Phytochemical screening was performed on the extracts using standard protocols and Thin Layer Chromatography (TLC).
2.6.1. Characterization reactions in tubes
Crude ethanol extracts of Opilia amentacea were analyzed for the presence of secondary metabolites using standard procedures as described by Ciulei (1982).
The principle is based on the ability of functional groups of these compounds to react with specific chemical reagents to give characteristic reactions.
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Preparation of ethanolic extract solutions
Crude ethanolic extract (1 g) was dissolved in 5 mL of ethanol 96% and 50 mL of distilled water was added. A part of the mixture at 20 mg/mL was hydrolyzed in an acid medium according to the following procedure:
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Hydrolysis of extract solutions
30 mL of the mixture (20 mg/mL) was mixed with 15 mL of 10% chlorhydric acid in a 100 mL ground-neck flask containing a few glass beads. The flask's content was brought to boil under reflux for 30 min. After cooling, the hydrolyzed extract was extracted using liquid/liquid partitioning with 2 x 10 mL dichloromethane in a 100 mL separator funnel. The collected organic phases were dried on anhydrous sodium sulfate, filtered on Whatman n°5 paper, and concentrated under reduced pressure in the rotary evaporator.
The concentrated organic extracts were dispatched in test tubes to characterize O-heteroside compounds such as steroidal and triterpene glycosides, flavonic, anthracenic, coumarinic derivatives and cardenolides through their genins.
The non-hydrolyzed solutions of ethanolic extract were used to screen phenolic compounds, reducing compounds, saponosides, alkaloid salts and anthocyanins.
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Screening for Tannins
1 mL of the ethanolic extract solution was diluted with 1 mL of distilled water in a test tube. After homogenization, two drops of a 2% ferric chloride solution were added to the content of the test tube. The appearance of a bluish-black or intense green color indicated the presence of tannins [
20].
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Screening for Flavonoids (Shibata or cyanidin reaction)
1mL of ethanolic extract solution was placed in a test tube containing some magnesium clippings. 0.5 mL of concentrated hydrochloric acid (37%) was added. The appearance of a red or orange color after bubbling of the mixture indicated the presence of flavonols and flavanones, respectively.
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Screening for Leucoanthocyans
1 mL of ethanolic extract solution was mixed with 0.5 mL of hydrochloric butanol solution (Bate Smith reagent) over a boiling bath water. The immediate appearance of a red color indicate a positive test for leucoanthocyans.
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Screening for anthracenosides/anthraquinones (Bornträger reaction)
1 mL of the hydrolyzed organic extract was evaporated in a water bath. The dry residue was dissolved with 0.5 mL of dichloromethane (DCM). 0.5 mL of a 25% ammonia solution was added. The mixture was shaken vigorously and allowed to stand. The formation of a cherry red color in the aqueous phase indicates the presence of anthracenosides/anthraquinones.
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Screening for sterols and terpenoids (Liebermann Burchard Reaction)
1 mL of the hydrolyzed organic extract was evaporated in a water bath. The residue was dissolved in 0.5 mL of dichloromethane (DCM) and 0.5 mL of acetic anhydride.
The mixture was homogenized, followed by carefully adding 1 mL of concentrated sulfuric acid. The formation at the interface of the two liquids of a reddish-brown or reddish-purple ring with a greenish or purplish supernatant, was a positive indication for the presence of steroids and terpenoids.
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Screening for coumarins and derivatives (Feigl reaction)
1 mL of the hydrolyzed organic extract was evaporated in a water bath. The residue was dissolved in 1 mL of hot distilled water, shaken, and dispatched into two non-fluorescent hemolysis tubes (T and E). Distilled water (0.5 mL) was added to T-tube (negative control), and 0.5 mL of a 10% ammonia solution in the E-tube (sample). The contents of both tubes (T and E) were observed under ultraviolet radiation at 254 nm and 365 nm. The intense greenish or bluish fluorescence in tube E indicates the presence of coumarins and derivatives.
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Screening for saponosides by the foam test
2 mL of ethanolic extract solution was mixed with 2 mL of distilled water in 16 mm diameter test tubes. The tubes were capped with parafilm and then shaken vigorously for 5 min. The development of foam column with a height of at least 1 cm and persistent for 5 min indicates the presence of saponosides.
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Screening for cardiotonic heterosides
1 mL of the hydrolyzed organic extract was evaporated in a water bath. The residue was dissolved in 1 mL of 50% ethanolic solution and dispatched into two non-fluorescent hemolysis tubes (T1 and T2). 0.5 mL of Baljet reagent was put into T1 and 0.5 mL of Kedde reagent was put into T2. 0.5 mL of a 1N NaOH solution was added to both tubes that were shaken and left to stand. The appearance of an orange (T1) and purplish (T2) color indicates the presence of cardiotonic heterosides.
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Screening for reducing compounds
To the ethanolic extract solution, diluted ½ with distilled water, was added 1 mL of Fehling's reagent (I+II). The mixture was heated over a boiling water bath. The appearance of a brick-red precipitate attests the presence of reducing compounds.
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Screening for alkaloids
10 mL of alcoholic extract solution was placed in a beaker and concentrated on a hot plate. The residual aqueous solution was diluted with 10 mL of alkalinized distilled water (pH = 8-9) with NaOH solution. The alkaline extract solution was extracted by liquid/liquid partitioning with 2 x 10 mL of DCM in a separatory funnel.
The organic phases were evaporated to dryness. The dry residue (alkaloid bases) was triturated with 0.5 mL of a 2% hydrochloric acid solution (alkaloid salts). The acid solution was dispatched into two watch glasses (V1 and V2). To the content of V1 were added two drops of Draggendorf's reagent (potassium iodobismuthite), and to V2, two drops of Mayer's reagent (potassium mercuri-ioduro). The appearance of precipitates of red or orange-yellow (V1) and whitish-yellow (V2) color attests the presence of alkaloids.
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Thin Layer Chromatography (TLC)
Phytochemical analysis of Opilia amentacea extracts (OCFe, OCET and OCER) was also performed on 20 cm × 20 cm silica gel 60 F254 TLC plates (Macherey-Nagel, Germany).
Extracts dissolved in 96°C ethanol at the concentration of 10 mg/mL were used for thin layer chromatographic analysis. The mobile phase used consisted of ethyl acetate, methanol and distilled water (77; 13; 10). The mobile phase prepared was placed in TLC migration chambers.
Sample solutions were applied on the TLC plates using the capillary microtubes. Typically, 10 µL volumes of samples were applied. The distance between deposit points was 1 cm. Distances from the bottom edge was 1.5 cm, and 1 cm from the side edges. Plates were dried after development using a hairdryer and immersed vertically in the migration chambers containing mobile phases. The migration was performed over a path of 8 cm at 0.5 cm from the superior edge. After migration, TLC plates were dried at room temperature.
The dried TLC plates were observed under white light and ultraviolet light at 254 nm and 365 nm. Secondary metabolites on the plates were revealed using 10% ferric chloride (FeCl3) for tannins; vanillin sulfuric acid for steroidal and triterpenic glycosides, anisaldehyde sulfuric acid for saponosides and Neu's reagent for flavonoids groups The spraying of particular chemical reagents revealed the investigated secondary metabolite groups before observation.