Plant collection
Plant collection was done from Quaid-i-Azam University, Islamabad in January–February 2017. The plant was identified by its native name and then confirmed by senior plant taxonomist; Syed Afzal Shah, Department of Plant Sciences, Quaid-i-Azam University, Islamabad. Voucher specimen (036245) was deposited at the Pakistan Herbarium, Quaid-i-Azam University, Islamabad.
Preparation of extract
The aerial parts of the plant were washed away to remove dust particles and dried under shade for few weeks. The fully dried plant material was then ground to powder and sieved through 60-mesh topology Willy Mill to get fine powder of same particle size. Extraction was carried out by mixing 1.2 kg of plant aerial part powder with 3 l of commercial methanol at 25 °C for 48 h. Filtration was performed by using Whatman No.1 filter paper. The filtrate was further processed in rotary vacuum evaporator for evaporation and obtained the methanol extract (BPM). Distilled water was used to suspend a part of BPM and then it was passed to liquid–liquid partition. The solvents were used in order of n-hexane (non-polar), chloroform, ethyl acetate (polar solvent), butanol (polar solvent). Fractions of these solvents were separated accordingly and named as BPH (hexane fraction), BPC (chloroform fraction), BPE (ethyl acetate fraction) and BPB (butanol fraction). The residue after last fraction was also collected and termed aqueous fraction and abbreviated as BPA. Each fraction was dried, weighed and stored for further pharmacological observations.
Qualitative phytochemical analysis
Qualitative screening of B. populneus methanol extract along with its fractions was performed to identify the active phytochemicals like flavonoids, phenols, tannins, alkaloids, saponins, terpenoids, coumarins, anthocyanins and anthraquinones.
Assessment of phenols
For qualitative assessment methodology of Harborne [37] was followed. An amount of 1 mg of each sample was taken and 2 ml of distilled water and 10% ferric chloride was added in it. The confirmation sign of phenols presence was formation of green or blue color.
Assessment of flavonoids
Alkaline reagent test This test was performed by following the protocol of Trease and Evans [38]. Each sample (1 mg) was added in 1 ml of 2 N sodium hydroxide. The confirmation sign of flavonoids presence was formation of yellow color.
FeCl3 test Few drops of FeCl3 solution were added in 1 ml of each extract. Existence of flavonoids was indicated by formation of blackish red precipitate [39].
Assessment of coumarins
Each sample (1 mg) was endorsed to react with 1 ml sodium hydroxide (10%). Formation of yellow color in test sample was an indication of the presence of coumarins [37].
Assessment of saponins
Froth formation with distilled water Each sample (2 mg) was mixed with 2 ml of distilled water in the test tube. After this accumulation, the test sample was mixed vigorously for almost 15 min. The formation of a soapy layer indicated the presence of saponins in test samples [37].
Emulsion test with olive oil A volume of 1 ml of each sample was poured in test tubes followed by addition of 5–6 drops of olive oil and shaken vigorously to form a stable froth. Formation of an emulsion was the confirmatory sign of saponin presence [39].
Assessment of tannins
FeCl3 test To 1 mg of each sample, 2 ml of 5% ferric chloride was added. Appearance of greenish black or dark blue color was the indication of tannins presence [38].
Alkaline reagent test A volume of 2 ml of 1 N NaOH solution was added in 2 ml of each plant extract. Appearance of yellow to red color showed the presence of tannins [39].
Assessment of terpenoids
Each sample (0.5 mg) was taken in the test tube and 2 ml of each chloroform and concentrated sulphuric acid was added to plant samples. Presence of terpenoids was indicated by the formation of brown layer in the middle of other two layers [38].
Assessment of anthraquinones
To 1 mg of each sample, hydrochloric acid diluted to 2% was added. The appearance of red color was the confirmatory sign of anthraquinone presence [37].
Assessment of anthocyanin and betacyanins
Each sample (1 mg) was taken in the test tube and followed by the addition of 2 ml of 1 N sodium hydroxide. The test sample was boiled at 100 °C for about 10 min. Anthocyanin presence was indicated by the formation of bluish green color while yellow color formation was indicative of betacyanin presence [38].
Assessment of alkaloids
Mayer’s test Each sample (2 ml) was allowed to react with conc. HCl and a special reagent named Mayer’s reagent. Formation of white precipitates or appearance of green color was indication of alkaloids presence [38].
Hager’s test Few drops of Hager’s (Saturated picric acid solution) reagent were added to 2 ml of each plant extract. Formation of bright yellow precipitates specified the manifestation of alkaloids [39].
Assessment of glycosides
Keller Killanis’ test To 1 ml of each plant extract, 1 ml glacial acetic acid was added and left to cool down. After cooling two drops of FeCl3 were added and 2 ml of concentrated H2SO4 along the walls of test tube was dispensed carefully. Development of reddish brown colored ring at the intersection of two layers indicated the presence of glycosides [39].
Assessment of sterols
Salkowski test To 2 ml of each of the plant extracts, 5 ml of chloroform was added and then 1 ml concentrated H2SO4 was carefully dispensed along the walls of the tube. The appearance of reddish color in the lower layer indicated the existence of sterols [39].
Assessment of vitamin C
DNPH test Dinitrophenyl hydrazine was dissolved in concentrated sulphuric acid and allowed to react with 1 ml of plant sample. Appearance of yellow precipitates indicated the presence of vitamin C in test samples.
Assessment of proteins
Xanthoproteic test According to this procedure, 1 ml of each plant sample was treated with few drops of conc. nitric acid. Presence of proteins in test samples was indicated by the formation of yellow color.
Biuret test An amount of 0.5 mg of each plant test solution was taken and equal volume of sodium hydroxide solution (40%) was added to it. After that few drops of 1% CuSO4 solution was added. Appearance of violet color in test samples manifested protein presence.
Assessment of steroids and phytosteroids
A volume of 1 ml of chloroform and few drops of concentrated sulphuric acid were added to 1 ml of plant test sample. Formation of brown-colored ring indicated steroids presence whereas appearance of bluish-brown colored ring marked the presence of phytosteroids in the test samples.
Assessment of phlobotannins
To 1 ml of each plant sample few drops of 10% ammonia solution were added. Formation of pink-colored precipitates showed the existence of phlobatannins in samples.
Assessment of triterpenoids
A volume of 1 ml of Libermann-Buchard Reagent (conc. H2SO4 + acetic anhydride) was added in 1.5 ml plant test samples. Triterpenoids were determined by the appearance of bluish-green color in the test samples.
Assessment of quinones
A volume of 1 ml of each plant sample was allowed to react with 1 ml concentrated sulphuric acid. Appearance of red color manifested the occurrence of quinones.
Assessment of oils and resins
Filter paper test Each plant sample was applied on filter-paper and checked for the establishment of transparent appearance which was a positive sign for the presence of oils and resins in respective test samples.
Quantitative spectrophotometric phytochemical analysis
Various fractions of B. populneus were evaluated spectrophotometrically, employing standardized procedures for the quantification of chief phytochemical constituents including phenols, flavonoids, and tannins.
Total phenolic content (TPC)
Determination of total phenolic content was done by spectrophotometer [40]. A volume of 1 ml of each sample was mixed with 2 ml of Phenol Folin–Ciocalteu mixture following 9 ml of pure deionized water in a volumetric bottle having capacity up to 25 ml. After shaking, 10 ml of 7% Na2CO3 was added. Vigorous stirring was practiced following instant dilution of the final mixture with pure deionized water making final volume up to 25 ml. After keeping the final mixture at 23 °C for at least 90 min, the optical density was checked at wavelength of 750 nm. The whole assay was repeated thrice for ensuring accuracy against the standard gallic acid. TPC was expressed as mg GAE (gallic acid equivalents)/gram dry weight extract/fraction.
Total flavonoid contents (TFC)
The spectrophotometric technique is the easiest and affordable technique for finding out the flavonoid contents within a plant [41]. The reaction mixture was made in a test tube by the scheduled addition of 0.3 ml plant sample, 0.15 ml of NaNO2 (0.5 mol/l) along with 0.3 M AlCl3·6H2O and 3.4 ml methanol (30%). The assortment was kept for 5 min and then 1 ml of 1 M NaOH was mixed in it. At 506 nm wavelength the optical density of the reaction mixture was detected using rutin as standard for comparison using concentrations 0–100 mg/ml.
Total tannin content (TTC)
Procedure of Van Buren and Robinson [42], was followed for the quantification of tannin content with slight modifications. According to this procedure, 500 mg plant sample was soaked in 50 ml distilled water. The sample was placed on mechanical shaker for 1 h and then filtered. The filtrate was made up to the mark in volumetric flask (50 ml). A volume of 2 ml of FeCl3 (0.1 M) and potassium ferricyanide (0.008 M) prepared in HCl (0.1 N) was mixed with 5 ml of the above filtrate. The absorbance was recorded at 200 nm via spectrophotometer against standard curve of gallic acid and the results were quantified as mg of gallic acid equivalents (GAE)/gram of dry plant extract [43].
Quantitative non-spectrophotometric phytochemical analysis
Brachychiton populneus fractions were quantified employing standardized non-spectrophotometric procedures for the presence of alkaloids, terpenoids, flavonoids and saponins.
Quantification of alkaloids
Alkaloids were quantified by following the procedure of [37]. A volume of 7.5 ml of 10% acetic acid prepared in ethanol was added to 10 mg of each plant sample. The sample assortment was covered and endorsed to stand for 4 h time interval. The mixture was then filtered and the subsequent filtrate was concentrated on a water-bath to reduce its volume up to one-fourth of its original volume. The extract sample was then finally precipitated by adding conc. NH4OH drop wise. The solution was endorsed to settle and the precipitates were collected after filtration. The residue obtained after washing with dilute NH4OH was completely dried and finally weighed to calculate the alkaloid percentage in respective plant samples.
Quantification of flavonoids
Flavonoid content was quantified by following the methodology of Krishnaiah et al. [44]. Plant sample (100 mg) was extracted repeatedly with 80% aqueous methanol (10 ml) at room temperature followed by filtration through Whatman-42 filter paper (125 mm). The filtrate was left for complete evaporation in water-bath after transferring into a crucible for complete dryness. The sample was weighed after constant weight obtained.
Quantification of saponins
Saponins were quantified following the methodology of Obadoni and Ochuko [45]. An amount of 100 mg of each plant sample was dispersed in 15 ml of aqueous ethanol (20%). The suspension was heated on water bath at 55 °C for 4 h with constant stirring. The mixture was filtered followed by re-extraction with another 15 ml of aqueous ethanol (20%). Both the extracts were combined and concentrated to 4 ml on water-bath at 90 °C. Then 2 ml of di-ethyl ether was added to the concentrate in a separating funnel. Aqueous layer was collected whereas ether layer formed was discarded. The sample was purified by repeating the above process. Finally 5 ml of n-butanol was added and n-butanol combined extracts were washed twice with 1 ml of 5% aqueous NaCl. The remaining solution was completely evaporated on water-bath. A constant weight of the sample was obtained after placing it in oven and saponin content was quantified as %age yield of plant sample.
Quantification of terpenoids
Plant sample (100 mg) was soaked in alcohol and placed for 24 h at room temperature. Next day the extract sample was and the filtrate was thoroughly extracted with petroleum ether. The ether extract obtained was treated as the total terpenoids in the sample [46].
Antioxidant capacity determination assays
For antioxidant potential determination seven different assays were performed to assess the antioxidant prospective against various free radicals and by different mechanisms of action. Extracts, fractions and positive standards (ascorbic acid, Rutin, catechin and gallic acid) 1 mg were liquefied in 1 ml analytical methanol or DMSO. These solutions were further serially diluted to 1000, 500, 250, 125, 62, 31.25, 15.62 µg/ml. In all assays, same dilutions of samples and standards were used; while standards were altered according to the requirement of assay.
DPPH (1, 1-diphenyl-2-picryl-hydrazyl) radical scavenging assay
The DPPH foraging competencies of damaging effects of the free radicals were evaluated by following the methodology of Brand-Williams et al. [47] with slight modifications. The stock solution of DPPH was prepared by dissolving 0.24 g of it in 100 ml of methanol and kept at 20 °C for further use. The stock solution was further diluted with methanol to optimize its absorbance (0.908 ± 0.02) at 517 nm. Now 100 µl of plant samples was mixed with 900 µl of DPPH aliquot and incubated for 15 min at room temperature in dark. Optical density was checked at wavelength of 517 nm by running Ascorbic acid as standard. Antioxidant capacity was determined by following Eq. 1:
$$ {\text{Free radical scavenging activity }}\left( {\text{\%}} \right) = \left( {\frac{{{\text{Control absorbance}} - {\text{Sample absorbance}}}}{\text{Control absorbance}}} \right) \times 100 $$
(1)
Hydroxyl radical scavenging assay
Hydroxyl free radicals scavenging potential of plant extracts was assessed by using the methodology accomplished by Halliwell and Gutteridge [48]. This technique involves mixing of 500 µl of 2.8 mM 2-deoxyribose, being prepared in phosphate buffer (50 mM) maintaining its pH 7.4, EDTA 0.1 M, 200 µl of 100 mM ferric chloride, 100 µl of 200 mM H2O2 and 100 µl of each plant sample in the reaction recipe. The reaction was initiated by the addition of 100 µl of 300 mM ascorbic acid and incubated at 37 °C for 1 h. After this 1 ml of 2.8% TCA and 1 ml of TBA (1% weight by volume) prepared in 50 mM NaOH were added to the reaction mixture. This whole recipe was heat treated for 15 min in water bath and then placed for cooling. Optical density was recorded at 532 nm. The hydroxyl radical scavenging activity was analyzed by following formula:
$$ {\text{Free radical scavenging activity }}\left( {\text{\%}} \right) = \left( {\frac{{1 - {\text{Sample absorbance}}}}{\text{Control absorbance}}} \right) \times 100 $$
Nitric oxide scavenging assay
Bhaskar and Balakrishnan [49] developed the methodology using Griess reagent to assess the antioxidant potential of plant samples. Equimolar quantity of napthylenediamine (0.1%) in distilled water and sulphanilamide (1%) in phosphoric acid (5%) was added to prepare griess reagent. 100 μl of 10 mM sodium nitroprusside being prepared in saline phosphate buffer was added to 100 μl of each plant sample. Then 1 ml of griess reagent was added to, reaction mixture, incubated for 3 h and analyzed spectrophotometrically at 546 nm by using ascorbate as a positive control. The percentage inhibition of nitric oxide radical formation was determined by following Eq. 1.
Chelating power assay
The iron (II) binding capability at multiple sites confers the antioxidant potential of plant samples following the methodology of Dastmalchi et al. [50]. A volume of 200 µl of plant sample was taken as plant aliquot, 900 µl of methanol and 100 µl of 2 mM FeCl2·2H2O was added to it and nurtured for 5 min. 400 µl of 5 mM ferrozine was added to initiate the reaction. The whole reaction mixture was further incubated for 10 min and then submitted to spectrophotometry at 562 nm by using EDTA as standard. The chelating power was determined by Eq. 1.
β-Carotene bleaching assay
β-Carotene bleaching activity of the plant was assessed by following the methodology suggested by Elzaawely et al. [51]. The format of this protocol was that a mixture of β-carotene was made by adding 2 mg of it with that of 10 ml chloroform followed by mixing 200 mg of Tween 80 and 20 mg of linoleic acid. The chloroform was evaporated out of the mixture by the help of vacuum and then 50 ml of distilled water was added to it, vigorously vortexed to get a uniform emulsion of β-carotene linoleate. Then 250 µl of freshly prepared emulsion was added to 30 µl of each plant sample and optical density was measured at time 0 h at wavelength 470 nm. The reaction mixture was kept at 45 °C for 2 h and the final optical density was measured again. Catechin served as standard in this assay. Inhibition of β-carotene was detected by slight alteration in the formula used by Mallet et al. [52]
$$ \% {\text{ inhibition }} = \, \left[ {\left( {{\text{A}}_{{{\text{A }}( 1 20)}} {-}{\text{ A}}_{{{\text{C }}( 1 20)}} } \right) \, /\left( {{\text{A}}_{{{\text{C }}(0)}} {-}{\text{ A}}_{{{\text{A }}( 1 20)}} } \right)} \right] \times 100 $$
where AA (120) is the antioxidant absorbance at t = 120 min, AC (120) is the control absorbance at t = 120 min, and AC (0) is the control absorbance at t = 0 min.
Reducing power assay
By the method of Landry et al. [53] reducing power activity was calculated. Plant extract 2 ml was mixed with 2 ml of 0.2 M phosphate buffer (pH 6.6) and 2 ml of potassium ferricyanide (10 mg/l) were mixed up and incubated at 50 °C for 20 min. Then 2 ml of trichloroacetic acid (TCA) (100 mg/l) was added to the solution. After this 2 ml of the above solution was picked up and diluted with 2 ml of pure H2O and 0.4 ml of FeCl3 (0.1%) in a test tube. Standard used in this assay was gallic acid. Optical density was measured after 10 min at 700 nm.
Phosphomolybdenum assay
The antioxidant capabilities of the plant sample was assured by phosphomolybdenum assay as per described in the methodology of Umamaheswari and Chatterjee [54]. Phosphomolybdenum reagent solution was prepared by mixing Na3PO4 (28 mM) and H2SO4 (0.6 M) with that of ammonium molybdate (4 mM). The reaction mixture is heated at 95 °C in water bath for 90 min taking a good care that it is fully covered with silver foil to avoid direct light exposure. After this heat treatment, the reaction mixture was cooled at room temperature for some time and submitted to spectrophotometric analysis at 765 nm. Ascorbic acid serves as a standard in this assay.
Statistical analysis
Experimental data results were conveyed in the form of mean ± standard deviation (SD) having triplicate analysis. The data was recognized and investigated by using the computerized GraphPad Prism (5.0) software to calculate the IC50 values. Statistix software 8.1 was used for further statistical analysis followed by applying ANOVA (One-way analysis variance) for the calculation of differences among various groups. The data is given as Additional file 1.
