Chemicals and reagents
In the present study, all the chemicals (hexane, ethanol, ethyl acetate, millipore water, conc. H2SO4, conc. HNO3, conc. HCl, picric acid, Ferric chloride, α-naphthol, NaOH, sodium acetate, acetic acid, pet ether, chloroform, diethyl ether, acetone, methanol, lead acetate, ammonia, distilled water, DPPH, DMSO, sodium phosphate, ammonium molybdate, ascorbic acid, acetic anhydride, magnesium chips) are purchased from Ranbaxy/HI-MEDIA and are of analytical grade used without purifying further.
Sample collection and preparation of the samples
The leaves and bark of Acacia ferruginea are collected from the village Nalligoundanpalayam (11012′19.2″ N 77017′55.3″ E), Avinashi, Tamil Nadu state, India. The plant is authenticated by the Botanical Survey of India, Coimbatore, Tamil Nadu, India (BSI/SRC/5/23/2018/Tech/2080). The pre-cleaned leaves and bark are dried under shade and pulverized for extraction.
Extraction
The dried powder samples of A. ferruginea (170 g) are sequentially extracted with solvents with increasing polarities (hexane, ethyl acetate, ethanol, 90% ethanol, and distilled water (each 1200 mL)) using a Soxhlet extractor. Solvent traces are removed by drying and are concentrated by simple distillation. The crude extracts are further dried using a rotary evaporator, and aqueous extracts are concentrated by lyophilization (Christ Alpha 1-2 LD plus) and stored at 40 C for further analysis.
Physicochemical studies
The fresh plant (leaves and bark) powder is used to determine the physicochemical behavior according to methods described in Indian pharmacopeia.
The pH of different solutions of bark and leaves (1% w / v and 10% w / v) of A. ferruginea material is measured using a Hanna pH meter [31]. Ash values (total, water soluble, and acid insoluble) are determined by Thiex et al. [32]. Dry matter, moisture content (LOD) [33], foreign matter [34], solubility, water-soluble and alcohol-soluble extractive values [35], fluorescence studies [36] are also determined. All tests are carried out in triplicate, with the findings expressed as mean and standard deviation, and the details are given in Additional file 1: SI-M1.
Metal content analysis
The polar and non-polar extracts of A. ferruginea are subjected to elemental analysis by adhering the extract to adhesive carbon tape, which is subsequently placed on aluminum specimen stubs and sputtered with a gold sputter coater. The samples are then analyzed using a TESCAN MIRA 3 Scanning Electron Microscope with a magnification of 30 kV and an accelerating voltage of 200 V to 30 kV [25, 37].
Qualitative phytochemical studies
Qualitative phytochemical analyses for all of the extracts are done by the standard procedures [38]. The extracts are tested for alkaloids, tannins, phenols, flavonoids, steroids, saponins (Additional file 1: SI-M2).
Quantitative studies
The total terpenoids, alkaloids, and phenolics are quantified from A. ferruginea leaves and bark by standard methods [39].
Quantification of phenolic compounds
At room temperature, 10 g of the plant sample is extracted several times with 80% aq. methanol (100 mL). Whatman filter paper No. 42 is used to filter the entire solution (125 mm). Then, the filtrate is moved to a crucible, dried over a water bath, and weighed to a constant weight [39].
Quantification of alkaloids
To the plant sample (5 g) is added, and 10% acetic acid in ethanol (200 mL) is added and left to stand (4 h). Then, it is filtered and concentrated for a quarter of an hour in a water bath. Drop by drop ammonium hydroxide is applied until the precipitation is complete [39]. The precipitate is cleaned with ammonium hydroxide, dried, and weighed, and the following calculations are made:
$$\% {\text{Alkaloid}} = W_{a} /W_{b} \times 100$$
Wa—weight of alkaloid; Ws—weight of sample.
Quantification of terpenoids
The dried plant extract (100 mg (Wa)) is immersed in 9 mL ethanol for 24 h. Then, it is extracted with petroleum ether (10 mL) using a separating funnel. The ether extract is separated, into pre-weighed beakers (Wb) and allowed to dry completely [39]. After ether is evaporated, the yield (%) of total terpenoid content is estimated using the following formula
$$\% {\text{Terpenoid}} = W_{a} - W_{b} /W_{a} \times 100$$
Antioxidant activity
Total antioxidant activity (TAA)
Sample preparation
The plant sample (10 mg) is dissolved in 1 mL DMSO solution and incubated (4000 C) for 24 h in a shaking incubator (100–120 rpm).
Phatak and Hendre's [40] procedure, with minor modifications, is used to validate total antioxidant activity employing the phosphomolybdenum method. The sample (0.5 mL) is diluted with the reaction mixture (0.5 mL of 28 mM sodium phosphate, 0.6 M H2SO4, and 4 mM ammonium molybdate reagent solution) and incubated (500 °C; 90 min) with a blank tube (without sample). The tubes are sterilized after incubation, and the absorbance is measured at 695 nm using a UV–visible spectrophotometer (Shimadzu, 1601). TAA (mg/g) is measured using ascorbic acid as the standard.
DPPH radical scavenging assay
The DPPH radical scavenging activity of the different extracts is assessed based on the method given by Chaves et al. [41] The diluted test solutions (25–200 μg/mL) of each extract are added to 0.3 mM DPPH in methanol solution to reach the final volume of 2 mL. An equal volume of DPPH and methanol served as a blank. After 30 min of incubation at room temperature, the resultant solutions appeared yellow rather than purple color. The colorimetric absorbance is fixed at 520 nm, and the percentage of inhibition is calculated using the formula below.
$${\text{Inhibition}}\% = A_{i} - A_{ii} /A_{i} \times 100$$
Ai—standard absorbance control; Aii—absorbance for extracts.
Spectral fingerprinting
UV
The extracts (2 mL/1000 ppm) are dissolved in their respective solvents, and the UV–Vis spectra are recorded in a double-beam UV spectrophotometer (Systronics U-2701) in the wavelength range of 200–800 nm. A shift reagent (NaOEt) is added to the polar extracts to observe the shift.
FT-IR
FT-IR spectral measurements are recorded in PerkinElmer FTIR00585 spectrophotometer in the range 4000–800 cm−1.
Fluorescence measurements
The fluorescence (excitation/emission) spectra of A. ferruginea extracts are measured using a fluorescence spectrophotometer (Agilent technologies, G9800A) with a wavelength range of 190–1100 nm. Absorption and fluorescence measurements are taken for the extract solution (2 mL/1000 ppm) using quartz cuvettes (path length: 1 cm) at room temperature.