Resorcinol

Plant collection and identification. Fresh pods of A. nilotica were collected in June 2008 from Potiskum, Yobe State, Nigeria. The pods were identified by a taxonomist in Department of Biological Sciences, University of Maiduguri, Maiduguri, Nigeria. The pods were air dried for three weeks under the shade and ground into fine powder.
Preparation of aqueous extract. Three hundred and fifty grams (350 g) of the powdered extract sample were exhaustively extracted with distilled water using reflux method. The crude aqueous extract was concentrated in vacuo and a brown colored extract weighing two hundred and sixty three grams (263 g) w/w was obtained. It was thereafter stored in a refrigerator at 4 ˚C until used.⁴ Fractionation of the aqueous pod extract. The method used for fractionation of A. nilotica pod powder has already been reported.^{5 (link),6} The crude aqueous pod extract was suspended in cold distilled water and then filtered using Whatman filter paper. The filtrate was thereafter subjected to fractionation using, chloroform, ethyl acetate and n-butanol. The fractionation with the organic solvents of different polarity was done until the organic layers were visibly clear to obtain ethyl acetate (58 g), n-butanol (25 g) soluble fractions and the residue (180 g). The product did not dissolve in chloroform, hence no product was obtained as shown in Fig. 1.
Phytochemicalanalysis of theextracts of A.nilotica. The aqueous extract and ethyl acetate, N-butanol and residual fractions of A. nilotica extracts were subjected to qualitative chemical screening for identification of various classes of active chemical constituents.^{7 , 9} Test for tannins (Ferric chloride test). Two millilitres (2 mL) of the aqueous solution of the extract were added to a few drops of 10% Ferric chloride solution (light yellow). The occurrence of blackish blue colour showed the presence of gallic tannins and a green-blackish colour indicated presence of catechol tannins.
Test for saponins (Frothing Test). Three millilitres (3 mL) of the aqueous solution of the extract were mixed with 10 mL of distilled water in a test-tube. The test-tube was stoppered and shaken vigorously for about 5 min, it was allowed to stand for 30 min and observed for honeycomb froth, which was indicative of the presence of saponins.
Test for alkaloids. One gram (1 g) of the extract was dissolved in 5 mL of 10% ammonia solution and extracted with 15 mL of chloroform. The chloroform portion was evaporated to dryness and the resultant residue dissolved in 15 mL of dilute sulphuric acid. One quarter of the solution was used for the general alkaloid test while the remaining solution was used for specific tests.
Mayer’s reagent (Bertrand’s reagent). Drops of Mayer’s reagent was added to a portion of the acidic solution in a test tube and observed for an opalescence or yellowish precipitate indicative of the presence of alkaloids.
Dragendorff’s reagent. Two millilitres (2 mL) of acidic solution in the second test-tube were neutralized with 10% ammonia solution. Dragendorff’s reagent was added and turbidity or precipitate was observed as indicative of presence of alkaloids.
Tests for carbohydrate (Molisch’s test). A few drops of Molischs solution was added to 2 mL of aqueous solution of the extract, thereafter a small volume of concentrated sulphuric acid was allowed to run down the side of the test tube to form a layer without shaking. The interface was observed for a purple colour as indicative of positive for carbohydrates.
Testsforcarbohydrate (Barfoed’stest). One milliliter (1 mL) of aqueous solution of the extract and 1ml of Barfoed’s reagent were added into a test-tube, heated in a water bath for about 2 min. Red precipitate showed the presence of monosaccharaides.
Standard test for combined reducing sugars. One milliliter (1 mL) of the aqueous solution of the extract was hydrolyzed by boiling with 5 mL of dilute hydrochloric acid (HCl). This was neutralized with sodium hydroxide solution. The Fehling’s test was repeated as indicated above and the tube was observed for brick-red precipitate that indicated the presence of combine reducing sugars.
StandardtestforfreereducingSugar (Fehling’stest). Two milliliters (2 mL) of the aqueous solution of the extract in a test tube was added into 5 mL mixture of equal volumes of Fehling’s solutions I and II and boiled in a water bath for about 2 min. The brick-red precipitate was indicative of the presence of reducing sugars.
Test for ketones. Two millilitres (2 mL) of aqueous solution of the extract were added to a few crystals of resorcinol and an equal volume of concentrated HCl, and then heated over a spirit lamp flame and observed for a rose colouration that showed the presence of ketones.
Testforpentoses. Two millilitres (2 mL) of the aqueous solution of the extract were added into an equal volume of concentrated HCl containing little phloroglucinol. This is heated over a spirit lamp flame and observed for red colouration as indicative of the presence of pentoses.
Test for phlobatannins (HCl test). Two millilitres (2 mL) of the aqueous solution of the extract were added into dilute HCl and observed for red precipitate that was indicative the presence of phlobatannins.
Test for cardiac glycosides. Two millilitres (2 mL) of the aqueous solution of the extract was added into 3 drops of strong solution of lead acetate. This was mixed thoroughly and filtered. The filtrate was shaken with 5 mL of chloroform in a separating funnel. The chloroform layer was evaporated to dryness in a small evaporating dish. The residue was dissolved in a glacial acetic acid containing a trace of ferric chloride; this was transferred to the surface of 2 mL concentrated sulphuric acid in a test tube. The upper layer and interface of the two layers were observed for bluish-green and reddish-brown colouration respectively as indicative of the presence of cardiac glycosides.
Test for steroids (Liebermann-Burchard’s test). The amount of 0.5 g of the extract was dissolved in 10 mL anhydrous chloroform and filtered. The solution was divided into two equal portions for the following tests. The first portion of the solution above was mixed with one ml of acetic anhydride followed by the addition of 1 mL of concentrated sulphuric acid down the side of the test tube to form a layer underneath. The test tube was observed for green colouration as indicative of steroids.
Test for steroids (Salkowski’s test). The second portion of solution above was mixed with concentrated sulphuric acid carefully so that the acid formed a lower layer and the interface was observed for a reddish-brown colour indicative of steroid ring.
Test for flavonoids (Shibita’s reaction test). One gram (1 g) of the water extract was dissolved in methanol (50%, 1-2 mL) by heating, then metal magnesium and 5 - 6 drops of concentrated HCl were added. The solution when red was indicative of flavonols and orange for flavones.
Testforflavonoids (pew’stest). Five millilitres (5 mL) of the aqueous solution of the water extract was mixed with 0.1 g of metallic zinc and 8ml of concentrated sulphuric acid. The mixture was observed for red colour as indicative of flavonols.
Test for anthraquinones (Borntrager’s reaction for free anthraquinones). One gram (1 g) of the powdered seed was placed in a dry test tube and 20 mL of chloroform was added. This was heated in steam bath for 5 min. The extract was filtered while hot and allowed to cool. To the filtrate was added with an equal volume of 10% ammonia solution. This was shaken and the upper aqueous layer was observed for bright pink colouration as indicative of the presence of Anthraquinones. Control test were done by adding 10 mL of 10 % ammonia solution in 5ml chloroform in a test tube.
Elemental analysis. The elemental content was determined using the standard calibration curve method.^{10 (link),11 (link)} Zero point (0.5 g) of air dried sample in an evaporating dish was placed in an oven at 80 ˚C and dried to a constant weight. The sample was placed in a weighing crucible and ashed at 500 ˚C in a hot spot furnance for three hours. The ashed material was prepared for the determination of trace element. A portion of zero point (0.5 g) of the ashed sample was digested by heating for two min with a mixture of 10 mL each of nitric acid (HNO₃), HCl and a perechloric acid in a 500 mL flask. The aliquot obtained from this mixture by filtration was mixed with a 10 mL of 2M HNO₃ and 30 mL of distilled water in a 100 mL volumetric flask. The volume was made up to zero mark with distilled water. Blank sample and standard solution for the various elements were similarly done. All samples placed in a plastic container and stored in a refrigerator maintained at 4 ˚C prior to analysis. Flame emission spectrometer (Model FGA-330L; Gallenkamp, Weiss, UK) was used to determine sodium (Na) and potassium (K) concentrations. Other elements, magnesium (Mg), calcium (Ca), iron (Fe), lead (Pb), zinc (Zn), manganese (Mn), cadmium (Cd), copper (Cu) and arsenic (As) were determined by atomic absorption spectrometry with (Model SPG No. 1; Unicam, Cambridge, UK) at the appropriate wave-length, temperature and lamp current for each element.¹²

All plant parts were extracted on the day of collection. The screening procedures were adapted from Wall et al.,[24 (link)] and Sofowora[25 ]. An extraction of each plant part was prepared by macerating a known weight of the fresh plant material in a blender with redistilled methylated spirit. Each extract was suction-filtered and the process repeated until all soluble compounds had been extracted, as judged by loss of colour of the filtrate. Extract from each plant part was evaporated to dryness in vacuo at about 45°C and further dried to a constant weight at the same temperature in a hot-air oven. A portion of the residue was used to test for plant constituents.
The test for tannins was carried out by subjecting 3 g of each plant extract in 6 ml of distilled water, filtered and ferric chloride reagents added to the filtrate. For cardiac glycosides, legal test and the Killer-Kiliani test[26 ] were adopted (0.5 g of extract was added to 2 ml acetic anhydrate plus H₂SO₄). The test for alkaloids was carried out by subjecting 0.5 g aqueous extract in 5 ml 1% HCl, boiled, filtered and Mayer's reagent added [26 ,27 ]. Cyanogenic glycosides were identified by subjecting 0.5 g extract in 10 ml sterile water, and were filtered. Sodium picrate paper was added to the filtrate and heated to boil. The extract was also tested for carbohydrates using resorcinol solution[24 (link)]. The extract was subjected to frothing test for the identification of saponin. Haemolysis test was further performed on the froted extracts in water to remove false positive results[25 ]. Fehling's solution was added to the extract and heated to detect reducing sugar. The extract was also tested for free glycoside bound anthraquinones[24 (link),25 ]. Five grams of extract was added to 10 ml benzene, filtered and ammonia solution added. The presence of flavonoids was determined using 1% aluminum chloride solution in methanol concentrated HCl, magnesium turnins, and potassium hydroxide solution[28 (link)].

Total amounts of sialic acids were quantified using the periodate-resorcinol assay. Briefly, cells were washed three times with ice-cold PBS and lysed in 250 μl PBS via freeze–thaw cycles in liquid nitrogen. All samples and the standard curve were oxidised with 5 μl of 0.4 M periodic acid for >10 min on ice. Then, 500 μl of the following solution were added to each sample and mixed by vortexing: 0.6% resorcinol, 0.25 mM CuSO₄, 36% H₂0, 44% concentrated HCl. Samples were incubated at 100 °C for exactly 15 min and allowed to cool down to room temperature afterwards, before adding 500 μl of tert-butanol. To remove any particular remnants, the samples were briefly centrifuged. OD₆₃₀ was measured in triplicate in a 96-well plate and sialic acid levels were calculated from the standard curve and normalized to the total protein amount of each sample.

A typical preparation of M_xO_y/SiO₂ oxide nanocomposites (where M is Cu, Mg, Mn, Ni and Zn) consists of three steps. In the first step, the homogeneous dispersion of silicon dioxide was prepared in the aqueous solution of the corresponding metal acetate, with an estimated ratio of the components under stirring at room temperature. The content of the metals was 3.0 mmol/g SiO₂. In the second stage, the dispersions were dried at a layer thickness of 4–7 mm at 130 °C for 5 h, then ground in a mortar and sieved through a sieve with a mesh size of 0.5 mm. In the third and final stage, all the powders obtained were calcined in air at 600 °C for 2 h. The reference sample of fumed silica was treated in the same three steps: homogenization of the aqueous dispersion, drying, grinding, sieving and calcination at the same temperature.
The modification of resorcinol–formaldehyde (RF) polymers by oxide nanocomposites was carried out by an in situ method by mixing resorcinol, formaldehyde and M_xO_y/SiO₂ nanocomposites or pristine SiO₂ at a weight ratio of 1:2:1 with stirring at room temperature. The unfilled resorcinol–formaldehyde control sample (RFR) was prepared by stirring resorcinol with formaldehyde at the same 1:2 weight ratio. All mixtures were hermetically sealed, placed in a thermostatic oven and treated at 50 °C for 4 days for complete curing and maturation of the RF resin. After gelling, a brown, solid polymer composite was obtained, which was dried at the same temperature for 18 h. All polymer composites were crushed and sieved to obtain a fraction of 0.2 to 0.5 mm. The polymer composites were labeled as RF/SiO₂, RF/Cu/SiO₂, RF/Mg/SiO₂, RF/Mn/SiO₂, RF/Ni/SiO₂, RF/Zn/SiO₂ and RF/Zn/SiO₂.
The carbonization of the samples was carried out in a tubular furnace under a nitrogen atmosphere (with a flow rate of 100 mL/min) by heating from room temperature to 800 °C at a heating rate of 5 °C/min and holding at the maximum temperature for 2 h. As-synthesized composites were designated as C/SiO₂, C/Cu/SiO₂, C/Mg/SiO₂, C/Mn/SiO₂, C/Ni/SiO₂ and C/Zn/SiO₂.