we can calculate the amount of ethanol present in the ethanolic extract to confirm the convulsant activity of the plant extract.
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Hi. If I were you I would have gone for the simplest "loss on drying" method for the purpose. Simply weigh a portion of the extract, allow it to dry at optimum temperature (that does not adversely affect/ degrade your constituents but evaporates the ethanol completely) and weigh again.
Making sure 1) not to lose any solid particulate matter in the process 2) Having some backup of your material to be tested.
In this way you can get the mass and the percentage composition of the ethanol lost/ contained in your extract.
Hope that helps. :)
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I have to prepare the aqueous extract of fresh leaves for my study.
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Soxhlet extraction is meant to recycle the same solvent for extracting a given amount of crude drug. As most of the organic solvents are costly, Soxhlet extraction is preferred unless and until the phytoconstituents are not thermolabile. In that case also if there is proper control of heating process, it would be very useful. As water is available at ease, these process is not followed with water usually. Moreover the boiling point of water is high so it will also take a long time for the process to carry on effectively. It is always better to boil the drug with water or macerate it for 24 hours to get the aqueous extract. If you are afraid of loss of constituents by boiling you can skip boiling. Keeping the drug immersed in water with continuous stirring can also be useful. Depends a lot on the phytochemical nature of your drug.
Regards
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I could do the extraction with hot water and acidifying it with H2SO4 to yield a dark brown precipitate. I am confused with how to make it mono ammonium salt? also how to get the white MAG
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Does anybody know answer?
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I am trying to isolate the iridoid glycoside fraction from a root plant extract. The best option I think is methanol extraction but I would appreciate if some has some better suggestiosn.
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I took the absorbance of the extract and sample with procedures as given in the OYAIZU 1986 method (attached here). Is there any formula to calculate frap in this method? I carried out the AlCl3 method for flavonoids. What formula should I use? Anyone with ideas, please help me out!
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Respected Sir,
Please try with the following method by Eom et al., 2007.
Eom, S.H., Cheng, W.J., Hyoung, J.P. Kim, E.H., Chung, M.I., Kim, M.J., Yu, C.Y. and Cho, D.H. 2007. Far infrared ray irradiation stimulates antioxidant activity in Vitis flexuosa Thunb. Berries. Kor. J. Med. Crop Sci. 15: 319.323.
Estimation of Flavonoids
The total flavonoids content was determined according to Eom et al. (2007). An aliquot of 0.5 ml of sample (1 mg/ml) was mixed with 0.1 ml of 10% aluminium chloride and 0.1 ml of potassium acetate (1 M). In this mixture, 4.3 ml of 80% methanol was added to make 5 ml volume. This mixture was vortexed and the absorbance was measured spectrophotometrically at 415 nm.
Does storage at -80 degrees affect enzyme activity in plants?
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I am trying to obtain antioxidants and several other enzyme activities from plant materials which are preserved at - 80 degree for some time, but am unable to achieve fruitful results. Any suggestions?
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I do not know what would be a specific effect, if any, on the particular enzymes that you are handling, but in principle, -80 storage does not inactivate enzymes. I have used -80 stored plant material many times to isolate antimicrobial enzymes, and there is no problem. Of course, the storage should have used liquid nitrogen to begin with, and there should have been no interim thawing of the plant material. If this is ok, then you need to look at everything else more carefully: buffers, technique, reagents etc etc...in case you are not getting any enzyme activity, you need to look at the complete protocol, but if you are seeking a change in enzyme activity, like an increase, and not getting one, maybe your treatments do not induce a change!
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Mr. Ramesh. If you are interested in the colloidal particles,go for centrifugation and then decant the supernatant liquid. If you are interested in the filterate you use the filter aids in the funnel like bentonite,charcoal,silcagel etc.Instead of funnel you can even use a small column using the same filter aids.
I
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I've prepared my sample with the aid of ultrasonicator followed by centrifugation at 8000rpm for 10 mins. The sample was not as expected and didn't show any antibacterial effect. Can somebody recommend any other efficient procedure for analysing antibacterial properties?
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Dear Abhin
You have not mentioned the species of Origanum which a genus of Mint (Lamiaceae) family. The most common plant Origanum vulgare (Oregano) is a perennial herb, sometimes called wild marjoram, and its close relative Origanum majorana known as sweet marjoram. Oregano reported to be antiseptic as well as a cure for stomach and respiratory ailments. Due to a high content of phenolic acids and flavonoids, Oregano exhibit antioxidant activity. It has also shown antimicrobial activity against strains of the food-borne pathogen. Since your extracts in polar solvents (methanol & Water) are not showing any significant antibacterial activity, there is possibility of finding antimicrobial activity in the extracts derived from less polar or non-polar solvents. Being a plant of mint family, chance of the presence of terpenoid constituents in the plant are there. Terpenoids, which are normally extracted in less polar solvents are also reported to be antimicrobial. Hence, you may also attempt bioassay of pet. ether/benzene extracts of the plant.
Good Luck
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I'm planning to work with plant extracts and their antimicrobial activities. Apart from basic disc diffusion assay, MIC and MBC, what other important parameters or analysis I can use that would make my study stronger to justify and eventually leading to publication of a research paper?
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I very we'll agree with Junwei Zhu. Once the plant extracts are screened, the extracts can be chromatographed on TLC and if some promising, phenolics or alkaloids appear, they can be eluted and further checked using HPLC and structural elucidation can be done using NMR or XR-D
How to identify and quantify the known compound from plant samples?
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Which is the best method? Column chromatography or hplc?
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For identification and quantitation, HPLC is OK. In case of natural products, a preliminary TLC identification will be fine. But if your focus is on isolation of the compound, then I would suggest column chromatography and subsequent TLC and then HPTLC of the isolated compound with standard to quantify and confirm its identity.
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Kinetic study and/or total antioxidant activity study of antioxidants from different types of tea
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Hi John
Very good colleague of mine (First author) has a number of different assays for analysis and he has published only few in this article.
Vuong,, Q; V., Nguyen, V. V., Golding, J. B., & Roach, P. D. (February 2011). "The content of bioactive constituents as a quality index for Vietnamese teas.". International Food Research Journal, 18 (1).
How to isolate, purify and characterise bioactive compounds?
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Extraction
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You have to follow the following steps 1) Extraction (by Soxhlet extraction or by Closed Vessel microwave-Assisted Extraction system) 2) Removal of solvent by rotatory evaporator 3) Designing bioassays to evaluate the dose mortality response of the extracts 4) In case of encouraging results better to use solvents with different polarity. 5) Extraction and analysis of extracted effective extracts by GC-MS 6) Authentication of the major peaks by standards 7) Designing the dose mortality or bio assays of your interests of each peak against your target in order to evaluate the activity of each fraction. Best of Luck
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In my lab, hydroxyl radical scavenging activity of plant extract was checked based on Klein et al, 1992. but for us the absorbance value for samples increased more than the control.
My question is
1. why is the absorbance of the sample more? we used DMSO for sample preparation. is it because of that?
2. what has to be used as blank? we used Nash reagent as blank, is this right?
please clear my doubts..... it will be more useful for me.....
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Hi,
1. DMSO is a potent scavenger of free radicals and its presence in your samples should have resulted in fewer radicals, which will eventually form fewer adducts that will be detected as less absorbance. In fact, we use DMSO as standard when we test for OH scavenging activity.
2. Normally, our blank is made up of the solvent/reagent used in sample prep., and I suspect the difference between your blank (Nash reagent) and the solvent used for sample pep (DMSO) may be the cause of your abnormal readings. Try blanking with the solvent/reagent in which your sample is dissolved.
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On alloxan-diabetic mice..
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Dear Isaac
A significant number of Journals publishing research articles on pharmacological and toxicological aspects. You can find out a suitable journal in view the findings of your work . A few are listed here-under:
1. Journal of Medicinal Plant Research
2. Journal of Pharmacognosy and Phytochemistry
3. International Journal of Pharmacognosy and Phytochemical Research
4. Journal of Pharmacology and Toxicology
5. Journal of Ethnopharmacology
6. Asian Pacific Journal of Tropical Biomedicine
7. Asian Journal of Experimental & Biological Science
8. Journal of Diabetes and Metabolism
9. Journal of Endocrinology and Metabolism
10. Evidence-Based Complementary and Alternative Medicine
11. Nigerian Journal of Physiological Sciences
12. International Journal of Biochemistry Research & Review
Good Luck
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Can we measure phenolic Glycoside in plant extract? After we extract that compound, how can we quantify it?
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HPLC is one of best way to measure those compounds if you have the standard compound of the Arbutin.
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Can anyone suggest a procedure for determining the total phenols content?
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Dear Bhuvana
The simplest and well recognized method for estimation of phenolics is the Folin-ciocalteau method. This spectrophotometric method is based on the reaction of phenolics with Folin reagent under alkaline conditions. Try this method.
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I want to assay antioxidant properties in plant phenolic extracts. How can I make various concentrations (0.5-5 mg ml) of the extract?
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1- First, a Beaker or a crucible should be brought, weighed (Assume that the weight is 100 g)
2- Transfer the extract to the Beaker
3- The solvent used in the extraction process shall be disposed of by evaporation of until the dry matter is obtained as far as possible
4- The beaker is weighed after full evaporation of the solvent (Assume that the weight is 110 gm)
5- The weight of the extract (the weight of the beaker with the extract - the weight of the empty beaker) = 110-100 = 10 g
6- The extract is dissolved in the appropriate solvent Ethyl alcohol is often used and an emulsifier may be added (the amount of the solvent is calculated to obtain the desired concentration(
To prepare different concentrations:
1- The extract is dissolved in the minimum amount of suitable solvent and then a quantitative transfer is done to a volumetric flask with a final size of 100 ml (the appropriate size for the quantity of the extract).
The concentration of the extract is calculated as follows
10gm/100ml = 10×103mg/100ml= 100 mg/ml
Different concentrations could be prepared from that stock solution
To prepare 5 mg / ml with a final volume of 100 ml
V1×C1 (before dilution) = V2×C2 (after dilution)( the concentration which we would to prepare )
? ×100 = 5×100
V1= 5×100/100= 5 ml
Take 5 ml of stock solution (Conc 100mg/ ml) and supplement with appropriate solvent to 100 ml in volumetric flask.
To prepare 0.5 mg / ml with a final volume of 100 ml
? ×100 = 5×100
V1= 0.5×100/100= 0.5 ml
Take 0.5 ml of stock solution (Conc 100mg/ ml) and supplement with appropriate solvent to 100 ml in volumetric flask.
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I did total phenolic, flavonoid content, DPPH, ABTS, metal chelating and FRP of potato peel and ginger extract. Ginger extract having high phenolic and flavanoid content shows high metal chelating and ABTS radical scavenging activity but low DPPH radical scavenging activity and FRP. Potato peel extract having low phenolic and flavanoid content shows high DPPH radical scavenging activity and FRP but shows low metal chelating and ABTS radical scavenging activity. In the case of ginger extracts, after reaching the particular concentration, it reduces again. Is it possible to optimize the concentration using linoleic acid model system?
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First question that comes to mind when reading Your post is - how do You define high and low antioxidant activity (in comparison to what is it high, how do You express it) - because there are many lapses in that even in published papers. If something shows high FRAP it usually should show high chelating activity. It is also impossible to have high total flavonoids and low chelating activity since they are analyzed by their ability to create colored chelat complexes with aluminium cations. In general You should find the feature about plant extract that is most important for Your product. In fish products most important issues might be oxidation of fat or oxidation caused by metals from cans. Than You should pick one assay that will measure activity mostly related with the changes You are trying to prevent. Beta-carotene/Linoleic acid assay is good to present ability to prevent oxidation of unsaturated fats, therefore it should be really good for Your purpose. I suggest to optimize concentration (based on the analysis) to the level at which it prevents oxidative changes in as much unsaturated fat as fish product You want to preserve contains.
Here are some papers that might be useful:
Koleva, I. I., van Beek, T. A., Linssen, J. P. H., Groot, A. d. and Evstatieva, L. N. (2002), Screening of Plant Extracts for Antioxidant Activity: a Comparative Study on Three Testing Methods. Phytochem. Anal., 13: 8–17. doi: 10.1002/pca.611
Y. S. Velioglu , G. Mazza , L. Gao , and B. D. Oomah. Antioxidant Activity and Total Phenolics in Selected Fruits, Vegetables, and Grain Products J. Agric. Food Chem., 1998, 46 (10), pp 4113–4117 DOI: 10.1021/jf9801973
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I have repeated the experiment 3 times.
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HPLC-MS can be a good alternative solution if available, in agreement with comments of the previous colleagues, you have to check other colorimetric methods or using even TLC to get a preliminary view.
Good luck
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Plant biology.
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For hexanic plant extracts, you can use a combination of hexane:ethyl acetate or toluene:ethyl acetate in the ratios ranging from 6:4 to 9:1. You will surely get good separation on TLC plates. Use anisaldehyde-sulfuric acid reagent for spraying the plates followed by heating at 110 C for 10 minutes.
Regards
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Information needed for a research oriented project...
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May be this information is useful to you:
Indian Murraya koenigii (curry leaf) essential oil containing β-caryophyllene, caryophyllene oxide, α- and β-phellandrenes and eugenol inhibited quorum sensing and biofilm formation in the pseudomonads by restraining cell attachment at a concentration of 0.02% in Pseudomonas aeruginosa PA01:
Bai AJ, Vittal RR. Quorum sensing and anti-biofilm activity of essential oils and their in vivo efficacy in food systems. Food Biotechnol 2014; 28 (3): 269-92.
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We report very nice results of any activity done by using herbal extracts. Do we ever quantify the amount of solvent remaining after the extraction of active constituents. For e.g. I am doing antibacterial study and I have done isolation using Ethanol. Are my results really of the extract or of the amount of ethanol remaining in the extract even after drying? How to validate/ ensure 100% removal of solvent?
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Why don't you evaporate the extract in a rotavapor and remove all the solvent traces from the extract. You can also spray dry the extract to remove the traces of the solvent present in it. Always it is better to powder the extract and then dissolve or disperse it in appropriate vehicle for assessing its activity using the vehicle as a blank control for comparison. If these is not possible, then it is better to include a solvent control group which will rule out all the possibilities of solvent traces being responsible for the observed activity. I hope this will help you.
Regards
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I would want to know which is the best extraction method that could yield almost all the constituents present in the plant. At the same time the best solvent system to use for that method.
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Attached, is a thesis on a related topic. I hope you find it helpful.
Regards
Mohammed
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I also want someone to suggest ways of extracting alkaloid and flavonoid from a plant material.
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Dear Rolayo,
This depends on the 'fluffiness' of the dried, powdered plant material. The best way is to use the volume of solvent that completely soaks the powdered plant material and add excess to completely immerse. Check the volume of the solvent used. After draining after first extraction, use the same volume for second and third extractions.
To determine the number of extractions required, check the dry material obtained in each extraction. Mostly 3-4 extractions should be sufficient..
For flavonoids: Extract in methanol or Methanol: water (70: 30 or 80: 20) and then defat the concentrated extract in n-hexane. discard the n-hexane extract and use the defatted residue for analysis/purification of flavonoids.
For alkaloids: Extract in methanol. concentrate to dryness. resuspend in dilute sulphuric acid (should be <1 N). The acid extract is partitioned with ethyl acetate. Discard ethyl acetate extract. The acid extract should be basified with ammonium hydroxide to pH8.0 and then partitioned with ethyl acetate. The ethyl acetate extract is concentrated to dryness to yield alkaloid fraction. If you want to extract quarternary ammonium salts, basify the (basic layer) to a further pH of 11.0 with sodium hydroxide solution and then reextract in ethyl acetate or appropriate solvent which will yield quarternary salts.
Regards,
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What is the basis for selecting the minimal dose of plant extract for calculating lethal dose for an animal. Many cite previous literature to be the reason for choosing a specific dose, but is there any systemic method or is it just random?
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Acute toxicity (LD50) study
Acute toxicity study was carried out using the method
of (Lorke, 1983). In the first phase, nine rats randomly
divided into three groups of three rats per group were
given 10, 100 and 1000 mg extract/kg body weight orally
(via a cannula), respectively. The rats were observed for
signs of adverse effects and death for 24 h and then
weighed daily for 14 days. In the second phase of the
study, the procedure was repeated using three rats
randomly divided into three groups of one rat each, given
1600, 2900 and 5000 mg extract/kg body weight,
respectively. The rats were also observed for signs of
toxicity, mortality and
weighed for 14 days
(Tijani et al., 1986)
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Cytotoxicity of drugs is carried out in cell lines.
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Lung (A549), prostate (PC-3 and DU-145), neuroblastoma (IMR-32), breast cancer (MCF-7), ovary (IGR-OV-1), acute lymphoblastic leukemia (HL-60), leukemia (THP-1), liver (HEP-2), colon (Colo-205, HCT-15, Caco-2), and cervix (Hela) are some of the cell lines used to study the cytotoxicity of herbal drugs using in vitro cytotoxicity assays like SRB and MTT.
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Hi,
i am performing TLC for seperation of alkaloids from the extract..But, alkaloid of my interest was run along with mobile phase and resolution is also poor , hence creating problem in its isolation.
What can i do to obtain better results?
I have used CHCl3:Ethyl acetate:methanol(4:5:0.5), it gives poor results...Currently , i m using Chloroform:Methanol (24:1).
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Pl you try Toluene:EtOAc:MeOH:ammonia solution in the ratio of 40:40:7 to 9 ml: 2-4 drop ammonia solution. I hope you will get good separation. Still you have any problem you can consult Plant drug analysis book.
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16 answers
TLC, terpenoids, spray reagent, phytochemistry, TLC
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First of all you need to search in literature the plants you're working with to have an idea of what type of mono- or diterpene you have. Depending on the terpene type and dyeing reagent, you will obtain different colours, but in the majority of cases they are not specific, e.g.:
1) 0.5 mL anisaldehyde in 50mL glacial acetic acid plus 1mL conc. H2SO4; brown spots for diterpenes (Phytochem Anal. 2012;23(2):184-9)
2) vanillin reagent (50 mL of reagent grade ethanol, 0.3 mL of
reagent grade sulfuric acid, and 1 g of vanillin (‡98.5% HPLC
Grade), and then slowly heating on a hot plate; pinkish-purple spots (not specific, because you can obtain this colour with different terpene types) J Forensic Sci. 2009 May;54(3):612-6.
3)vanillin-H2SO4 solution (1 g vanillin dissolved in 100 mL 1% H2SO4) and heated at 85°C on a plate heater; dark blue spot for linalool, purple or blue for cineol & caryophyllene, depending on the concentration (Chem Cent J. 2012; 6: 46).
In order to differentiate between mono- or diterpene, you have to carry out other analysis like NMR ou GC.
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I am currently working on plant extract. Do we need to do dose fixation or dose dependent studies?
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Plant extracts taken for biological studies are normally based direct human trial experiences provided in ancient systems of medicines (either through published literature or personal experiences of clinical practioners of traditional systems).
While working on plant extracts on animal models it is always advisable initially to work on dose response curve and select one dose for detailed studies (invitro and invivo studies)
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I've tried to extract rosmarinic acid using a few methods and I wasn't successful.
I used the method of the British Phamacopeia and it doesn't function, solvent extraction (using Methylene Chloride after Ethyl Acetate and Methanol) but in comparison to the standard in TLC the molecule doesn't appear (movil phase: Methylene Chloride:Ethyl acetate:acetone:formec acid (10:80:25:8,5) )
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Only methanol should do the job :-) dont confuse yourself
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4 answers
.
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Automatic solvent extraction system (ASE), Super critical fluid extraction (SFC) and microwave assisted extraction techniques are some latest technique for the extraction of bio-active compounds from plant.
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Which is the most suitable method for isolating protein in plant extract?
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Really depends on the type of protein and how stable it is. Besides standard precipitation methods and liquid chromatography mentioned earlier, we have found that heating the extract can be very useful to drop out RuBisCO and other contaminants. 50 C at pH 5 for 30 minutes works well if your protein is stable under this condition. If you got to higher pH, you will likely have to raise the temperature- for some proteins we use 70 C for 30 minutes at pH 8.
How do plant extracts reduce the blood sugar level of glucose induced hyperglycemic mice while doing antihyperglycemic test ?
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While evaluating medicinal plant's extract on glucose tolerance in glucose induced hyperglycemic mice, we use glibenclamide as standard drug. If the plant extract reduces blood sugar level of mice, by exactly what mechanism does this happen? Could we say that the glibenclamide mechanism & plant extract mechanism both work as the same way or anything else?
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There is considerable diversity in the mechanisms of action of antdiabetic plants. Some act by increasing the release of insulin. Others by increasing insulin sensitivity. There are others that act by decreasing basal hepatic glucose production, thereby reducing fasting plasma glucose. Some block enzymatic degradation of complex carbohydrates in the small intestine, thereby lowering postprandial glucose. There are several other mechanisms as have been highlighted already by some contributors. This shows that antidiabetic plants do not operate by a single mechanism. The use of glibenclamide as reference drug in diabetic work may not be sufficient to decipher the actual mechanism of action of antidiabetic plants, except the plants operate like sulfonylureas. I suggest you go through the review article by Ivorra et al. In this article, the different mechanisms of actions of several antidiabetic plants were highlighted [ Ivorra MD, Paya M and Villar A (1989) A review of natural products and Plants as potential antidiabetic drugs. Journal of Ethnopharmacology 27:243-275.
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Can anyone recommend on the extraction protocol of phytosterols?
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The mentioned publication by Caligani et al. seems to be just what you need. Phytosterols are easily isolated after alkaline saponification of the oil sample. What do you ask actually
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I have methanol plant extract passed through silica gel column and I need to separate each class of compounds like alkaloids, flavinoids, terpinoids. Any suggestions?
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Thank you Dr. Alaa but I need to take fractions for further uses
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Does anybody know any assay using extracts of a resistant plant extract to control disease in susceptible plants of the same species?
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Fadel, I'd like to extract from Theobroma cacao plants and my idea is to verify if applying extracts from resistant plant varietes it could enhancing resistance in susceptible ones. Once is suposed that elicitors would be present in those extracts, am I wrong? Don't worry about english Igor, mine is terrible so!!
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How to overcome the formation of fungus on aqueous plant extract solutions?
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Dear Sudheer
I will suggest you to use a desiccator instead of these chemicals for storing the extract. If possible, first spray dry the extract and get it in a powder form. Otherwise also you can keep it as such. Just try to remove as much solvent as you can by keeping it on a water bath. Concentrate it sufficiently and store in a normal desiccator with any of the following desiccant chemicals (this list is not limited, many more are available):
1. silica gel (the beads in little packets, you might have seen very often)
2. sodium hydroxide
3. calcium chloride (anhydrous)
The desiccant will need to be replaced after it has absorbed all of the water that it can hold. Some chemicals will liquefy when this occurs so that you will know they need to be replaced (e.g., sodium hydroxide). Otherwise, you'll just need to switch out the desiccant when it starts to lose its effectiveness.
If you store properly in this way, it will never catch fungus. This is just a simple in-house method. Try and see the result.
Regards
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how you define a plant extract? what is its difference with an essential oil? Can you give some references?
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Plant extracts are usually solvent extracted and essential oils distilled but things are getting a bit blurred of late with both being referred to as plant extracts. For a good accurate guide as to what is an essential oil written by an Industry (raw aromatic materials) expert go to http://www.users.globalnet.co.uk/~nodice/ and look at the Essential Oil pages.
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How could we purify this product from Lathyrus sativus extracts to use as standard on HPLC?
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Put the Extract through Dowex-50-X8H+ and wash column with Distilled Wtaer
Collect fractions.Concentrate Ninhydrin +ve Fractions and add acetone
Store in Refrigerator. Almost pure ODAP separates. Redissolve in minimum volume of water and adjust PH to 7 with Dilute LiOH . Centrifuge the solution and carefully adjust pH to 6.8 with Glacial acetic acid. Analytically Pure ODAP will crystallize out in time.All the Best
How to increase Folin-Ciocalteu's selectivity and specificity?
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95 answers
I'm trying to perform a Folin-Ciocalteu reaction on a plant extract to determine its phenolic content. Due to interferences from other reducing substances, this method is not that specific for phenolics but it's the only one I have available now. I've heard that if we adjust some reagents concentrations (Na2CO3, etc) we would get a more precise reaction. Is this right or are there other means to get round the problem? Thank you.
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I have been working on a method for HTP analysis of total polyphenols using Folin reagent. I came across an article by Magalhaes et al titled "Rapid microplate high-throughput methodology for assessment of Folin-Ciocalteu reducing capacity". This article gets into the chemistry behind why Na2CO3 works. They suggest using NaOH to prevent phenolate ion regeneration which occurs with Na2CO3. The authors state that the oxidation of polyphenols occurs faster when the alkalinity of the medium increases and when the phenolic compounds are not protonated. The NaOH increases the reaction kinetics. There is also an in depth analysis of the interferences with this method. This is a great read and will be helpful to your search!
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Can anyone advise on how to do analysis of tylophorine content in crude extract of tylophora indica by HPLC and which column & mobile phase is most suitable?
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According to best of my knowledge these are alkaloids. These may be present in S-tylophorine was dextrorotatory and the (–)-isomer had the R configuration. You have separate in Chiral HPLC column because these are soluble in chloroform. You have try with normal phase HPLC system.
Check its solubility again. Then I can give you HPLC method. Try...
Gudluck
:)
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We want screen medicinal plants of Bangladesh and we have rats, mice, rabbits. We are looking for simple methods.
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I would start out by making sure that your compound does not reduce MTT on its own as some plant extracts do this. Incubate your compound with MTT for 2 hours and check to make sure that purple colour does not develop. If your compound does not reduce MTT, then start with the MTT assay. The acid phosphatase assay can also be used. The LDH-release assay will tell you whether your compound is causing cell lysis, but secondary necrosis can happen if your compound is left on your cell for too long. To differentiate between apoptosis and necrosis, use the Annexin V/PI assay, but again, make sure that your compound is not inherently fluorescent (many are). You can begin to tease out the mechanism if you desire, or do preliminary in vivo work. I would start by testing your compound in tumour xenografts in immune-deficient mice. Intratumoural injections can be done if you want to do a proof of principal preliminary study.
You can also submit your compound to NIH and they will screen it against a library of cancer cell lines. If you do it yourself, be sure to include normal cell cultures, such as mammary epithelial cells, PBMCs, human umbilical vein endothelial cells, dermal fibroblasts, etc, and calculate the EC50 for both cancer cells and normal cell cultures. Good luck!
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I extracted horse gram (soaked for 3 days in EtOH, H2O , HCl), with consequent evaporation and lyophilisation. Initially the product (tar like in consistency and colour) was soluble in DMSO but after a week (stored at room temp) I'm finding it highly difficult to solubilise it. Any suggestions?
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Reduce or totally avoid HCl in soaking mixture to devoid of tar color.
Lyophilise more diluted extract rather highly concentrated extract, you will get interesting results for better solubility.
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I have to study antiulcer activity on the plant extract. The problem that I am facing is that chlorophyll also has antiulcer effects.
So to have a confirmation about the antiulcer potential of the plant, I need to separate chlorophyll from the plant extract. Can you suggest a solution?
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Better treat extract with activated charcoal ( adjust quantity by trials) and filter the extract ,as chlorophyll has high affinity for charcoal.
The chlorophyll free filtrate then can be concentrated , evaporate the solvent totally.
Even if reminent of chlorophyll is left can be removed by subsequent column chromatography
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We have been thinking to purify the plant compound by Ion exchange chromatography but one of our senior colleague suggested HPLC. So what would be the right choice?
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Dear Jwala,
I have worked with the protease from Euphorbia hirta latex and found no problem in purifying with anion exchange column, i guess You can go for Ion exchange....
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Can anyone help me with the isolation of terpenoids, especially triterpenoids, from MeOH extract?
Also, what is the solvent system for TLC and Column Chromatography?
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For triterpenoids and its glycosides from methanolic extracts-go with ChCl3:MeOH:Water(65:35:5) for TLC and Chcl3:Methanol for CC. For Spraying reagent-LB reagent and will give purple spots upon heating.
You should read-Natural Products Isolation edited by Richard J. P. Cannell.
Gudluck
:)
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43 answers
What identification tests (reagents, chemicals) are used for flavonoids?
What precautions should be taken for isolation of flavonoids by Column chromatography?
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Flavonoids can be isolated from the ethanolic extract of plant material. It will be appropriate to fractionate the ethanolic extract with ether, ethylacetate and butanol respectively . Firest immerse the extract into water and fractionate with ether or pet ether or chloroform and then with ethyl acetate and butanol sequentially. Distiilled the respective solvent. Flavonoids will come in the ethyl acetate extract and butanol extracts. Butanol extract may contail flavonoid glycosides with 2 and more than two sugars. With the help of TLC flavonoids can be visualised by sprying different reagents. With Sulphuric acid yellow spots and with alcoholic feerric chloride bluish grey spots. By column chromatography using silica gel 100-230 mesh isolation is possible using chloroform and methanol mixture. After isolation of the compounds in pure state and by crystallization with the help of NMR and Mass spectral studies one can identify the flavonoids. Hope this information will be sufficient. If require any further information freely can ask.
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I am working on a fruit, "breadfruit", to see how I can extract enzymes from it.
Experts are amazed with the fast enzymatic activities of this fruit and I was wondering if there's a simple extraction method for crude enzyme and before further identification.
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Hi all friends
i am asking for a procedure for liquid- liquid fractionation for a polar(methanol extract) thermolabile plant extract , i want to fractionate the extract to 5-6 fractions to ease the isolation process
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hey if u r interested in a alkaloid extraction with difeerent solvents system .... then u can first mix the methanol extract with water and petroleum ether in equal amount n fractionate it... the ether layer will contain waxes, terpeoinds, the aqueous phase will contain amino acids, alkloids, sugar molecule. then acidify with .2 N HCl then extract with chloroform the aqueous phase is basified with ammonia till ph 11 then again extract with chloroform the chloroform phase will contain alkaloids. for more information u can refer Harbone - phytochemical extraction
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Cell culture
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You can use Human Dermal Fibroblast Cell (HDF) or Human Keratinocytes cell can be used to assay for wound healing effects of plant extracts.
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3 answers
Sa
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Have you done the phytochemical profiling of the extract? Which type of constituents you are expecting from the plant.? Is there any previous literature for your plant regarding isolation and chemical characterization? On the basis of chemical tests, you should firstly find out the categories of constituents present. Then only you can proceed for isolation of individual components.
Gud Luck
Regards
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I need help with the HPLC standard for the saponin.
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Thanks for your contribution
How to separate volatile oil from a mixture of oil-water after extraction of oil using clevenger apparatus?
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34 answers
I have recently extracted volatile oil from some plant material and now I have a mixture of both oil and water. Could you please suggest what is the best method to separate pure oil from this mixture of water and oil. From the literature I know that I need to use propane but I don't have a concrete reference for that.
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Pass the oil through a bed of sodium sulphate in a funnel. Water will adsorbed by the sodium sulphte and oil will pass. or put the oil-water mixture in -20C. Water will freeze but not oil. Gudluck :)
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I found it difficult to dissolve PE and Chlorofrm extract to dissolve in normal saline. At the same time I cannot use other solvents that interfere with RBC membrane
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How about Ethanol or n-Butyal Alcohol.
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I am trying to dissolve my methanolic extract in water, I have tried vortexing and sonication, but there is a waxy material. This becomes suspended on sonication and then clumped together again on shaking, it dissolves only in methanol.
What is this clumpy material?
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here's the paper you have asked about using GC/MS and LC/MS for your plant extract
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After extraction of a drug from plant materials using solvent, what rare conditions should be maintained for storage and how to select a solvent or vehicle for dissolving drug (plant extract) before the administration to the animal? Which is the best route for administration of drug while studying anti cancer activity of plant extract ?
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After extraction using the relevant solvent system, dry the extract to evaporate the solvent and remove any moisture (freeze drying, if possible). Store the dried extract in a vacuum dessicator in a dark, cool and dry place till further use.
To prepare the drug for dosing (if not soluble in water directly) triturate required quantity of dried extract with suspending agent (0.5-1% Na-CMC) prepared in water. If required, use a few drops of surfactant such as Tween 80. If possible, avoid using DMSO for preparation of drug for dosing.
Herbal extract are usually absorbed when given via oral route. However, if the extract fails to show activity (sometimes due to poor absorption), i.p. route should also be tried as it bypasses the intestinal barrier and may prove effective.
Hope this helps with your work.
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I am planning to add a plant extract into cells then harvest and carry out molecular assays. How do I treat my plant extracts before adding to the flask containing cells? Is it by using DMSO?
Secondly, should I add at confluence and wait for 24 hours or slightly before confluence?
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Use medium if possible, but if your compound is not water soluble, then DMSO will be your best bet. Try to keep the concentration below 0.2%. DMSO has its own biological activities at higher concentrations and can either kill your cells or protect them by acting as an antioxidant. Just include a DMSO control in your assay, and if you're doing a colorimetric assay (e.g., MTT) make sure your compound doesn't reduce MTT (some do), and if it's FACS based (Annexin V/PI), make sure your compound doesn't cause a shift on its on (most I've studied do).
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If so, how must we apply? Must we use small sprayer or larger pulverizators? Droplet diameter of mixture and biogradable herbal active ingredient? It's fotolysis? Side effects of this mixture? How do we survey these side effects?
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Dear Mustafa
To the best of knowledge and memory I have not read any paper on the effect of essential oils on bedbugs. I am attaching a review on insecticidal properties of essential oils and few other papers.
In response to another query, I have attached several papers on this subject. Follow the thread:
Good luck.
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Proanthocyanidins in peanuts were reported to be similar with those found in cranberries. In some references, ethyl acetate was used. How do I remove ethyl acetate and produce a pure proanthocyanidin extract?
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Thanks for the suggestion. Do you have a protocol in isolating proanthocyanidins using Sephadex resin?
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Has anyone had experience doing disk diffusion and minimal inhibitory tests for plant extracts? The results of my MIC test contradict the disk diffusion test results. In MIC, it shows positive results in the highest concentration of the plant extract, where in disk diffusion only three concentrations show positive results against the bacteria. I used three types of gram-positive and three of gram-negative bacteria for the tests.
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Diffusion methods are attractive because of their simplicity and low cost, but they are, like all agar-based methods, labour and timeintensive. On the basis of the higher MIC values obtained when the disk diffusion method was used in our experiments, it appears that this diffusion method could not always be a reliable method for screening the antimicrobial activity of plant extracts. The absence of an inhibition zone did not necessarily mean the compound was inactive, especially for less polar compounds, which diffuse more slowly into the culture medium. The diffusion assay is not suited to natural antimicrobial compounds that are scarcely soluble or insoluble in water and thus their hydrophobic nature prevents uniform diffusion through the agar media. For quantitative determination of antibacterial activity the agar dilution method is more appropriate, where antibacterial activity of plant extracts was shown at lower concentrations compared to the disk diffusion method. The agar dilution and broth microdilution methods produced comparable results and a good level of agreement only for gram-positive bacteria. Our evaluation included colorimetric determination using TTC or INT, and ATP determination by bioluminescence measurement. Our results showed that the broth microdilution method with ATP measurement is a rapid and accurate way of testing antimicrobial efficiency for all the tested bacteria, including campylobacters. As the microdilution method by TTC or INT produced comparable results and cost may restrict the use of ATP indicator, we suggest using INT for quantitative antimicrobial determination for normal growing bacterial strains and ATP for microaerophilic species like Campylobacter spp. This method may be an acceptable alternative for quantitative determination of bacterial susceptibility to plant extracts.
For more detail information please read the manuscript: Evaluation of diffusion and dilution methods to determine the antibacterial activity of plant extracts (Journal of Microbiological Methods 81 (2010) 121–126) or contact me directly.
Anja Klančnik
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I am Final year student of Ebonyi State University in department of Biochemistry I am embarking on my project now.
Which way is the best method to use if ethanol is to be used to get gross extract from Guava leave?
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Hi. Try using soxhlet extraction method.
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. However, so far I have been unsuccessful. I tried to dissolve them in pure DMSO, or 5% dichloromethane
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I work with murine lymphoma cells and when i have to use non polar extracts i dissolved them in EtOH 10% supporting me with a vortex and a sonicator. At this concentration, the EtOH shows only about a 15% of citotoxicity and decreases as i make serial dilutions. I hope this info helps you.
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There are plenty of papers involving phytochemical analysis for certain plant species. Moreover, target compounds isolated different plant organs produced in vitro or collected from different locations ex vitro etc.... Without testing the presence of certain compounds in plants on TLC plate prior to HPLC, how much our analysis would be reliable even our referance substances come across with such peaks! of the extract?...
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Please refer to the publication by Nancy Zabe et al. Methanol and acetonitrile-water mixtures have been traditionally used for a number of years for the extraction processes for eg extraction of aflatoxin from corn grains and peanuts. However, there is a widespread concern regarding the health hazards of acetonitrile and methyl alcohol Methyl alcohol if accidentally ingested can cause permanent blindness. Moreover, both acetonitrile and methyl alcohol incur hazardous waste disposal charges. Ethyl alcohol can be used as a substitute for methanol in a rapid method for total aflatoxin determination using immunoaffinity column chromatography with flourescence detection (AflaTest). The ethyl alcohol extraction method was shown to meet the same performance specifications for precision, accuracy and limit of detection as the methyl alcohol extraction method. It was also shown By Nancy Zabe et al that the results from the rapid method using ethyl alcohol and water extraction correlated with an HPLC method using methyl alcohol extraction. The authors concluded that ethyl alcohol may be substituted to methanol as a solvent for rapid determination of aflatoxin thereby reducing the hazardous waste generation. (Please refer to the following: Nancy Zabe, Kedist Ayalew and Stephen Powers, Ethanol Extraction Method for a Rapid Test for Aflatoxin in corn. VICAM, Chapter 17, pp 297-305, 2008)
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The combination of reverse phase and open column chromatography is much needed for isolating active compounds from polar fractions. Can these resins be used in purification columns like that of the C18 columns, which on the other hand are not apt for open columns?
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These resin (Diaion-HP-20, Amberlite-XAD-4,8,7,16 etc.) are quite good for preliminary purification of polar compounds like phenolics. When you will get your fraction after these resins then you can further purify your individual compounds in C-18 column and go for characterization.
Gud luck
:)
Can someone suggest a method to stabilize nanoparticle formulation of plant extracts?
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I find that the yield I obtain, even using higher concentrations of extract, to be low and I'm finding it extremely difficult to stabilize the particles.
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Plant extracts with diverse combinations of phyto-compounds give non reproducible results even in traditional formulations ( due to many reasons ,but primarily due to different plant /herbs sources collected from different areas). For conjugating drug-encapsu- lated nanoparticles with targeting ligands, you can try SPRAY DRIED plant extracts ( as they have almost uniform particle sizes) Useful ref: 1. Encapsulated plant extract (Gelsemium sempervirens) poly (lactide ... www.ncbi.nlm.nih.gov/pubmed/20511672Shareby SS Bhattacharyya - 2010 - To test the hypothesis if nanoparticle-encapsulated extract (now onwards to be ... and thereby improve bioactivity, we formulated nanoparticle encapsulation based on ... and F68 (polyoxyethylene-polyoxypropylene) was used as a stabilizer
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Suggest how to crystallize from a plant ethyl acetate extract purified compound?
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For crystallization of any chemical compound you can choose either of the two methods
1. Single solvent-Dissolve your compound (mix of two compounds) in a solvent in which it is sparingly soluble. Dissolve the compound by heating or any other mechanical way. Leave the solution in a cool environment. If the compound is of crystalline nature crystals will be formed.
2. Mix solvent- Dissolve your compound (mix of two compounds) in a solvent in which it is easily soluble. Then add drop by drop a solvent in which it is insoluble. when turbidity appears on first drop leave it in a cool environment for crystallization.
I hope it works for you......
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I would like to extract chemicals from the plant Solanum nigrum. Which is the best solvent to use and why?
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This strongly depends on the chemicals you want to extract. You should give more details on that.
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I am working with Verbenaceae family plants. What is the best solvent system to run crude extract on TLC?
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depending on which are the major components you are interested in.
for terpenoids toluene : ethyl acetate (4:1) or benzene : ethyl acetate (5:1)
for alkaloids CHCl3/MeOH/AcOH (18:1:1, v/v/v), and Dragendorff as a revealing reagent
for coumarins n-BuOH/AcOH/H2O (4:1:5, v/v/v), and acetate of lead (5%) and alcoholic KOH (5%) as a revealing reagent
for flavonoids n-BuOH/AcOH/H2O (4:1:5, v/v/v), and AlCl3 (0,5 g/100 mL of EtOH) as a revealing reagent while for tannins use FeCl3 (10% in MeOH/H2O, 1:1, v/v)
for anthocyanins HCl/ formic acid/water, (19.0/39.6/41.4 v/v/v )
hope it helps
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For different species of bacteria
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You must find correlation between absorbance (optical density OD) and CFU/ml. During growth phase, take samples of bacterial culture in regular interval. Measure optical density of samples and make serial dilution. Plate diluted samples (typicaly from 10-5 to 10-7 dilution) on agar plates in triplicate and after incubation count colonies. Calculate CFU/ml. Make graph correlating CFU/ml to OD. Then you can deduce OD of your sample.
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I am analyzing extracts of the Euphorbiaceous plant Jatropha curcas, which is rich in diterpenoids, using GC-MS. I have found repeatedly a compound with retention time of 27.2 min, obtained from an methanolic extract of calluses. That extract was fractionated by column chromatography; fractions were tested for terpenoids with p-anisaldehyde (always was positive). My library cannot identify the compound and it is associating the mass spectrum with Stilbene, but with a low probability (28%). I want to say that I am not chemist, but biotechnologist. Could any of you give tips to interpret the mass spectrum? I am not interested to identify the exact structure, but only the kind of compound.
Please find attached a Word file with details.
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Always check the Retention Index or compare the mass spectrum to a standard as a secondary form of identification - some plants are known to contain stilbenoids, but there is always the chance that a stilbene derivative could also be due to side-reactions of the phenyl portion of your column stationary phase bleeding at high temperatures or with dissolved oxygen....are you using a VS-5 column?
Do not rely on % matches too much, they are only a mathematical representation of fitting a structure to a mass spectrum.
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I am concerned with the study of insecticidal efficacy of certain plant extracts, the plants chosen are mostly wild herbs and shrubs. I collect the plants for the experiment during their flowering stage, however recently it was suggested that it is important to determine the plant age before collection.
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It would probably be useful to know, also the date the plant was collected. One should also note if the plant was damaged. Some compounds are produced as a result of stress, such as being attacked by insects. If insects attack plants only during a certain part of the year, the plant probably won't expend energy producing insecticidal compounds during the rest of the year. In addition, plants may be more vunerable to certain insect pests at different portions of their growth cycle and the plant mave have evolved to produce protective compounds only during the vunerable portion of their life.
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The saponins were detected by FTIR and a FOAM assay. But I cannot understand why the Baljet reagent is false for Lactones, in my phytochemical screening, if the saponins have groups of Lactones. Is the Baljet reagent specific?
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Baljet reagent probably does not work for all lactones. It is reported to work less efficiently for bufadienolides than for cardenolides (I don't know if it works for saturated lactones). You may try a general reagent :
Hydroxylamine - iron(III) chloride for lactones, esters, amides and anhydrides of carboxylic acids.
Solution a: Dissolve 20 g hydroxylammonium chloride in 50 ml water, make up to 200 ml with ethanol. Store the solution in the refrigerator.
Solution b: Dissolve 50 g potassium hydroxide in as little water as possible and make up to 500 ml with ethanol.
Spray solution I: Mix equal parts of a and b and filter off the precipitated potassium chloride. Place the solution in the refrigerator (stable for about 2 weeks).
Spray solution II: Dissolve 10 g finely powdered iron(III) chloride in 20 ml 36% hydrochloric acid. Shake with 200 ml diethyl ether until a homogenous mixture is obtained. The solution II is stable for some time only well sealed.
Procedure: Spray with I, dry at room temperature and spray with II.
Literature:
V.P. Whittaker, S. Wijesundera, Biochem. J. 51, 348 (1952).
How to crystallise an isolated plant extract for NMR studies?
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I have performed column chromatography of plant extracts and now want to go with NMR studies.Could anyone please let me know that how I can crystallise these plant extracts?
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Why do you have to crystallize them? NMR does not have to be solid dissolved in NMR solvent... If the extract is soluble in NMR solvent of your choice you are good to go. However, to get crystals - there is a general path way of finding two miscible solvents, where one will dissolve the extract, while the other one will not. Then, dissolve the extract in one and let the solvent interchange in closed container. In other words, in small vial (say 20 ml) you dissolve extract in 5 ml of solvent and put the vial into a 200 ml jar filled with 20 ml of the other solvent. Close tight and let it sit for a day or more. That's the simplest way if evaporation/drying is not enough for good quality crystals.
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Is it possible to block the fusion of the E protein of dengue virus with heparin receptors by using natural products like extract of basil leaf or papaya etc?
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In theory, it is possible providing there are molecules that binds heparin receptor in the compounds that you are testing for. However, other factors should also be considered, for example, the concentrations, amount, half-life etc. of the molecules or binding with or without the presence of dengue viral particles.
One needs to consider the effects on human cells when heparin receptor is blocked as these receptors are also functions in cellular responses such as Wnt/wingless pathways, some cytokine pathways (IL8, IFNg etc). And they also involve in cell proliferation and thus have a very wide cellular function in vivo.
Just my 2 cents
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I want to measure the chlorophyll content of leaves of rice that will be collected from fields. After 4 to 5 hrs from the time of collection, I have to measure the chlorophyll content in laboratory. I need a full,detailed procedure for this, including the amount of leaf sample, proportion of chemicals, time duration and instruments.
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A simple method is using dimethyl sulfoxide (DMSO) for chlorophyll extraction according to Barnes et al. (1992) and Shinano et al. (1996).
1) Barnes JD, Balaguer L, Manrique E, Elvira S, Davison AW (1992). A reappraisal of the use of DMSO for the extraction and determination of chlorophylls a and b in lichens and higher plants. Environ Exp Bot 32: 83–100
2) Shinano, T., Lei, T.T., Kawamukai, T., Inoue, M.T., Koike, T., and Tadano, T. Dimethylsulfoxide Method for the Extraction of Chlorophylls a and b from the Leaves of Wheat, Field Bean, Dwarf Bamboo and Oak, Photosynthetica, 1996, vol. 32, pp. 409–415
Two or three similar discs (about 0.8 cm in diameter) from each leaf are weighed and then incubated with 3 ml DMSO in a glass tube at 65°C until the tissue become colorless. The absorbance at 664.9 and 648.2 nm of the DMSO extract is determined with a spectrophotometer and the chlorophyll a and b concentrations of the leaves are calculated according to the below equations of Barnes et al. method
Chl a = 14,85 x A664.9 – 5,14 x A648.2 (μgr Chl a/ml)
Chl b = 25,48 x A648.2 – 7,36 x A664.9 (μgr Chl b/ml)
Finally, after the appropriate calculations you can express the chlorophyll concentration as:
mgr chl/gr fresh weight or
mgr chl/cm2 of leaf surface