Potassium hydroxide

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

Endogenous lipids from mouse liver and heart were detected and quantified using several techniques. FC was quantified using straight-phase HPLC and ELS detection as previously described10 (link). Quantification was made against an external calibration curve. This chromatographic set-up was also used to fractionate DG. Quantification of CE, TG, SM, and phospholipids (all from the total extract) and DG (fractionated from the HPLC) was made by direct infusion (shotgun) on a QTRAP 5500 mass spectrometer (Sciex, Concord, Canada) equipped with a robotic nanoflow ion source, TriVersa NanoMate (Advion BioSciences, Ithaca, NJ)11 (link). For this analysis, total lipid extracts, stored in chloroform:methanol (2:1), were diluted with internal standard-containing chloroform/methanol (1:2) with 5mM ammonium acetate and then infused directly into the mass spectrometer. The characteristic dehydrocholesterol fragment m/z 369.3 was selected for precursor ion scanning of CE in positive ion mode12 (link). The analysis of TG and DG was performed in positive ion mode by neutral loss detection of 10 common acyl fragments formed during collision induced dissociation13 (link). The PC, LPC and SM were detected using precursor ion scanning of m/z 184.114 (link), while the PE, phosphatidylserine (PS), phosphatidylglycerol (PG) and phosphatidylinositol (PI) lipid classes were detected using neutral loss of m/z 141.0, m/z 185.0, m/z 189.0 and m/z 277.0 respectively15 (link)16 (link). For quantification, lipid class-specific internal standards were used. The internal standards were either deuterated or contained diheptadecanoyl (C17:0) fatty acids.
Ceramides (CER), dihydroceramides (DiCER), glucosylceramides (GlcCER) and lactosylceramides (LacCER) were quantified using a QTRAP 5500 mass spectrometer equipped with a Rheos Allegro quaternary ultra-performance pump (Flux Instruments, Basel, Switzerland). Before analysis the total extract was exposed to alkaline hydrolysis (0.1M potassium hydroxide in methanol) to remove phospholipids that could potentially cause ion suppression effects. After hydrolysis the samples were reconstituted in chloroform:methanol:water [3:6:2] and analyzed as previously described17 (link).
For the recovery experiments the tissue samples were spiked with non-endogenously present lipids (or endogenous lipids spiked at relatively high levels) and could therefore all be detected by lipid class specific scans using the shotgun approach. In the recovery experiment we therefore also included the PA and phosphatidylcholine plasmalogen (PC P) lipid class, which we could not measure endogenously using our current analytical platform. Due to poor ionization efficiency, FC was derivatized and analyzed as picolinyl esters according to previous publication18 (link). See Table 1 for details. With some exceptions, lipids are annotated according to Liebisch et al.19 (link).

Skin scrapings were obtained from lesions compatible with mange, encompassing both healthy and injured tissue, and were processed in a 10% KOH solution for 60 min at 37 °C. Subsequently, they were observed under the microscope (20× and 40×) for the detection of S. scabiei. Identification of mites was performed according to the keys and descriptions of Wall and Shearer [25 ].