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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SECONDARY METABOLITES
ALKALOIDS
Extraction
Being bases, alkaloids are normally extracted from plants into a weakly acid (1M HCl or 10% acetic acid) alcoholic solvent and are then precipitated with concentrated ammonia. These steps may be repeated or further purification can be achieved by solvent extraction. Such relatively crude extracts can be tested for the presence of alkaloids by applying various reagents meant for these secondary metabolites which represent the largest single class of secondary plant substances. The extraction of alkaloids is based, as a general rule, on the fact that they normally occur in the plant as salts and on their basicity, in other words on the differential solubility of the bases and salts in water and organic solvents. The plant material often contains substantial quantities of fats, and also waxes, terpenes, pigments, and other lipophilic substances which may interfere with extraction procedure, for example, by causing the formation of emulsion. These technical problems can be more or less completely avoided by a preliminary defatting of the powdered drug. Petroleum ether and hexane are well suited for this step: alkaloids are soluble in these solvents only in exceptional cases, when medium is neutral.The methods for the isolation of alkaloids are based on the fact that they can be extracted under neutral or basic conditions (after basification of plant material to pH 7-9 with ammonia, sodium carbonate, or sodium bicarbonate), as free base with organic solvents (e.g., dichloromethane, chloroform, ethers, ethyl acetate, alcohols) and as protonated base with polar solvent (water, alcohols) under acidic conditions (after acidification to pH 2-4 with diluted acids like phosphoric acid, sulphuric acid, citric acid)³. Keeping all these things constant, two processes are used routinely for the removal of alkaloids from plants. Solvent extraction in alkaline medium The powdered material is moistened with water and mixed with lime which combines with acids, tannins and other phenolic substances and sets free the alkaloids (if they exist in the plant as salts). Extraction is then carried out with organic solvents such its ether or petroleum spirit to take free bases. The concentrated organic liquid is then shaken with aqueous acid and allowed to separate. Alkaloid salts are now in the aqueous liquid, while many impurities (usually neutral) remain behind in the organic liquid. The operation is repeated as many times as necessary until the organic phase no longer contains any alkaloids The aqueous solutions of the alkaloid salts, combined, and washed with a non polar solvent (hexane, diethyl ether). These are alkalinized with a base in the presense of an organic solvent and not miscible the alkaloids as bases precipitate and dissolve in the organic phase. The extraction of the aqueous phase continues until the totality of the alkaloids has gone into the organic phase. The purification step may be carried out, like the previous one and depending on the quantity. Finally, the organic solvent containing the alkaloids as bases is decanted, freed from possible traces of water by suitable drying agent and evaporated under reduced pressure. A dry residue is left. Extraction in acidic medium: The powdered material is extracted with water or aqueous alcohol containing dilute acid. Pigments and other unwanted materials are removed by shaking with chloroform or other organic solvents. The free alkaloids are then precipitated by the addition of excess sodium bicarbonate or ammonia and separated by filtration or by extraction with organic solvents. This technique can be used to extract quaternary ammonium saltsThe alkaline medium ensures that the alkaloids are in their free base form. Medium polarity alkaloidal bases can be extracted using such organic solvents as chloroform, dichloromethane or diethyl ether. The second method (first treatment with aqueous acid) alkaloidal salts are formed, which are ionized and therefore soluble in aqueous media. The alkaloid then can be recovered as free base by making the environment basic of aqueous extract2,6,7.
Estimation of alkaloids
There is no universal method which can be applied to quantify analytically all the classes of alkaloids. Thus alkaloids in the free base form are difficult to crystallize whereas their salts crystallize comparatively easily. A rapid, easy, and simple spectrophotometric method was developed for the estimation of total alkaloids precipitated by Dragendorff's reagent in plant materials. It is based on the formation of yellow bismuth complex in nitric acid medium with thiourea. The yellow-colored complex formed obeys Lambert-Beer's law in the concentration range of 0.06-50 micro g/ml with λ at 435 nm. Using this method, the alkaloidal percentage of certain alkaloids (ajamalicine, papaverine, cinchonine, piperine, berberine) and some plant materials containing alkaloids (Berberis aristata, Solanum nigrum, and Piper longum3,8,9.
Thin Layer Chromatography (Silica gel 60 F 254 pre-coated TLC plates)
Toluene-ethyl acetate-diethylamine (70:20:10) is suitable for most of the drugs. Most of the alkaloids are separated on silicic acid. Aluminium oxide-precoated TLC plates may also be employed. With Dragendorff reagent alkaloids spontaneously give orange-brown color, which is usually stable in visible light. Dragendorff reagent followed by spay with sodium nitrite can also be tried to intensify the color developed by former reagent. Extract dried tissue from the plant with 10% acetic acid in ethanol, leave to stand for few hours. Concentrate the extract to one-quarter of the original volume and alkaloids are then precipitated by drop wise addition of concentrated NH4OH. Former is collected by centrifugation, washing with 1%NH4OH. Residue is then dissolved in ethanol or chloroform. Commonly employed and most informative solvent systems and other requirement are as under: Methanol: Con. NH4OH (200:3) and n-butanol-aqueous citric acid (on sodium citrate-buffeted paper) Detection of the spots: Presence of alkaloids is ascertained by any fluorescence in UV light and then by application of following spray reagents separately: Dragendorff, iodoplatinate and Marquis 5,11
FLAVONOIDS
EXTRACTION
The mostly cited method for the removal of crude saponins and flavonoid mixture from the plants share few steps in common especially if these secondary metabolites are being processed for separation using column chromatography. First extraction is done using methanol followed by suspending the dried residue in water. This solution is first made devoid of lipid content (waxes, chlorophyll and fats) by fractionating with n hexane or petroleum ether. This defatted material is finally fractionated in separatory funnel by ethyl acetate and n butanol (liquid – liquid extraction) to produce fractions rich in flavonoids and saponins respectively flavonoids are mainly water soluble compounds and can be extracted with 70% ethanol and remain in aqueous layer during partition with petroleum ether. They usually occur bound to sugars as glycosides).Flavonoids in their glycoside forms are water-soluble but when they are to be isolated from leaves then polar solvents may not be useful as the former are mostly present as aglycones. Lipophilic Flavonoids of superficial leaf can be extracted by solvents of moderate polarity followed by such solvents as hexane or petroleum ether. The glycosides can be extracted using acetone or lower alcohols like ethanol or methanol or hydroalcoholic mixture to ensure maximum extraction. After removing the volatile solvent, aqueous remnant is submitted to liquid liquid extrcation successively with petroleum ether, diethyl ether and ethyl acetate to get waxy materials, free aglycone and major chunk ok glycosides respectively. When aglycone has at least one free phenolic group, they dissolve in alkaline hydroxide solution. Boiling water can be used sometimes, while extracting glycosides, to inactivate glycosidase enzyme responsible for enzymatic degradation of glycosides. Flavonoids (particularly glycosides) can be degraded by enzyme action when collected plant material is fresh. It is thus advisable to use dry, lyophilized, or frozen samples. When dry plant material is used, it is generally ground into a powder. For extraction, the solvent is chosen as a function of the type of flavonoid required. Polarity is an important consideration here. Less polar flavonoids (e.g., isoflavones, flavanones, methylated flavones, and flavonols) are extracted with chloroform, dichloromethane, diethyl ether, or ethyl acetate, while flavonoid glycosides and more polar aglycones are extracted with alcohol–water mixtures. Glycosides have increased water solubility and aqueous alcoholic solutions are suitable. The bulk of extractions of flavonoid-containing material are still performed by simple direct solvent extraction. Powdered plant material can also be extracted in a Soxhlet apparatus, first with hexane, for example, to remove lipids and then with ethyl acetate or ethanol to obtain phenolics. This approach is not suitable for heat-sensitive compounds. A convenient and frequently used procedure is sequential solvent extraction. A first step, with dichloromethane, for example, will extract flavonoid aglycones and less polar material.
A subsequent step with an alcohol will extract flavonoid glycosides and polar constituents. Certain flavanone and chalcone glycosides are difficult to dissolve in methanol, ethanol, or alcohol–water mixtures. Flavanone solubility depends on the pH of water-containing solutions. Flavan-3-ols (catechins, proanthocyanidins, and condensed tannins) can often be extracted directly with water. However, the composition of the extract does vary with the solvent whether water, methanol, ethanol, acetone, or ethyl acetate. For example, it is claimed that methanol is the best solvent for catechins and 70% acetone for procyanidins¹,². Free flavonoid aglycones exuded by plant tissues (leaf or root) may be washed from the surface with non-polar solvents, such as methylene chloride, ethyl ether, or ethyl acetate. However, more polar glycosidic conjugates dissolve in polar solvents (methanol and ethanol), and these organic solvents are applied for extraction procedures in Soxhlet apparatus. Mixtures of alcohol and water in different ratios are applied for the extraction of flavonoids and their conjugates from solid biological material (plant or animal tissues and different food products). The extraction efficiency may be enhanced by the application of ultracentrifugation or pressurized liquid extraction (PLE), a procedure performed at elevated temperature ranging from 60°C to 200°C Supercritical fluid extraction with carbon dioxide also may be used procedures have to be carefully adjusted because of the possibility of thermal degradation of the flavonoid derivatives6,11,12.
Estimation
Several liquid chromatographic (LC) methods with UV-Vis absorption (Gil et al., 1995; Hertog et al., 1992; Blouin and Zarins, 1988) or diode-array ultraviolet (DAD-UV) (Mouly, 1998; Mateos, et al., 2001; Bramati, et al., 2002), fluorescence (Hollman et al.,1998) and mass spectrometric (Raffaelli et al., 1997; Justesen, 1998) have been developed for the analysis of flavonoids. Merken und Beecher (2000) have reviewed the various HPLC and sample preparation methods that have been employed to detect and quantify flavonoids. Being phenolic in nature, they change color when treated with base or with ammonia. This property of flavonoids is exploited for detection in solution or on chromatogram. Flavonoids contain conjugated aromatic systems and they show intense absorption bands in the UV and visible regions of the spectrum. Aluminum chloride colorimetry was used for flavonoids determination (Chang et al., 002). In this method, plant analyte is (0.5 ml of 1:10 gm/lit) mixed with 1.5 ml of methanol, 0.1 ml of 10% aluminum chloride, 0.1 M potassium acetate and 2.8 ml of distilled water. After keeping the mixture at room temperature for 30 min, the absorbance of the reaction maximum was measured at 415 nm with a double beam UV/visible spectrophotometer (Perkin Elmer, USA). The calibration curve is prepared by using quercetin solutions at concentrations from 12.5 to 100 μg of quercetin per ml of methanol5,11.
THIN-LAYER CHROMATOGRAPHY
Flavanoids are generally present in plants bound to sugar as glycosides and any one flavonoid aglycone may occur in a single plant in several glycosidic combinations. Therefore while analyzing such glycosides, it is better to keep things simple by analyzing aglycone portion first by performing hydrolysis of flavonoids. Detecting agent: NP/PEG reagent Observation: Intense fluorescence is produced in UV-365 nm. PEG increases the sensitivity. The fluorescence behavior is structure dependent. (Yellow, green, orange)5,11.
Solvent Systems for TLC of Flavonoids on Silica Gel
Flavonoid aglycones:
EtOAc–i-PrOH–H2O, 100:17:13,
EtOAc–CHCl3, 60:40,
CHCl3–MeOH, 96:4,
Toluene–CHCl3–MeCOMe, 8:5:7,
Toluene–HCOOEt–HCOOH, 5:4:1,
Toluene–EtOAc–HCOOH, 10:4:1,
Toluene–EtOAc–HCOOH, 58:33:9,
Toluene–EtCOMe–HCOOH, 18:5:1,
Toluene–dioxane–HOAc, 90:25:4.
Flavonoid glycosides:
n-BuOH–HOAc–H2O, 65:15:25,
n-BuOH–HOAc–H2O, 3:1:1,
EtOAc–MeOH–H2O, 50:3:10,
EtOAc–MeOH–HCOOH–H2O, 50:2:3:6,
EtOAc–EtOH–HCOOH–H2O, 100:11:11:26,
EtOAc–HCOOH–H2O, 9:1:1,
EtOAc–HCOOH–H2O, 6:1:1
EtOAc–HCOOH–H2O, 50:4:10,
EtOAc–HCOOH–HOAc–H2O, 100:11:11:26,
EtOAc–HCOOH–HOAc–H2O, 25:2:2:4,
THF–toluene–HCOOH–H2O, 16:8:2:1,
CHCl3–MeCOMe–HCOOH, 50:33:17,
CHCl3–EtOAc–MeCOMe, 5:1:4,
CHCl3–MeOH–H2O, 65:45:12,
CHCl3–MeOH–H2O, 40:10:1,
MeCOMe–butanone–HCOOH, 10:7:1,
MeOH–butanone–H2O, 8:1:1.
Flavonoid glucuronides :
EtOAc–Et2O–dioxane–HCOOH–H2O, 30:50:15:3:2,
EtOAc–EtCOMe–HCOOH–H2O, 60:35:3:2,
Flavanone aglycones CH2Cl2–HOAc–H2O, 2:1:1.
Flavanone glycosides:
CHCl3–HOAc, 100:4,
CHCl3–MeOH–HOAc, 90:5:5,
N-BuOH–HOAc–H2O, 4:1:5 (upper layer), Chalcones
EtOAc–hexane, 1:1, Isoflavones
CHCl3–MeOH, 92:8, CHCl3–MeOH, 3:1,
Isoflavone glycosides:
N-BuOH–HOAc–H2O, 4:1:5 (upper layer).
Dihydroflavonols:
CHCl3–MeOH–HOAc, 7:1:1
Biflavonoids:
CHCl MeCO Me–HCOOH, 75:16.5:8.5,
Toluene–HCOOEt–HCOOH, 5:4:1.
Anthocyanidins and anthocyanins
EtOAc–HCOOH–2 M HCl, 85:6:9,
N-BuOH–HOAc–H2O, 4:1:2,
EtCOMe–HCOOEt–HCOOH–H2O, 4:3:1:2,
EtOAc–butanone–HCOOH–H2O, 6:3:1:1.
Proanthocyanidins:
EtOAc–MeOH–H2O, 79:11:10,
EtOAc–HCOOH–HOAc–H2O, 30:1.2:0.8:8.
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SAPONINS
Extraction
Generally defatted plant material is extracted with methanol or ethanol or aqueous alcoholic solution. Concentrated alcoholic extract is then fractionated with water saturated n butanol. From n butanol phase saponins are then precipitated by addition of ether. Being water soluble, they can be extracted with it, generally at boiling point. Although an aqueous medium is a good choice for subsequent freeze drying, it also promotes the hydrolysis of the bidesmosides. Alternate procedure is to use alcohols of hydro alcoholic solutions. Before this defatting is advisable. But polar solvents will extract most of the other plant metabolites besides saponins. Therefore concentrated polar extract is fractionated using n butanol, because the later solubilizes the saponins, which are then precipitated by adding diethyl ether. Further crude saponins may be purified.
Terpenoids/sterols
a) Triterpenoids
b) Isolation:
Dried tissues are first defffated with ether and then extracted with methanol. The concentrated methanol extract can be examined directly or after hydrolysis to liberate aglycone, if glycosides are present.
The most common spray reagents are
1) Car price reagent (20% SbCl3 in chloroform): A range of colors are produced visible in both day light and UV, on heating sprayed with SbCl3-CHCl3 and heating to 110 °c for 10 seconds.
2) Liebermann-Burchard reagent: On spraying plate with this reagent and heating it at 110 °c for a while, a range of colors are produced.
3) H2SO4 alone or diluted with water or methanol also gives many colored spots on spraying and heating.
Solvent systems are:
Hexane: ethyl acetate (1:1)
Chloroform-methanol (10:1)
A less used choice is n- butanol-2M NH4OH(1:1)
Petroleum ether-ethyl formate-formic acid (93:7:0.7) 1,5,11.
Acid hydrolysis of the saponin and absolute configuration of monosaccharide
Compound 1 (5 mg) was heated with 1.5M HCl–CH3OH (10 ml) under reflux for 7 hours. The reaction mixture was diluted with water and extracted with CHCl3. Sapogenin was detected in the CH2Cl2 layer by TLC. Half of the H2O layer was neutralized with K2CO3and concentrated, and then subjected to HPLC-MS analysis with authentic samples, and arabinosyl (19.3 min), xylosyl (17.2 min), and glucosyl (15.6 min) were detected by comparison of the retention times with the standards. Another half of the H2O layer was concentrated to dryness, and dissolved in pyridine (1 ml). Hydroxylamine hydrochloride (5 mg) was added to pyridine solution. The resulting mixture was heated at 90 °C for 30min, and acetic anhydride (0.5 ml) was added in and heated at 90 °C for another 30min. The reaction solution was then concentrated to dryness, and the residue was suspended in dichloromethane (concentration, 1 ml) and subjected to GC analysis. The absolute configurations of the glucose units in compounds 1 were confirmed as L-arabinosyl (6.60min), D-xylosyl (6.70 min), and D-glucosyl (8.67min) by comparison of the retention times with standard samples¹.
Estimation of total saponins
A physico-chemical method for saponine determination is given by Morales and curl (1938). Three gram of the air dried powdered sample is taken in a 250 ml round bottom flask and extracted with methanol (90% v/v) refluxing it for half an hour. The same extraction process is to be repeated thrice and the solvent is distilled off. Now the soft extracted left over is treated with 25 ml petroleum ether (60-80°c) by refluxing it for half an hour. Solvent is the cooled and removed by decantation. The left soft extracted is then treated with a mixture of 25 ml chloroform and 25 ml ethyl acetate. The solvent were poured off after cooling keeping the soft extract in the same flask. The soft extract was again dissolved in 25 ml of methanol, filtered (whatman no. 42) and conc. To 5ml this methanolic extracts was then added to 25 ml. acetone (with constant stirring) to precipitate glycoside. The precipitate was then filtered, collected and dried to constant weight to get saponins
THIN LAYER CHROMATOGRAPHY
Chloroform-methanol-water (13:7:2)
Chloroform- glacial acetic acid-methanol-water (64:32:12:8)
Detection: Vanillin sulphuric acid or anisaldehyde sulphuric acid
Blue colored spot with Liebermann Burchard reagent: if triterpenes are there pink to red color will be the indication of these, while a blue green color corresponds triterpenes steroidal saponins. Sapogenins are obtained by hydrolysis of crude saponins. Briefly, inorganic acid is used to hydrolyse the concentrated extract, followed by neutralization of the medium. Precipitates are dried and then extracted with petroleum ether. This is then applied on TLC plates trying following solvent systems: acetone-hexane (4:1), chloroform-carbon tetrachloride-acetone (2:2:1), chloroform-acetone (4:1), chloroform-ethyl acetate (1:1). Detecting agent antimony chloride is produces pink to purple colored spots after heating at 1100c for 10 minutes. Saponins are much more polar owing to their attachemnet with sugar and they are more easily separated by paper chromatography or by TLC on Cellulose 5,6,10.
TANNINS
Extraction
Dried plant material is finely powdered. The extract is then prepared by stirring sample water for 3 h at room temperature. The crude extract obtained is filtered with a paper filter (Whatman No. 1), and concentrated using a rotary evaporator at 50°C, followed by the addition of 3 ml of water. The extract is run through the Sephadex LH-20 column to purify the total tannins, using 50% methanol, followed by 70% acetone (Asquith and Butler, 1985; Hagerman and Butler, 1994). The acetone isolate is collected and rotary evaporated under the same conditions. The final concentrate (MPT) is dissolved with 35% acetone and kept in 4°C until use. (Jamunaa Ambikabothya, Halijah Ibrahima, Stephen Ambub, Srikumar Chakravarthib, Khalijah Awangc, Jaya Vejayand)¹.
Phenolic compounds
Due to multiplicity of hydroxyl functions, phenols tend to be relatively polar and dissolve in aqueous alcohol. Being weak acid, they may also be extracted or partitioned into aqueous alkali as phenolate salts³.
Extensive polymerization (reactions by the action of polyphenol oxidases) develops brown color in damaged plant material when exposed to the air and in certain extracts.²,³.
Estimation of total phenols
Total phenols are determined by using the Folin Ciocalteu reagent . A dilute extract is or gallic acid (the phenolic compound commonly used as the standard) is mixed with the Folin Ciocalteu reagent (5 ml of the reagent diluted ten fold with distilled water) and aqueous Na2CO3 (4 ml, 1 M). The mixtures (each with a separate extract representing leaves, stems or roots) are allowed to stand for 15 min and total phenols are determined by colorimetry at 765 nm. The standard curve is prepared using 0, 50, 100, 150, 200 and 250 mg/lit –1 solutions of gallic acid in methanol/water (50:50 v/v). The total phenol value was expressed in terms of gallic acid equivalent (mg/g of dry mass), which is a commonly used reference value.
Estimation of total polyphenols
The total poly phenolic content of the herbal extracts was estimated according to the method given by Singleton and Rossi (1965). Ten milligram of standard tannic acid was dissolved in 100 ml. distilled water in a volumetric flask (100g/ml. of stock solution). From this stock solution, 0.5 to 2.5 ml. of aliquots were pipetted out in to 25ml. volumetric flask. 10 ml. of distilled water and 1.5 ml. of folin ciocalteu’s reagent were added to each of the above volumetric flasks. Four ml of 20% sodium carbonate solution was added after 5 min. and the volume was made up to 25 ml. One gm. of powdered material was extracted with 95% ethanol (thrice), filtered with a 1,2 micron filter paper and was adjusted to 5o ml. with 95% ethanol in a flask. From this stock solution the same steps were repeated as given below for the preparation of standard. Percentages of total phenolics were calculated from calibration curve of tannic and total phenolics were expressed as mg tannic acid equivalents/gm (mg TAE/g) of dry weight.
Conclusion
Plant secondary metabolites are currently the subject of much research interest, but their extraction as part of phytochemical or biological investigations presents specific challenges that must be addressed throughout the solvent extraction process. Successful extraction begins with careful selection and preparation of plant samples, and thorough review of the appropriate literature for indications of which protocols are suitable for a particular class of compounds or plant species. During the extraction of plant material, it is important to minimize interference from compounds that may coextract with the target compounds, and to avoid contamination of the extract, as well as to prevent decomposition of important metabolites or artifact formation as a result of extraction conditions or solvent impurities. This chapter presents an overview of the process of plant extraction, with an emphasis on common problems encountered and methods for reducing or eliminating these problems. In addition to generally applicable extraction protocols, methods are suggested for more or less selectively extracting specific classes of compounds, and phytochemical methods are presented for detection of classes of compounds commonly encountered during plant extraction, including selected groups of secondary metabolites and interfering compounds. Plants and microorganisms produce complex mixtures of natural products, and the election of the best protocol for an efficient extraction of these substances is not a simple task. ‘‘Classic’’ solvent-based procedures (e.g., maceration, percolation, Soxhlet extraction, extraction under reflux, steam distillation) are still applied widely in phytochemistry despite the fact that they lack reproducibility and are both time- and solventconsuming. This is principally because they only require basic glassware and are convenient to use for both initial and bulk extraction. Accelerated solvent extraction is a newer instrumental technique. While it offers some advantages over conventional methods (mainly efficiency and reproducibility), it is best suited for initial rather than bulk extraction. It has found a wider application in industry (where large numbers of extracts have to be produced in an efficient and reproducible way) rather than in academia. To date, mainly plant and microbial sources have been investigated for their metabolites. However, it is important to remember that researchers are only beginning to explore other biotopes (e.g., the marine environment, insects) and that many plants and microorganisms have not yet been characterized. Moreover, several species among the bacteria known are yet to be cultured under laboratory conditions. This leaves much scope for the potential discovery of novel and/or useful natural products in the future¹.
REFERENCES
1. Sarkar Satyajit D.,Latif Zahid, Gray Alaxander I., “Natural product isolation” ; second edditon; published by Springer; Page no-117.
2. Mukherjee D. Pulok K.; “Quantity Control Herbal drug An approach evaluation of Botnicals” ; first edition ; Business Horizones New Delhi India 2010 ; Page no-256,259.
3. Haughton Peter, Raman Amla: “Laboratory Handbook for the fractionation of natural Extracts”, first edditon; Published by Springer; Page no-41.
4. Phytochemistry; Anthocyanins from red flowers of camellia cultivar ‘Dalcha’ Jain-Bin Li Fumio Hashimoto Keiichi Shimizu, Yusuke Sakta. 2008 . Page no. 69.
5. Harborne J.B. “Phytochemical Methods Aguide to modern technique of plant Analysis; Ed. Third eddion: Published by champan & Hall London 1998; Page no.11.
6. Brunton Jean “ Pharmacognosy Phytochemistry Medicinal Plants; second eddion; Published by Levoisier 1999; Page no. 781, 793, 796,
7. Evans, William C; Trease & Evans Pharmacognosy; Ed 6th Saunders Published by Elsevier 2009. Pg no. 337, 353.
8. Sreevidya N, Mehrotra S. Spectrophotometric method for estimation of alkaloids precipitable with Dragondroff’s reagent in plant matarials. J AOAC. 2003 Nov.; 86.
9. Agarwal, S.S., Paridhavi M.; Herbal drug Technology; Ed1st; Universities Press India Pvt. Ltd. (India) 2007; Page no. 354.
10. Hildebert H., Wagner, Sabine Bladt; “ A thin Layer Chromatography Atlas”; published by springer; Page no. 45.
11. Kiran Alice and K.V. Peter; “Horiculture Sciences Series”; Volume second; New India Publishing House; Page no. 23
12. Gokhale Kokate; Text book of Pharmacognosy; Ed. 1st; Published by Nirali Prakashan; Page no.15-25.
13. Andersen and Markham; Chemistry, Biochemistry, and Applications; Ed 20th; 2005. Page no. 28.9
14. Chang C, Yang M, Wen H, Chem J Estimation of total flavonoid contents in plants by two complementary colorimetric methods. J. Food Drug Anal, (2002). Page no.178-182.
15. Ali Mohammad; Text Book of Pharmacognosy, 2e; second eddion ; ;Page no-46.
16.McDonald S, Prenzler PD, Autovich M, Robards K. Phenols content and antioxidant activity of olive extracts. Food Chemistry, (2001) Page no. 73-84.
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