Ferric chloride

Antioxidant (DPPH and ABTS radical scavenging, reducing power (CUPRAC and FRAP), phosphomolybdenum, and metal chelating (ferrozine method)) and enzyme inhibitory activities [cholinesterase (ChE) Elmann’s method], tyrosinase (dopachrome method), α-amylase (iodine/potassium iodide method), and α -glucosidase (chromogenic PNPG method)) were determined using the methods previously described by Zengin et al. (2014) (link) and Dezsi et al. (2015) (link).
For the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay: Sample solution (1 mg/mL; 1 mL) was added to 4 mL of a 0.004% methanol solution of DPPH. The sample absorbance was read at 517 nm after a 30 min incubation at room temperature in the dark. DPPH radical scavenging activity was expressed as millimoles of trolox equivalents (mg TE/g extract).
For ABTS (2,2′-azino-bis(3-ethylbenzothiazoline) 6-sulfonic acid) radical scavenging assay: Briefly, ABTS+ was produced directly by reacting 7 mM ABTS solution with 2.45 mM potassium persulfate and allowing the mixture to stand for 12–16 in the dark at room temperature. Prior to beginning the assay, ABTS solution was diluted with methanol to an absorbance of 0.700 ± 0.02 at 734 nm. Sample solution (1 mg/mL; 1 mL) was added to ABTS solution (2 mL) and mixed. The sample absorbance was read at 734 nm after a 30 min incubation at room temperature. The ABTS radical scavenging activity was expressed as millimoles of trolox equivalents (mmol TE/g extract) (Mocan et al., 2016a (link)).
For CUPRAC (cupric ion reducing activity) activity assay: Sample solution (1 mg/mL; 0.5 mL) was added to premixed reaction mixture containing CuCl₂ (1 mL, 10 mM), neocuproine (1 mL, 7.5 mM) and NH₄Ac buffer (1 mL, 1 M, pH 7.0). Similarly, a blank was prepared by adding sample solution (0.5 mL) to premixed reaction mixture (3 mL) without CuCl₂. Then, the sample and blank absorbances were read at 450 nm after a 30 min incubation at room temperature. The absorbance of the blank was subtracted from that of the sample. CUPRAC activity was expressed as milligrams of trolox equivalents (mg TE/g extract).
For FRAP (ferric reducing antioxidant power) activity assay: Sample solution (1 mg/mL; 0.1 mL) was added to premixed FRAP reagent (2 mL) containing acetate buffer (0.3 M, pH 3.6), 2,4,6-tris(2-pyridyl)-S-triazine (TPTZ) (10 mM) in 40 mM HCl and ferric chloride (20 mM) in a ratio of 10:1:1 (v/v/v). Then, the sample absorbance was read at 593 nm after a 30 min incubation at room temperature. FRAP activity was expressed as milligrams of trolox equivalents (mg TE/g extract).
For phosphomolybdenum method: Sample solution (1 mg/mL; 0.3 mL) was combined with 3 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The sample absorbance was read at 695 nm after a 90 min incubation at 95°C. The total antioxidant capacity was expressed as millimoles of trolox equivalents (mmol TE/g extract) (Mocan et al., 2016c (link)).
For metal chelating activity assay: Briefly, sample solution (1 mg/mL; 2 mL) was added to FeCl₂ solution (0.05 mL, 2 mM). The reaction was initiated by the addition of 5 mM ferrozine (0.2 mL). Similarly, a blank was prepared by adding sample solution (2 mL) to FeCl₂ solution (0.05 mL, 2 mM) and water (0.2 mL) without ferrozine. Then, the sample and blank absorbances were read at 562 nm after 10 min incubation at room temperature. The absorbance of the blank was sub-tracted from that of the sample. The metal chelating activity was expressed as milligrams of EDTA (disodium edetate) equivalents (mg EDTAE/g extract).
For ChE inhibitory activity assay: Sample solution (1 mg/mL; 50 μL) was mixed with DTNB (5,5-dithio-bis(2-nitrobenzoic) acid, Sigma, St. Louis, MO, United States) (125 μL) and AChE [acetylcholines-terase (Electric ell AChE, Type-VI-S, EC 3.1.1.7, Sigma)], or BChE [BChE (horse serum BChE, EC 3.1.1.8, Sigma)] solution (25 μL) in Tris–HCl buffer (pH 8.0) in a 96-well microplate and incubated for 15 min at 25°C. The reaction was then initiated with the addition of acetylthiocholine iodide (ATCI, Sigma) or butyrylthiocholine chloride (BTCl, Sigma) (25 μL). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (AChE or BChE) solution. The sample and blank absorbances were read at 405 nm after 10 min incubation at 25°C. The absorbance of the blank was subtracted from that of the sample and the cholinesterase inhibitory activity was expressed as galanthamine equivalents (mgGALAE/g extract) (Mocan et al., 2016b (link)).
For Tyrosinase inhibitory activity assay: Sample solution (1 mg/mL; 25 μL) was mixed with tyrosinase solution (40 μL, Sigma) and phosphate buffer (100 μL, pH 6.8) in a 96-well microplate and incubated for 15 min at 25°C. The reaction was then initiated with the addition of L-DOPA (40 μL, Sigma). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (tyrosinase) solution. The sample and blank absorbances were read at 492 nm after a 10 min incubation at 25°C. The absorbance of the blank was subtracted from that of the sample and the tyrosinase inhibitory activity was expressed as kojic acid equivalents (mgKAE/g extract) (Mocan et al., 2017 (link)).
For α-amylase inhibitory activity assay: Sample solution (1 mg/mL; 25 μL) was mixed with α-amylase solution (ex-porcine pancreas, EC 3.2.1.1, Sigma) (50 μL) in phosphate buffer (pH 6.9 with 6 mM sodium chloride) in a 96-well microplate and incubated for 10 min at 37°C. After pre-incubation, the reaction was initiated with the addition of starch solution (50 μL, 0.05%). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (α-amylase) solution. The reaction mixture was incubated 10 min at 37°C. The reaction was then stopped with the addition of HCl (25 μL, 1 M). This was followed by addition of the iodine-potassium iodide solution (100 μL). The sample and blank absorbances were read at 630 nm. The absorbance of the blank was subtracted from that of the sample and the α-amylase inhibitory activity was expressed as acarbose equivalents (mmol ACE/g extract) (Savran et al., 2016 (link)).
For α-glucosidase inhibitory activity assay: Sample solution (1 mg/mL; 50 μL) was mixed with glutathione (50 μL), α-glucosidase solution (from Saccharomyces cerevisiae, EC 3.2.1.20, Sigma) (50 μL) in phosphate buffer (pH 6.8) and PNPG (4-N-trophenyl-α-D-glucopyranoside, Sigma) (50 μL) in a 96-well microplate and incubated for 15 min at 37°C. Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (α-glucosidase) solution. The reaction was then stopped with the addition of sodium carbonate (50 μL, 0.2 M). The sample and blank absorbances were read at 400 nm. The absorbance of the blank was subtracted from that of the sample and the α-glucosidase inhibitory activity was expressed as acarbose equivalents (mmol ACE/g extract) (Llorent-Martínez et al., 2016 (link)).
All the assays were carried out in triplicate. The results are expressed as mean values and standard deviation (SD). The differences between the different extracts were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference post hoc test with α = 0.05. This treatment was carried out using SPSS v. 14.0 program.

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

Determination of total phenolic content (TPC): Amount of TP were assessed using the Folin-Ciocalteu reagent [25 ]. Briefly, the crude extract (50 mg) was mixed with Folin-Ciocalteu reagent (0.5 mL) and deionized water (7.5 mL). The mixture was kept at room temperature for 10 min, and then 20% sodium carbonate (w/v, 1.5 mL) was added. The mixture was heated in a water bath at 40 ^oC for 20 min and then cooled in an ice bath; absorbance was read at 755 nm using a spectrophotometer (U-2001, Hitachi Instruments Inc., Tokyo, Japan). Amounts of TP were calculated using gallic acid calibration curve within range of 10-100 mgL^-1(R² = 0.9986). The results were expressed as gallic acid equivalents (GAE) g/100g of dry plant matter. All samples were analyzed thrice and the results averaged. The results are reported on dry weight basis (DW).
Determination of total flavonoid contents (TFC): The TFC were measured following a previously reported spectrophotometric method [26 (link)]. Briefly, extracts of each plant material (1 mL containing 0.1 mg/mL) were diluted with water (4 mL) in a 10 mL volumetric flask. Initially, 5% NaNO₂ solution (0.3 mL) was added to each volumetric flask; at 5 min, 10% AlCl₃ (0.3 mL) was added; and at 6 min, 1.0 M NaOH (2 mL) was added. Water (2.4 mL) was then added to the reaction flask and mixed well. Absorbance of the reaction mixture was read at 510 nm. TFC were determined as catechin equivalents (g/100g of dry weight). Three readings were taken for each sample and the results averaged.
Determination of reducing power: The reducing power of the extracts was determined according to the procedure described earlier [27 ], with a slight modification. Concentrated extract (2.5-10.0 mg) was mixed with sodium phosphate buffer (5.0 mL, 0.2 M, pH 6.6) and potassium ferricyanide (5.0 mL, 1.0%); the mixture was incubated at 50 ^oC for 20 min. Then 10% trichloroacetic acid (5 mL) was added and the mixture centrifuged at 980 g for 10 min at 5 °C in a refrigerated centrifuge (CHM-17; Kokusan Denki, Tokyo, Japan). The upper layer of the solution (5.0 mL) was decanted and diluted with 5.0 mL of distilled water and ferric chloride (1.0 mL, 0.1%), and absorbance read at 700 nm using a spectrophotometer (U-2001, Hitachi Instruments Inc., Tokyo, Japan). All samples were analyzed thrice and the results averaged.
DPPH^. scavenging assay: 1, 1^’–diphenyl–2-picrylhydrazyl (DPPH) free radical scavenging activity of the extracts was assessed using the procedure reported earlier [28 (link)]. Briefly, to extract (1.0 mL) containing 25 μg/mL of dry matter in methanol, freshly prepared solution of DPPH (0.025 g/L, 5.0 mL) was added. Absorbance at 0, 0.5, 1, 2, 5 and 10 min was measured at 515 nm using a spectrophotometer. The scavenging amounts of DPPH radical (DPPH^.) was calculated from a calibration curve. Absorbance read at the 5th min was used for comparison of radical scavenging activity of the extracts.
Determination of antioxidant activity in linoleic acid system: The antioxidant activity of the tested plant extracts was also determined by measuring the oxidation of linoleic acid [28 (link)]. Five mg of each plant extract were added separately to a solution of linoleic acid (0.13 mL), 99.8% ethanol (10 mL) and 0.2 M sodium phosphate buffer (pH 7, 10 mL). The mixture was made up to 25 mL with distilled water and incubated at 40 ^oC up to 360 h. Extent of oxidation was measured by peroxide value applying thiocyanate method as described by Yen et al. [27 ]. Briefly, ethanol (75% v/v, 10 mL ), aqueous solution of ammonium thiocyanate (30% w/v, 0.2 mL), sample solution (0.2 mL) and ferrous chloride (FeCl₂) solution (20 mM in 3.5% HCl; v/v, 0.2 mL) were added sequentially. After 3 min of stirring, the absorption was measured at 500 nm using a spectrophotometer (U-2001, Hitachi Instruments Inc., Tokyo, Japan). A control contained all reagents with exception of extracts. Synthetic antioxidants butylated hydroxytoluene (BHT) was used as a positive control. Percent inhibition of linoleic acid oxidation was calculated with the following equation: 100 – [(increase in absorbance of sample at 360 h / increase in absorbance of control at 360 h) × 100], to express antioxidant activity.

This test was done to confirm the presence of tannins in a given sample. Here, 1 ml of each extract was diluted with 1 ml distilled water, followed by the addition of 2 drops of ferric chloride. A transient greenish to black color indicates the presence of tannins¹⁴ .