Estimation of phytochemical constituents and in vitro antioxidant potencies of Brachychiton populneus (Schott & Endl.) R.Br.

DPPH radical scavenging activity

The IC₅₀ values of DPPH radical scavenging activity of B. populneus extract/fractions are shown in Table 4. Best values for IC₅₀ were exhibited by BPA (46.51 ± 2.1 µg/ml) followed by BPE (48.32 ± 2.1 µg/ml), BPB (63.38 ± 3.4 µg/ml), BPM (143.7 ± 2.7 µg/ml), BPC (259.6 ± 3.3 µg/ml) and BPH (461.7 ± 1.5 µg/ml). The observed order of IC₅₀ of different fractions was BPA < BPE < BPB < BPM < BPC < BPH. The DPPH radical scavenging activity of extract and its various fractions showed significant correlation with TPC (R² = 0.8529**, p < 0.01) and TFC (R² = 0.8567**, p < 0.01) (Table 5). All the fractions showed higher IC₅₀ values than ascorbic acid (29.57 ± 1.1 µg/ml). Concentration dependent activity was observed as illustrated in Fig. 3.

Sample	DPPH scavenging	Hydroxyl radical scavenging	Nitric oxide scavenging activity	β-carotene bleaching inhibition activity	Iron chelating activity
	IC50 (μg/ml)
BPM	143.7 ± 2.7c	345.6 ± 2.1c	130 ± 2.9c	148.8 ± 2.3c	761.5 ± 1.9c
BPH	461.7 ± 1.5a	618.3 ± 4.0b	180.4 ± 3.3a	347.3 ± 3.6a	> 1000
BPC	259.6 ± 3.3b	764.8 ± 2.5a	165.5 ± 2.7b	244.8 ± 2.8b	> 1000
BPE	48.32 ± 2.1e	180.5 ± 3.6e	82.7 ± 3.08d	77.9 ± 1.5e	336.7 ± 1.8e
BPB	63.38 ± 3.4d	255.0 ± 2.2d	121.2 ± 2.5c	115.3 ± 2.1d	605.4 ± 2.3d
BPA	46.51 ± 2.1e	144.3 ± 3.2f	46.49 ± 2.7e	40.04 ± 3.1g	249.8 ± 2.8f
Rutin	–	110.7 ± 1.7g	–	–	–
Ascorbic acid	29.57 ± 1.1f	–	16.4 ± 1.6f	–	–
Catechin	–	–	–	58.4 ± 2.8f	–
EDTA	–	–	–	–	177.2 ± 2.8g

Values are presented as mean ± SD (n = 3). Means with different superscript (a–g) letters in the rows are significantly (p < 0.01) different from each other

BPM: B. populneus methanol extract; BPH: B. populneus n-hexane fraction; BPC: B. populneus chloroform fraction; BPE: B. populneus ethyl acetate fraction; BPB: B. populneus butanol fraction; BPA: B. populneus aqueous fraction

Antioxidant activity	Correlation R2
	TFC	TPC
DPPH scavenging activity	0.856**	0.852**
Hydroxyl radical scavenging activity	0.888**	0.721*
Iron chelating assay	0.988***	0.872**
Nitric oxide scavenging activity	0.949***	0.988***
β-Carotene bleaching activity	0.956***	0.876**
Total antioxidant activity	0.941***	0.977***
Total reducing power assay	0.978***	0.953**

TFC: total flavonoid content; TPC: total phenolic content

Column with different *, **, *** are significantly different at p < 0.05, p < 0.01 and p < 0.001

Hydroxyl radical (•OH) scavenging activity

All the extract/fractions of B. populneus scavenged •OH radicals and prevented 2-deoxyribose breakdown in this assay. A concentration-dependent pattern was observed for hydroxyl radical scavenging activity (Fig. 3). Lowest IC₅₀ values were shown by BPA and BPE (144.3 ± 3.2 μg/ml and 180.5 ± 3.6 μg/ml) respectively followed by BPB (255.0 ± 2.2 μg/ml), BPM (345.6 ± 2.1 μg/ml) while the highest IC₅₀ was observed for BPH and BPC (618.3 ± 4.0 μg/ml and 764.8 ± 2.5 μg/ml) respectively. IC₅₀ values of different fractions were significantly different from the used standard rutin (110.7 ± 1.7 μg/ml). Overall pattern of BPA < BPE < BPB < BPM < BPH < BPC was observed (Table 4). A good correlation (R² = 0.7216*, p < 0.01) was observed with TPC as well as (R² = 0.8881**, p <0.01) with TFC (Table 5).

Nitric oxide (NO⁻) scavenging activity

In the present study, the lowest IC₅₀ value for nitric oxide scavenging activity was observed for BPA (46.49 ± 2.7 μg/ml) and BPE (82.7 ± 3.08 μg/ml) followed by BPB (121.2 ± 2.5 μg/ml), BPM (130.0 ± 2.9 µg/ml), BPC (165.5 ± 2.7 μg/ml) and BPH (180.4 ± 3.3 μg/ml) as compared to standard ascorbic acid (16.4 ± 1.6 µg/ml) as shown in Table 4. The % inhibition pattern is shown in Fig. 3. A highly significant (p < 0.01) correlation of IC₅₀ was observed with TPC (R² = 0.988***, p < 0.001) and TFC (R² = 0.9494***, p < 0.001) (Table 5).

β-Carotene scavenging activity

The BPA of B. populneus showed the lowest IC₅₀ value (40.04 ± 3.1 μg/ml) as compared to other fractions viz. BPE (77.9 ± 1.5 μg/ml), BPB (115.3 ± 2.1 μg/ml), BPM (148.8 ± 2.3 μg/ml), BPC (244.8 ± 2.8 μg/ml) and BPH (347.3 ± 3.6 μg/ml). All the fractions showed higher IC₅₀ values than catechin (58.4 ± 2.8 μg/ml) while IC₅₀ value of BPA was lower than catechin as shown in Table 4. The concentration dependent bleaching power pattern observed is shown in Fig. 3. The IC₅₀ values showed significant correlation with both TPC (R² = 0.8764**, p < 0.01) and TFC (R² = 0.9566***, p < 0.001) (Table 5).

Iron chelating activity

IC₅₀ values for Iron chelating activity of different fractions of B. populneus are given in (Table 4). The best IC₅₀ value for iron chelating activity was exhibited by BPA (249.8 ± 2.8 µg/ml) followed by BPE (336.7 ± 1.8 µg/ml), BPB (605.5 ± 2.3 µg/ml) and BPM (761.6 ± 1.9 µg/ml) while BPC and BPH showed the higher IC₅₀ values (> 1000 µg/ml). IC₅₀ value of standard EDTA was 177.2 ± 2.8 µg/ml as shown in Table 3. Significant correlation of IC₅₀ (R² = 0.8721**, p < 0.01) was observed with TPC and also with TFC (R² = 0.9888***, p < 0.001) as listed in Table 5. The  % inhibition of iron chelating assay is depicted in Fig. 3.

Phosphomolybdenum assay

Total antioxidant capacity of various extract/fractions was determined by phosphomolybdate method and expressed as equivalents of ascorbic acid (mg/g of extract) at 500 μg/ml sample as shown in Fig. 4a. Maximum antioxidant activity was shown by BPA (851.6 ± 2.2 mg ascorbic acid equivalents/g sample) followed by BPE (759.03 ± 2.28 mg ascorbic acid equivalents/g sample), BPB (685.4 ± 0.86 mg ascorbic acid equivalents/g sample), BPM (685.4 ± 205 mg ascorbic acid equivalents/g sample), BPC (592.5 ± 1.37 mg ascorbic acid equivalents/g sample) and BPH (555.6 ± 1.1 mg ascorbic acid equivalents/g sample) and was found to decrease in the order of BPA > BPE > BPB > BPM > BPC > BPH. The assay showed highly significant (p < 0.001) correlation with TPC (R² = 0.9774***) and TFC (R² = 0.9412***) (Table 5).

Reducing power activity

Brachychiton populneus aqueous fraction showed the highest reducing power (988.34 ± 2.1 mg ascorbic acid equivalent/g sample) measured at 500 μg/ml of extract concentration followed by BPE (956.2 ± 1.71 mg ascorbic acid equivalents/g sample), BPB (933.6 ± 3.04 mg ascorbic acid equivalents/g sample), BPM (885.4 ± 2.06 mg ascorbic acid equivalents/g sample), BPC (850.19 ± 2.6 mg ascorbic acid equivalents/g sample) and BPH (822.3 ± 1.8 mg ascorbic acid equivalents/g sample) as shown in Fig. 4b. There was exhibited a significant correlation (p < 0.001) with both TPC (R² = 0.9534***) and TFC (R² = 0.9783***) (Table 5).

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.

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].

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].

DPPH (1, 1-diphenyl-2-picryl-hydrazyl) radical scavenging assay

Hydroxyl radical scavenging assay

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

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.