Solvent polarity mediates phytochemical yield and antioxidant capacity of Isatis tinctoria

Sample collection

Isatis tinctoria (woad) plants were collected from the mountains of district Lower Dir (34°50′43.19″N, 71°54′16.43″E), Khyber Pakhtunkhwa (KP), Pakistan in March 2014. This district was selected because of the natural habitat for the woad plants. Plants were immediately transferred to Molecular Systematics and Applied Ethnobotany Lab (MOSAEL) at Quaid-I-Azam University (QAU), Islamabad, Pakistan. By the help of taxonomist at QAU, the plants were confirmed as Isatis tinctoria (Ullah et al., 2017). The plants were washed with tap water to remove the dust. Four parts woad plant (branches, flowers, leaves, and roots) were separately shade-dried (indoor not exposed to sunlight) at room temperature, ground into a fine powder by using FGHGF 2,500 g grinder. The powder sifting was done via strainer (2 mm pore size) for uniform particle size and stored at 4 °C.

Solvent selection and combination

Solvents (n-hexane, chloroform, ethyl acetate, acetone, ethanol, methanol, and water) were selected and a combination (1:1, v/v, n-hexane-ethyl acetate, n-hexane-ethanol, methanol-chloroform, methanol-ethyl acetate, methanol-acetone, acetone-water, and methanol-water) of these solvents were employed for the extraction. These solvents were selected and combined on the bases of solvent polarity index and miscibility according to HPLC Solvent Guide, Solvent Miscibility and Viscosity Chart adapted from Paul Sadek in 2002, which was previously adapted by various researchers (Menkiti, Agu & Udeigwe, 2015; Ullah et al., 2017; Chinchu & Kumar, 2018).

Extraction

For the extraction efficiency, 20 g powder of branches, flowers, leaves, and roots were separately soaked in 1 L canonical flasks containing 500 mL solvent (14 different flasks with different solvent for each plant part). The flasks, containing soaked plant powder, were sealed with a cotton plug and aluminum foil and placed on a shaker (BT1010 Benchmark Scientific Orbi-Shaker XL, NJ-USA) at room temperature for 24 h. After 24 h of incubation in the shaker, the flasks were transferred to a sonicator (SONICA Ultrasonic Sonication, Meizhou, Guangdong, China) for 5 min. The sonicated suspension was strained through a sterilized cheesecloth followed by filtration through Whatman filter paper grade 1. The filtrate was first evaporated using rotary evaporator (R-300 Rotary Evaporator; Buchi, Flawil, Switzerland), followed by vacuum drying at 0.06 atm pressure (Rocker 300C Vacuum Pump; Rocker, Kaohsiung, Taiwan) at room temperature.

The extraction efficiency of the solvent was calculated using the formula; ${\displaystyle \text{\%}\mathrm{efficiency}=\left[\frac{W2-W1}{W3}\right]\times 100}$

where,

W1 = Weight of empty bottle

W2 = weight of bottle + Extract

W3 = Weight of powder used for extraction

Four milligrams of each extract was dissolved in 1 mL DMSO (Dimethyl sulfoxide) for further analysis.

Total phenolic content (TPC) assay

To investigate the role of solvent polarity on the TPC, 20 µL of each sample (14 different extracts from branches, flowers, leaves, and roots each, 400 µg/mL DMSO), 20 µL of gallic acid (1 mg/mL, as positive control), and 20 µL DMSO and methanol (as a negative control) were added to 96-wells microplates, followed by the addition and mixing of 90 µL of the Folin-Ciocalteu’s reagent (10 times diluted, 100 mmol/L) to each plate via multi-channel micropipette. The plates were incubated at room temperature for 5 min. Finally, 90 µL sodium carbonate (7% w/v) was added to each well and mixed properly followed by 90 min incubation at room temperature (Chandra et al., 2014). Readings were carried out at 630 nm wavelength via microplate reader (Biotek ELX 800; Biotek, Winooski, VT, USA). Results were calculated and expressed as gallic acid equivalent (GAE) µg/mg of extract (Al-Duais et al., 2009).

Total flavonoids content (TFC) assay

To investigate the role of solvent polarity on the TFC, aluminum chloride colorimetric method (Chang et al., 2002) was modified for the microplate method. Briefly, 20 µL of each sample (400 µg/mL), 20 µL of quercetin (1 mg/mL, as positive control), and 20 µL DMSO and methanol (as negative) were added to 96 wells plate, followed by the addition and mixing of 10 µL of aluminum chloride (10 g/L) in distilled water, 10 µL of potassium acetate (98.15 g/L) and 160 µL of distilled water via multi-channel micropipette. The plates were incubated at 37 °C for 30 min. The readings were taken at 450 nm via microtiter plate reader (Biotek ELX800; Winooski, VT, USA). The results were expressed as quercetin equivalent (QE) µg/mg of extract.

Ferric reducing antioxidant power (FRAP) assay

To investigate the impact of solvent polarity on the total reducing power of extracts, potassium ferricyanide trichloroacetic acid method was used (Benzie & Strain, 1996) with some modifications and adaptation for microplate method (Athamena et al., 2019). Ferric reducing antioxidant power (FRAP) will be written as total reducing power (TRP) beyond this point. Eppendorf tubes were labeled, 40 µL sample was added to each tube followed by 50 µL (0.2 mol/L) sodium phosphate dihydrate (Na₂HPO₄. 2H₂O) buffer, 50 µL 1% potassium ferricyanide (K₃Fe (CN)₆), and 50 µL 10% trichloroacetic acid. The mixture was centrifuged at 3,000 rpm for 10 min. After centrifugation 166.66 µL of the supernatant of each sample were added to 96 well plates followed by 33.3 µL ferric chloride (FeCl₃, 1%). The readings were taken at 630 nm via microtiter plate reader (Biotek ELX800; Biotek, Winooski, VT, USA). DMSO was used as negative control and ascorbic acid (1 mg/mL) as a positive control. Results were expressed as ascorbic acid equivalent (AAE) µg/mg of extract.

Total antioxidant capacity (TAC) assay

To evaluate the possible role of solvent polarity in the TAC of the extracts, the phosphomolybdenum method (Benzie & Strain, 1996) was used with some modifications and adaptations to microplate method (Lahmass et al., 2018). Microplates were labeled accordingly, followed by the addition 20 µL of the samples, 20 µL ascorbic acid (as a positive control), 20 µL DMSO each in different wells. All the wells were loaded with 180 µL of the reagent (0.6 mol/L H₂SO₄, 28 mmol/L NaH₂PO₄, 4 mmol/L ammonium molybdate). The plates were covered and incubated at 95 °C for 60 min in a water bath. The sample was transferred to other plates after cooling. The readings were taken at 695 nm via microtiter plate reader (Biotek ELX800). Values were expressed as ascorbic acid equivalent (AAE) µg/mg of extracts (Lahmass et al., 2018).

DPPH free radical scavenging assay

To analyze the possible free radical scavenging capacity of all extracts, which are extracted with different polarity solvents and from different plant parts, the scavenging of free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), was investigated using 96-well microplates read by a microplate reader (Beara et al., 2009; Zhu et al., 2018). For the estimation of IC₅₀ values, four different concentrations (200 µg/mL, 100 µg/mL, 50 µg/mL, and 20 µg/mL) of each extract were prepared and 5 µL were loaded in wells of the microplates. All wells containing 5 µL sample, 5 µL DMSO, and 5 µL methanol as negative control were loaded with 195 µL of freshly prepared DPPH solutions (25 µg/mL) and mixed by pipetting thoroughly. Plates were incubated in the dark at room temperature for 30 min. Readings were taken at 515 nm via microtiter plate reader (BioTek Elx800). DPPH free radical scavenging percentage was identified for each concentration and log IC₅₀ values were calculated for all extracts (Fezai, Mezni & Rzaigui, 2018; Zhu et al., 2018).

Statistical and graphical analysis

The current investigation was performed in triplicate, with at least two similar repeats. The basic analysis and the graphs were prepared using MS Excel. The figures were prepared using MS PowerPoint. The different letters indicate a significant difference on the basis of one-way ANOVA with Tukey’s test. ANOVA and the Pearson’s correlation were performed using Minitab 16 software (Wakeel et al., 2018a; Wakeel et al., 2018b.).

Extraction efficiency

The crude extract quantity, purity, and quality greatly depend on the plant part used and the solvent used for the extraction (Ullah et al., 2017; Jacotet-Navarro et al., 2018). To investigate the impact of solvent type and plant part on the bioactive extraction process, seven solvents (n-hexane, chloroform, ethyl acetate, acetone, ethanol, methanol, and water) were selected. Furthermore, to achieve more variety in polarity index, one ratio one (1:1, v/v) combinations of solvents (n-hexane-ethyl acetate, n-hexane-ethanol, methanol-chloroform, methanol-ethyl acetate, methanol-acetone, acetone-water, and methanol-water) were also prepared. Different solvents extracted different quantity of crude extract from branches, flowers, leaves, and roots. The overall extraction efficiency of branches was higher and lower in roots as compared to flowers and leaves. The maximum extraction efficiency for flowers, branches, leaves, and roots was 40, 39, 37 and 13% respectively (Fig. 1A). The extraction efficiency of acetone-water (1:1, v/v) was very high and ethyl acetate, n-hexane alone or in combinations was very low for all plant parts (branches, flowers, leaves, and roots) as compared to other solvents (Fig. 1A). Although the amount of crude extract was different in each part, there was a significantly positive correlation between branches, flowers, leaves, and roots based on the Pearson’s correlation (Fig. 1B).

Total phenolic contents (TPC)

Solvent type and polarity play an important role in TPC. Different plant parts have a different level of TPC (Lesjak et al., 2011). The extractions were carried out with 14 different solvents (Figs. 2A–2D). TPC level significantly increased with increasing solvent polarity with a few exceptions. Methanol-ethyl acetate in branches and leaves, methanol and acetone-water in flowers, n-hexane-ethanol in roots were the most efficient solvents (Figs. 2A–2D). Chloroform, n-hexane, and n-hexane-ethyl acetate showed the minimum level of TPC in all four parts (Figs. 2A–2D).

Total flavonoid contents (TFC)

Solvent type and polarity index, plants species and plant parts play an important role in the TFC level in the extracts (Do et al., 2014). In contrast, to the TPC level, the TFC level was quite low and it was not dependent on the solvent polarity (Figs. 3A–3D). Based on the TFC level, chloroform and ethyl acetate in branches (Fig. 3A); methanol-ethyl acetate, methanol-chloroform, and n-hexane-ethyl acetate in flowers (Fig. 3B); chloroform in leaves (Fig. 3C); and ethyl acetate, acetone, and n-hexane-ethanol in roots (Fig. 3D) were the most efficient solvents. The extracts, extracted with water and methanol-water were found to be the most inefficient solvent with minimum TFC level in all plant parts (Figs. 3A–3D). Except for chloroform extract in leaves, which is 143 µg/mg QE (Fig. 3C), the rest of the plant parts showed less than 90 µg/mg QE (Figs. 3A, 3B, and 3D).

Ferric reducing antioxidant power

The solvent type and polarity, as well as plant parts, play an important role in the total reducing power (Do et al., 2014; Ullah et al., 2017). The TRP level increased with increasing solvent polarity index, except for water, which has the maximum polarity index among the selected solvents (Figs. 4A–4D). Methanol-ethyl acetate in branches and leaves; methanol and acetone-water in flowers; and n-hexane-ethanol in roots were the most efficient solvents (Figs. 4A–4D). Chloroform, n-hexane, n-hexane-ethyl acetate were the most inefficient solvents based on the total reducing power in all plant parts used in the current study (Figs. 4A–4D).

Total antioxidant capacity (TAC)

The method of extraction, selection of plant parts, and use of appropriate solvent for the extraction greatly influence antioxidant capacity (Musa et al., 2011). In the current study, phosphomolybdate method was adapted to evaluate the impact of solvent polarity on the total antioxidant capacity in four different parts of woad. Phosphomolybdate method is adopted by a large group of researchers for the evaluation of antioxidant capacity and is considered one of the most authentic methods used for TAC (Chekroun-Bechlaghem et al., 2019). TAC of extracts varied significantly among different solvents and plant parts, which indicates that each solvent with specific polarity can isolate specific compounds that have a specific antioxidant capacity. Acetone in branches and roots (218 µg/mg and 226 µg/mg respectively, Figs. 5A and 5D), methanol-ethyl acetate in flowers and leaves (217 µg/mg and 193 µg/mg respectively, Figs. 5B–5C), and methanol-chloroform in leaves (193 µg/mg, Fig. 5C) showed the maximum TAC.

DPPH free radical scavenging capacity

Extraction techniques, solvent type, and polarity, plant part selected for extraction mediate antioxidant and free radical scavenging capacity (Chekroun-Bechlaghem et al., 2019). DPPH free radical method has been extensively used for different natural and synthetic compounds antioxidant and free radicals scavenging potential (Zhu et al., 2018; Chekroun-Bechlaghem et al., 2019). First of all, the percent inhibition of DPPH free radicals was identified in four different concentrations (200, 100, 50, and 20 µg/mL) of each extract. Based on the percent inhibition log IC₅₀ values were calculated for each solvent in the respective plant parts. The log IC₅₀ value was significantly different in all solvents, which indicates that each solvent extracted some specific type of secondary metabolites that could carry different scavenging level of DPPH free radicals. Methanol in branches, ethyl acetate in flowers and leaves, and methanol-water in roots showed strong activity with minimum log IC₅₀ values (0.2, 0.04, 0.09 and 0.04 µg/mL respectively, Table 1).

Correlation

The Pearson’s correlation was performed for the relationship between TPC, TFC, TAC, and TRP in each plant part (branches, flowers, leaves, and roots) investigated in the current study. In branches, TPC showed significantly positive correlation with TRP, insignificant correlation with TAC, while significantly negative correlated with TFC. The correlation between TFC and TAC and TRP and TAC was insignificant, while the TFC and TRP was significantly negative correlated (Figs. 6A and 6B). In flowers, TPC showed significantly positive correlation with TRP and TAC, while insignificant correlation with TFC. The correlation of TFC with TAC was significantly positive, while with TRP it was insignificant. There was insignificant correlation between TAC and TRP (Figs. 6C and 6D). In leaves, the correlation of TPC with TRP and TAC was significantly positive, while with TFC, it was significantly negative. TFC showed significantly negative correlation with TRP, while no correlation with TAC. TRP and TAC showed significantly positive correlation (Figs. 6E and 6F). Finally, in roots, the TPC exhibited significantly positive correlation with TRP and TAC, while insignificant correlation with TFC. The correlation of TFC with TRP and TAC was insignificant. TRP showed significantly positive correlation with TAC (Figs. 6G and 6H). Taken together, it is concluded that TPC play an important role in the antioxidant capacity of woad plant extracts.