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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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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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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Hello,
Chapter 12 in " Research Methods in Plant Science: Vol. 1. Soil Allelochemicals (2012)" by Gleadow et al. ought to give some information for you. Be VERY careful if you need to handle cyanide salts and/or HCN since they are extremely toxic!
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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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Well, there are many ,kits' available in the market to test inhibition of certain compounds. You need to know what kind of compounds or markers are you targeting, so you will be able to find things in the internet. Cayman Chem is a good supplier to start with.
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Which solvent systems hould be used to make the plant extract. I am not having facility of reverse phase so by using silica how can I isolate the sesquiterpenes
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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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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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I got the results seen attached below before (up) and after (down) treating cells with plant extract. Can somebody help me understand why the treated cells have spaces within the monolayer. Does it suggest decrease in proliferation, death or?
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In several cases in my studies, I have noticed this effect and I feel that it is due to some stress and not decrease in proliferation or death.
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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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Dear 
i thank this artivle help you
Article in International Journal of Pharmacy and Technology 9(1):28936-28954 · February 2017
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I need help with the HPLC standard for the saponin.
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Try methods of these publications
1) Structure–biological activity relationships in triterpenic saponins: the relative activity of protobassic acid and its derivatives against plant pathogenic fungi;
2) Screening for feeding deterrent and insect growth regulatory activity of triterpenic saponins from Diploknema butyracea and Sapindus mukorossi
3) Synergistic/potentiation interaction between nematostatic constituents from Azadirachta indica, Madhuca indica and Sapindus mukorossi
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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 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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It is difficult to dissolve the methanolic fractions in normal saline, but that is a must for membrane stabilization work.
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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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Temperature can destroy plants chemical components at a certain temperature. I am trying to determine the best temperature that would help to retain all chemical components of a plant.
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Temperature not exceeding 40 degrees centigrade with air circulation is good for drying a plant material in an oven. However, drying temperature depends on the purpose for which you are going to use the material after drying.
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I would like to observe all secondary metabolites that are present in the crude extracts.
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the solvents are based on the compounds which we are going to isolate, based on this the solvents varies.
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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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Drying of plant extract
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use a lyophilizer.
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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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Toxicity of lantana camera: L.camara var. aculeatagiven in dose of 6g/kg body weight to guinea pig produces cholestasis, after euthanized the animal L.camara could not be in blood and urine samples and find in lower G.I.T and feces.
Single dose (1-3 mg/kg) of L.camara produces liver injury after injected i.v. in sheep due to triterpinene acid-lantadene-Aand chatracterised by transient rises in serum enzymes with or without hyperbilirubinemia. In higher dose produce hepatic necrosis and in lower doses produce cholestatic syndrome for several days.increase in hepatic and renal xanthine oxidase activity and causes rise in glutamicoxaloacetic transaminase acitivity, obstructive jaundice, photosensitisation when ingested L.camara in cattle, sheep, buffalo, guinea pig.
REFERENCE:
Sharma S, Sharma OP, Singh B, Bhat TK. Biotransformation of lantadenes,the pentacyclic triterpenoid hepatotoxins of lantana plant, in guinea pig. Toxicon 2000;38(9):1191-202.
Pass MA, Seawight AA, Lamberton JA, Heath JJ. Lantadene A toxicity in sheep: A model of cholestasis. Pathology 1979;11(1):89-94.
Sharma OP, Makkar HP, Dawra RK, Neggi SS. A review of the toxicity of L.camara in animals.Clin toxicol 1981;18(9):1077-911.
MC Kenzie RA. Bentonite as therapy for lantana camara poisoning of cattle. Aust Vet J; 1991;68(4):146-8.
Courtesy: www.pharmatutor.com - THE DETAIL STUDY OF LANTANA CAMARA PLANT FOR THEIR MEDICINAL IMPORTANCE -A REVIEW,HH SAPARIA*, M BAIDYA, AR MAHESH ,Krupanidhi College of Pharmacy, Chikkabellandur, Carmelaram Post,Banglore-560035, Karnataka, India.
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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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Try with extracting 0.1 M HCl solution.
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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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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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Hi, You could try following conditions: A fused silica capillary column DB-5MS ; the injector and interface temperature at 250 C and 300 C, respectively; oven temperature: from 70 to 315 C at a heating rate of 5 C/min; end of a heating program with an isothermal period of 10 min; carrier gas: helium at 1.0 ml/min; injection of samples (e.g. 10 mg of pure essential oil, dissolved in 1 ml of diethyl ether): a pulsed split mode (injection volume 1 ul; the flow 1.5 ml/min for the first 0.5 min and then 1.0 ml/min throughout the remainder of the analysis; split ratio 40: (1)). MS conditions : ionization voltge of 70 eV, acquisition mass range 35–650, scan time 0.32 s. This will give you excellent results for essential oils, but, unfortunately, GC/MS is not the best choice for plant solvent extract analysis. As many plant metabolites are not sufficiently volatile, only a (small) fraction of plant extract (volatile and semi-volatile compounds) is analyzable under GC/MS conditions. You could get somewhat better results if you rise temperature of injector and interface (be careful here what are the temperature limits of the column/instrument) and extend isothermal period at the end of program. Anyway, if you are only interested in volatile fraction of diethyl ether, hexane, CH2Cl2 etc. plant extracts, GCMS is acceptable. For "full" profile, it is better to use some other technique (eg. LCMS). I hope this was of some help...
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For antioxidant activity with DPPH.
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Preparation of Extract- their is various methods involved like SOXHLET, MACERATION methods, by increasing order of their polarity
(Pet ether, Methanol, ethyl acetate and aqueous) filter the solvent, Concentrate the solvent to 1/4th of the initial extract evaporate to dryness and subject to Antioxidant activity by DPPH or FRAP method.
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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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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 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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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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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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Methanol quality and extraction process (cold and hot extraction).
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I think so. Thank you Mr. Abraham Esobedo - Moratilla for you valuable information.
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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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With a reference link.
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Not sure if this is a perfect procedure as I have not performed this but you might find it useful... I got this from Nugroho et al. (2014)
Peroxynitrite-scavenging activity
An assay method described by Kooy et al. [19] was used to measure the peroxynitrite-scavenging activity of the compounds, extracts, and fractions from P. aviculare. The principle of this method is to monitor the intensity of highly fluorescent rhodamine formed from non-fluorescent DHR 123 under the presence of ONOO−. The concentration of DHR 123 was 5 μM. The samples were dissolved in 10% DMSO (concentration: 5 μg/mL). The final fluorescent intensity was measured with or without the treatment of 10 μM ONOO− in 0.3 N NaOH. The fluorescence intensity of the oxidized DHR 123 was measured at the excitation and emission of 480 nm and 530 nm using a microplate fluorescence reader FL 500 (Bio-Tek Instruments Inc., Winooski, VT, USA).
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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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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 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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i.e. Isolation of metabolites from natural sources.
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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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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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20 answers
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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21 answers
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 working plants; producing herb loaded nanoparticles. Is there a method for synthesis of oil loaded nanoparticles as I have obtained essential oils from plant extracts.
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8 answers
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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Corrosion inhibition using natural products or plant extract
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I am trying to find the solubility in water of a plant extract supported in glycerol. The plant extract is mostly insoluble in water but the glycerol is very soluble in water. How will I quantify the insoluble part? Any ideas?
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1 answer
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
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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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6 answers
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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4 answers
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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Dichloromethane and ethyl acetate dry extract dissolve in MeOH. For the TLC you can try toluene: ethyl formate: formic acid (5:4:1), the rosmarinic acid should be visible after few hours on the TLC plate, good luck
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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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Can anyone recommend on the extraction protocol of phytosterols?
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Dear Dr Singh,
you can see the publication: Galyautdinov et al
Leonard M.Khaliov
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6 answers
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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Yes, I think if you use HPLC u can get goods separation. u can see our article Osman et al., 2010 which available on research gate
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5 answers
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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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
:)
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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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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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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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5 answers
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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4 answers
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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U can also run the same solvent system of tlc ( but slightly reduced polarity eg. C:m:w 65:25:3 etc.) in the column provided u run the column at once without stopping it and collecting small fractions.
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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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Lipase
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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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Method I commonly would perform: Take a dried MeOH extract and cut it between water and EtOAc; then take that water cut (the very polar compounds fraction), and cut it again with n-BuOH (the polar & glycosylated ternenoid-like compounds fraction); take the EtOAc cut, dry it down, and cut it between n-hexane (the very non-polar compounds fraction) and 90% Aq. MeOH (the mostly the non-glycosylated ternenoid-like compounds fraction). Adjusting the pH of the aqueous phases can be useful as well, but one does run a greater risk (in some cases) of cross reactivity with the solvents and constituents.
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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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Synergetic effect-corrosion study
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6 answers
I'm working on a plant and checking its anti-proliferative activity.
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Extraction of what? Sugars, polysaccharides, lipids, terpernoids, phenolics, polyphenolics, alkaloids, saponins, proteins, DNA ect ect. It is apparent from the replies so far, most people think you mean DNA. You need to be far most specific in your question as there is not just one answer. Different classes of compound required very different extraction methods. Presumably if you are testing extracts for anti-proliferative activity in mamalian cell cultures, then the extracts need to be water soluble, or soluble in low concentations of a nonpolar solvent?
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9 answers
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 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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What is the effect of sodium fluoride on an enzyme found in animal tissue?
What are the parameters to find out the toxicity level of a particular individual?
How to separate volatile oil from a mixture of oil-water after extraction of oil using clevenger apparatus?
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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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12 answers
.
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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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.
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There are different protocols for the determination of PAL activity in plants. But I think it depends on which plant/system you are working.
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6 answers
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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2 answers
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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133 answers
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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When can we call wild plants medicinal plants?
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Any plant which provides health-promoting characteristics, temporary relief from symptomatic problems or has curative properties.
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Plants - herbs - grown for medicinal purposes, as opposed to growing them for culinary or ornamental purposes.
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Medicinal herbs are plants or parts of plants used for therapeutic or medical benefit.
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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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What is the variation in rosmarinic acid content in O. stamineus or other herbs?
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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).
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Medicinal plants are widely used in many parts of the world; due to traditional knowledge and popular use of certain plants specially by the rural community.
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Dear Yalem,
I got your point of consideration.
I am attaching herewith two articles which we follow for the standardization of medicinal plants.
In India Herbal companies like Himalaya Herbal drugs are standardizing their medicinal products. For eg. gassex for gastric problems
Liv 52 for Liver etc.
I am providing you the link of the company site too.
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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Super-critical Carbon Dioxide solvent extraction, essential oils, applied separations
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I have repeated the experiment 3 times.
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it happens sometimes but its rather impossible that a plant doesn't possess phenolic compounds. Moreover, the solvent in which the extracts is prepared is also important. Many a times reagents used or method used is not appropriate coz there are several interfering components. So, other chromatographic and spectral techniques is very important viz., TLC, HPLC, LC-MS, GC-MS and others.