EXTRACTION, ESTIMATION AND THIN LAYER CHROMATOGRAPHY OF FEW PLANT METABOLITES: A REVIEW

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ABOUT AUTHOR:
Dr. Amit Gangwal,
Associate Professor,
Smriti college of pharmaceutical education, Indore
gangwal.amit@gmail.com

INTRODUCTION
Plant remains to be the enviable source of molecules of therapeutic significance. Since antiquity, these bio resources have been in use for variety of diseases in different part of the world. Regardless of the type of plant, targeted ailment or other such parameters, the one step which is one of the most important and common is removal of the molecule or fraction or part there of from the plant biomass. Several new methods besides the usual organic solvent extraction have been developed over the last few years for the extraction of primary and secondary metabolites. These are alcohol extraction with various biocompatible solvents, recovery of carboxylic acids and antibiotics with reactive extraction, dissociation extraction, aqueous two-phase extraction, and supercritical and near critical fluid extraction. Extraction and re-extraction processes are integrated into a single step by emulsion liquid membrane and solid supported liquid membrane extractions.

REFERENCE ID: PHARMATUTOR-ART-1705

There are several extraction procedures or schemes (depending on various factors) for isolation of various plant constituents generally known as primary and secondary metabolites, nonetheless there are only one or two methods for scrupulous and perfect extraction of these metabolites. Irrespective of the plant or part thereof or activity or subsequent operation, these methods are sufficient to provide perfect extraction of various metabolites viz alkaloids, flavonoids, tannins, saponins, carbohydrates etc. In various publications, sometimes extraction schemes is not fully mentioned or not followed as mentioned in the pioneering text source or there is reporting of some modified process. There is a need of piled up information for the extraction, estimation and chromatography of some class of phytoconstituents, especially for the researchers interested in exploring a plant afresh or even for a routine assignment. This project is an attempt to compile and summarize the most relevant and time tested procedures for three basic operations while studying a plant from view point of phytochemistry or some allied reasons. To keep the text relevant and limited, barring few instances direct methods are given. Extensively cited and used procedures are being mentioned here. Many more procedures can be spotted in literature. Variation might be in starting solvent or fractionation schemes but in most of such cases ultimate steps usually remains same. This variation is intended because of subsequent steps, chiefly isolation of pure phytochemicals from crude extract employing range of solvents. Sometimes extraction is done to get rid of unwanted material for they hinder the removal of other metabolite or they are to be separated later in the extraction protocol or simply they are the problematic constituents in the sense they show false positive chemical presence or false biological activities. Therefore this project also describes the process to remove out rightly interfering compounds¹.

Extraction
Natural products may be obtained from the crushed biological material by extraction with a solvent such as petroleum ether, chloroform (trichloroniethane), ethyl acetate (ethyl ethanoate) or methanol. Several solvents of increasing polarity may be used. Thus lipid material (waxes, fatty acids, sterols, carotenoids and simple terpenoids) can be extracted with non-polar solvents such as petroleum ether, but more polar substances such as the alkaloids (mainly free bases) and glycosides are extracted with methanol, aqueous methanol or even hot water. Many alkaloids are present as their salts with naturally occurring acids such as tartaric acid. Polar solvents dissolve ionic solutes and other polar substances. There are many methods based on the technique or set up used but this project will explore only classical methods because such methods are easy, putative and can be implemented in most of the laboratories in limited setups. When it comes to extraction of phytoconstituents, the most widely employed method is extraction using a single solvent at atmospheric pressure which can be boiled owing to their azeotropic nature. Whether the compound(s) to be isolated is chemically undefined or not, it is important to have an idea about the relationship between the method applied and the properties of the substance extracted. A well known and time tested thumb rule is that “like dissolves like”. It means non polar solvents will remove non polar phytoconstituents and vice versa holds equally true. In most instances it is likely that that moderately polar phytoconstituents will be extracted2,3

Extractions can be either ‘‘selective’’ The initial choice of the most appropriate solvent is based on its selectivity for the substances to be extracted. In a selective extraction, Thus non polar solvents are used to solubilize mostly Lipophilic compounds (e.g., alkanes, fatty acids, pigments, waxes, sterols, some terpenoids, alkaloids, and coumarins). Medium-polarity solvents are used to extract compounds of intermediate polarity (e.g., some alkaloids, flavonoids), while more polar ones are used for polar compounds (e.g., flavonoid glycosides, tannins, some alkaloids). Water is not used often as an initial extractant, even if the aim is to extract water-soluble plant constituents (e.g., glycosides, quaternary alkaloids, tannins) 1,2,3. A crude natural product extract is generally an extremely complicated mixture of several compounds possessing varying chemical and physical properties. The fundamental strategy for separating these compounds is based on their physical and chemical properties that can be cleverly exploited to initially separate them into various chemical groups. However, in some cases, from the literature search of the related genera and families, it is possible to predict the types of compounds that might be present in a particular extract. This tentative prediction on the possible identity of the classes of compounds may help choose suitable extraction and partitioning methods, and solvents for extracting specific classes of compounds, for example, phenolics, saponins, alkaloids. Plant natural products are usually extracted with solvents of increasing polarity, for example, first n-hexane, diethylether, chloroform (CHCl3), to name a few, followed by more polar solvents, i.e., methanol (MeOH), depending on the chemical and physical nature of the target compounds.Alcoholic (MeOH or EtOH) extracts of plant materials contain a wide variety of polar and moderately polar compounds. By virtue of the co-solubility, many compounds, which are insoluble individually in pure state in MeOH or EtOH, can be extracted quite easily with these solvents. The concentrated extract is then extracted with an equal volume of n-hexane, usually three times, to give a fraction containing non-polar compounds, such as lipids, chlorophylls, and so on. The process is sometimes referred to as ‘‘defatting.’’ Although MeOH and n-hexane are not completely miscible, they are miscible to some extent. Sometimes, a small amount of water is added to MeOH to obtain a 95%-aqueous methanolic solution to get two distinct layers with similar volumes. The methanolic layer is evaporated to dryness and then dissolved in water. Occasionally it is not a solution, but a suspension. The solution (suspension) is partitioned between CHCl3, ethylacetate (EtOAc), and n-butanol (n-BuOH), successively. Partitioning with CHCl3 can be omitted depending on the chemical nature of the target compounds. Less polar compounds are present in the CHCl3 soluble fraction and polar compounds, probably up to monoglycosides, in the EtOAc-soluble one. The n-BuOH fraction contains polar compounds, mainly glycosides. Evaporation of the remaining water layer leaves polar glycosides and sugars as a viscous gum. However, separation by solvent partitioning cannot be always performed in a clear cut manner; overlapping of the compounds in successive fractions is usually found. When using EtOAc as an extraction solvent, especially the technical grade solvent, researchers must remember that it contains a trace amount of acetic acid (AcOH), which may cause a trans-esterification of acetyl group to the hydroxyl groups, and have a catalytic effect on labile functional groups or delicate structures. When the acetates of some compounds are isolated from the EtOAc-soluble or subsequent n-BuOH-soluble fraction, it is suspected that trans-esterification may have produced the acetates of the original compounds as artifacts. Chloroform is an ideal solvent for extracting alkaloids owing to its slight acidic nature, because alkaloids tend to be soluble in acidic media. When water layer is to be extracted thoroughly with n-BuOH, n-BuOH saturated with water is frequently used. Although n-BuOH is not miscible with water, 9.1ml of n-BuOH is soluble in 100ml of water at 250C.

Therefore, when the water layer is extracted with n-BuOH unsaturated with water many times, the volume of the water layer drastically decreases. Usage of unbalanced volumes of solvents sometimes causes unexpected partitioning of compounds. When saponins are the major target, it is advisable that the glycoside fraction (n-BuOH layer) is partitioned with a 1%-KOH solution to remove widely distributed phenolic compounds, such as flavonoids and related glycosides. Before concentrating the extract, the n-BuOH layer must be washed several times with water. In turn, re-extraction of the acidified alkaline layer gives a fraction rich in phenolic compounds. Some acylated saponins and flavonoids, present in plant extracts, are also hydrolyzed under alkaline conditions. Thus, at least a small-scale pilot experiment, such as tracing the fate of compounds by thin layer chromatography (TLC), is strongly recommended. However, this method is useful for the isolation of known alkali-resistant saponins on a large scale. Partitioning between Miscible Solvents Contrary to what has already been discussed earlier, miscible solvents are sometimes used for partitioning on addition of water. A plant material is extracted with MeOH and evaporated to obtain a residue. The residue is re-dissolved in 90% aqueous MeOH, and the resulting solution is extracted with n-hexane. This step seems to be similar to the previous partitioning example. In the next step, an appropriate amount of water is added to the 90%-aqueous MeOH to obtain an 80% aqueous solution, which is then extracted with CCl4 (MeOH and CCl4 are miscible). The final step is to make a 65%-aqueous MeOH solution with the addition of water, and the resulting solution is extracted with CHCl3 (MeOH and CHCl3 are miscible). Evaporation of the n-hexane, CCl4, and CHCl3 layers gives three fractions in order of polarity. Concentration of the 65%-aqueous MeOH layer gives the most polar fraction. This fraction is expected to contain glycosides as major constituents as well as a large amount of water-soluble sugars. preparation of detannnified extract: Defatted methanolic extract is partitioned with chloroform. The chloroform extract is washed with 1% NaCl to get extract tannin. Some authors have suggested the removal of crude saponins, from n butanol fraction of defatted menthol or alcohol or hydro-alcoholic extract, by precipitating with ethyl acetate². 

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