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

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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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