The analysis of new higher operative bioactive compounds and chemical functional group from herbal plants through UF-HPLC-DAD and Fourier transform infrared spectroscopy methods and their biological activity with antioxidant potential process as future green chemical assay

2.1 Pure sample preparation and extracts

The plant extract was prepared by taking 1 g of powdered dry sample and 10 ml of distilled water added to it. The sample was then mixed with the help of a mechanical shaker and filtered through filter paper. The plant sample then transferred to the separate bottle and the pure solution was kept in the refrigerator as described by (Ivanišová et al., 2020).

2.2 Sample elution

The pure solution of both plants samples was run in the ultra action process through UF-HPLC was used with linked of filtering membrane (Size 0.22 µm), of samples further the samples were diluted into a 1:10 V/V of the buffer with 12 mM of ABS solution was used and adjusted the pH 7.5 before running of the sample. The bioactive compound was eluted from the bioactive receptor complex on the dried filter plates over which MS binding samples were harvested. One hundred microliters of eluent (50% methanol, 50% ammonium formate buffer [final concentration 5 mM at pH 7, spiked with 2 mM [2H] DPCPX or [2H] ZM-241385 as internal standard, all HPLC grade) was applied to the filter plates which were then centrifuged for 1 min at 800g (2000 rpm) in a 5810 plate centrifuge (Eppendorf, Hamburg, Germany), while filter eluates were collected in 1.1 ml polystyrene deep 96-well plates (Brand Tech Scientific, Essex, CT, USA). This procedure was performed twice resulting in a total of 200 μL eluate for each sample. For standard curve samples, the same procedure was followed but for the presence of increasing concentrations (1–100 pM) of DPCPX or ZM-241385 in the eluent. After elution, 96-deep-well plates were sealed with rapid easy pierce film (Nacalai, San Diego, CA, USA) and stored at −20 °C before LC-MS-MS quantification (Hou et al., 2020).

2.3 New and innovative methods of UF-HPLC-MS apparatus used

The identification of mass spectrometers with (6530 UF-HPLC ALO, CA, USA) with chromatographic separation was employed on a new advanced column of ZORBAX ST C18 column the eddulent of HPLC mobile phase attached with electrospray ionization (ESI) source. The parametric conditions were capillary voltage 3600 V; nozzle voltage 1000v, the flow rate of drying gas “ 7L/Min” Skimmer voltage 66 V; RFOCT2 750, fragmentor voltage 136 V, The spectra data were obtained in full-scan MS mode was attached in the system, the data was observed with a diverse collision energy source of (19–46 eV) (Ahmed et al., 2018b).

2.4 Assay based binding of UF-HPLC-MS

The proper isolation screening of bioactive compounds was based on three steps of samples preparation washing, incubation, dissociation process was done on both herbal plants. Both samples were incubated with 20 mins in buffer solution at 38 °C the solution was boiled in a water bath. The auto ultrafiltration device (Millipore Crop, MA, USA) was attached to HPLC-MS. The millipore amicon ultra −0.6 ml, item UFC, 503207). The bioactive compounds were further evaluated with 1000 L of 50% methanol directly (Hou et al., 2020).

2.5 UF-HPLC- producer accuracy and method

The procedure of screening was manipulated according to the approach of previous research and consisted of three steps: incubation, washing, and dissociation. Briefly, 100 µL of tested SMD solution was incubated with 200 µL HSA (600 µM) and 200 µL buffer solution for 20 min at 37 °C. Meanwhile, denatured HSA solution (boiled for 15 min in a water bath) was used as the negative control in the same manner. The incubated solutions were then filtered through ultrafiltration devices (Millipore Corp., Billerica, MA, USA) with a 30 kDa molecule weight cut-off membrane (Millipore AmiconUltra-0.5 ml, item: UFC 503096) and centrifuged at 14,000g to separate the non-specific ingredients from the HSA-ligand complexes for 15 min at room temperature. The residues were then washed with 200 µL of buffer solution by centrifugation to remove the unbound components three times. The ligands showing specific binding to HSA were then released from the mixtures by elution with 400 µL 50% methanol (pH = 3) for 20 min, and then centrifuged at 14,000g for 15 mins at room temperature, a process which was repeated twice. The dissociated filtrates were combined and added to 1000 µL by 50% methanol and then directly analyzed (Li et al., 2020).

2.6 Fourier transform infrared spectroscopy (FTIR) analysis for compounds

The FTIR (Model/ Make 25 Germany) with the attached auto software for data and operational processes. The small amount of all leaves twigs flower and seed of both plants were made into a pellets KBr for FTIR analysis and thin film for pressure. All the samples were analyzed in triplicates with plain KBr pellets as blank. The spectral data were compared with a reference to identify the functional groups existing in the samples (Ahuchaogu et al., 2020).

2.7 DPPH radical scavenging assay

The scavenging activity was estimated using the method described by Ahmed et al. (2018a). 0.5 ml of various concentrations of the extracts (0.1–0.5 mg ml⁻¹) in methanol were added to 3 ml of DPPH (2,2-diphenyl-2-picrylhydrazyl) solution (0.004%) in methanol. After the mixture was incubated in darkness at room temperature for 30 min, the absorbance was recorded at 517 nm by UV spectrophotometer (Shimadzu Corporation, Kyoto, Japan). Inhibition of the free radical by DPPH (I) was calculated from the Eq. (3) described by (Chen et al., 2020) I = [(Ablank − A sample)/Ablank] × 100, (3) where A blank is the absorbance of the blank (containing all reagents except the extract or standard), and A sample is the absorbance of the extract or standard. The value of IC50 was determined from the plot of scavenging activity, drawn according to the concentration of the extract, which is defined as the total antioxidant activity necessary to decrease by 50% the initial DPPH radical concentration. The experiments were carried out in triplicate. Butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) were used as standard antioxidants (Loucif et al., 2020).

2.8 Ferric reducing (FRAP) assay

The ferric reducing antioxidant power of the extract was investigated by the method developed by Jones et al. (2020). Different amounts of the extracts (0.025–0.4 mg ml⁻¹) in methanol were mixed with 2.5 ml of phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of potassium ferricyanide (1%) [K₃ Fe(CN)₆]. The mixture was incubated at 50 ◦C for 20 min. The mixture was added to an aliquot of trichloroacetic acid (10%). Then, this mixture was centrifuged for 10 min. The obtained upper layer (2.5 ml) was transferred to another tube with 5 ml of distilled water and then 1 ml of ferric chloride (0.1%) was added to the obtained mixture. The absorbance was measured at 700 nm. The tests were run in triplicate.

2.9 ESR measurements from herbal plants

The measurements were performed using an ESR spectrometer (X-Band Bruker EMX 081, Rheinstetten, Germany) in Saraykoy Laboratory of TAEK (Turkish Atomic Energy Authority). The measurements were set as follows: the microwave power was 2 mW, the modulation frequency was 100 kHz, and the modulation amplitude was 0.5 G (Ahmed et al., 2018a).

2.10 ABTS radical scavenging assay by spectrophotometry

In this assay, the radical scavenging activities of extracts were determined by using the ferryl myoglobin/ABTS protocol (Fernández-Poyatos et al., 2020). The stock solutions of 500 mM ABTS diammonium salt, 400 mM myoglobin, 740 mM potassium ferricyanide, and 450 mM H2O2 were prepared in phosphate-buffered saline (PBS) (pH 7.4). Metmyoglobin (MbIII) was prepared by mixing equal amounts of myoglobin and potassium ferricyanide solutions. The reaction mixture (total volume 2 ml) contained the following substances (final concentrations in the reaction mixture): ABTS (150 mM), MbIII (2.5 mM), 16.8 ml of the sample, and 978 ml PBS. The reaction was initiated by adding 75 mM H2O2 (330 ml) and the lag time in seconds, before the absorbance of ABTS⁺ at 734 nm began to increase was recorded. The calibration curve was plotted with lag time in seconds versus the concentration of the standard antioxidants (L-ascorbic acid or Trolox).

2.11 Oxygen radical absorbance capacity assay

In the oxygen radical absorbance capacity (ORAC) assay, oxygen radical absorbance capacity was measured on the detection of chemical damage to b-phycoerythrin through the decrease in the fluorescence emission. The fluorescence was recorded every 5 min until the last reading was less than 5% of the 0 min reading. ORAC values were calculated in terms of mmol/g of fresh weight (Amirah et al., 2020).

2.12 Total phenolic contents

The amount of TP was calculated using Folin–Ciocalteu reagent. 0.5 ml of essential oil was mixed with 2.5 ml of 10% Folin–Ciocalteu reagent. The mixture was kept at room temperature for 10 min and then 2.0 ml of 75% sodium carbonate (w/v) was added. The mixture was vortexes for 15 s heated in a water bath at 40 °C for 30 min and then cooled in an ice-bath; finally, absorbance was taken at 765 nm. The amount of TP was calculated using a calibration curve for Gallic acid (20–100 ppm). The results were expressed as Gallic acid equivalents (GAE) per dry matter (Loucif et al., 2020; Barbouchi et al., 2020; Sengun et al., 2020).

2.13 Total antioxidant activity determination by ferric thiocyanate method (FTC)

The antioxidant activity of Adhatoda vasica and Calotropis procera was determined according to the ferric thiocyanate method (FTC) as described by Besbas et al. (2020). Then, the solution which contains different concentrations of plants extracts (from 15 to 45 μL/mL) solution in 2.5 ml of sodium phosphate buffer (0.04 M, pH 7.0) was added to 2.5 ml of linoleic acid emulsion in sodium phosphate buffer (0.04 M, pH 7.0). For this, 5 ml of the linoleic acid emulsion was prepared by mixing and homogenizing 15.5 μL of linoleic acid, 17.5 mg of tween-20 as an emulsifier, and 5 ml phosphate buffer (pH 7.0). On the other hand, 5 ml of control composed of 2.5 ml of linoleic acid emulsion, and 2.5 ml, 0.04 M sodium phosphate buffer (pH 7.0). The mixed solution (5 ml) was incubated at 37 °C in a polyethylene flask. The peroxide levels were determined by reading the absorbance at 500 nm in a spectrophotometer (Shimadzu, UV-1208 UV–VIS Spectrophotometer, Japan) after reaction with FeCl₂ and thiocyanate at intervals during incubation. In the FTC method, during the linoleic acid peroxidation, peroxides are formed and this leads to oxidation of Fe⁺² to Fe⁺³. The latter ions form a complex with thiocyanate and this complex has a maximum absorbance at 500 nm. The absorbance of the red color was measured at 500 nm until it reached a maximum value (Elmastaş et al., 2006). This step was repeated every 5 h. The percentage inhibition values were calculated at this point (30 h). High absorbance indicates high linoleic acid emulsion peroxidation. The solutions without oil were used as blank samples. The inhibition percentage of lipid peroxidation in the linoleic acid emulsion was calculated by the following equation: $$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{f}\mathrm{l}\mathrm{i}\mathrm{p}\mathrm{i}\mathrm{d}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{d}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}(\%)=100-(\mathrm{A}\mathrm{s}/\mathrm{A}\mathrm{c}\times 100)$$ where A_c is the absorbance of control reaction which contains only linoleic acid emulsion and sodium phosphate buffer and A_s is the absorbance in the presence of plant samples (Gülçin, 2006a,b).

2.14 Statistical analysis

The couple markers of all compounds confirmed after extractions process of each sample which prepared through software of graph Pad Prism 7.04. The developed methods are confirmed and aureate the measurement of all bioactive compounds confirmed through standards digital library. The data of HPLC-DAD was measured by the help of Chrom Gate v 3.31 Knauer software was used (Ahmed et al., 2018b).

3.1 UF-HPLC-MS methods for isolation of bioactive compounds from herbal plant

The ten critical bioactive compounds isolated from various parts of (leaves, flower, twigs, and seeds) both herbal plants are shown in Table1. The isolation of ten crucial bioactive compounds such as P-hydroxy-benzoic, Stictic acid, Usnic acid, Epigallocatechin gallate, Quercetin, Epigallocatechin, Catechin, Morin, Epicatechin, Naringenin measured in various parts of Adhatoda vasica and Calotropis procera. The higher range of Naringenin was measured (16 ± 0.41) in the leaves of Adhatoda vasica while the Quercetin and Epicatechin are showing the same value of (14 ± 0.02). The higher value of Quercetin, Stictic acid, Catechin Morin, Naringenin also displayed the same values in leaves extracts of Calotropis procera. The results showed that the flowers of Adhatoda vasica prove to be a rich source of important compounds in which Naringenin (15 ± 0.41) was in higher in the flowers. Similarly the Quercetin, Epigallocatechin contents were higher in leaves' extracts of Calotropis procera. The similar trend observed in twigs and seeds of Adhatoda vasica, however, the Epicatechin was higher in seeds of Calotropis procera. The chromatogram of ten important bioactive compounds isolated from the UF-HPLC-MS method is shown in Figs. 1 and 2.

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Fig. 1

Adhatoda vasica (a) and Calotropis procera (b) plants.

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Fig. 2

UF-HPLC method of various bioactive compounds isolated from (1) P-hydroxy-benzoic, (2) Stictic acid (3) Usnic acid (4) Epigallocatechingallate (5) Quercetin (6) Epigallocatechin (7) Catechin (8) Morin (9) Epicatechin (10) Naringenin.

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3.2 Functional groups analysis of bioactive compounds by Fourier transform infrared spectroscopy (FTIR)

The important functional group analysis of various parts of both herbal plants was studied with their properties presented in Tables 2 and 3. Free alcohol O—H Stretching and Intermolecular bonded alcohol O—H Stretching 3985.14 ± 5.51. 3285.14 ± 5.51 in leaves of Adhatoda vasica while lower values of the same parameter shown in the seed of Calotropis procera. The Intermolecular bonded alcohol O—H Stretching and Intramolecular bonded alcohol O—H Stretching 3285.14 ± 5.51 in leave of 51 in leaves of Adhatoda vasica while lower values of the same parameter shown in the seed of Calotropis procera. The higher ranges of C—H stretching alkane, C—H bending aromatic compounds overtone 3285.14 ± 5.51 in leaves of Adhatoda vasica however the flower and twigs showed second and third ranges of values. To C⚌N stretching imine/oxime or C⚌O stretching conjugated ketone or alkenes 3915.14 ± 5.51, O—H bending phenol 3915.14 ± 5.51 C—N stretching amine 3935.14 ± 5.51 in leaves of Adhatoda vasica followed by the flower twigs and least values of all three functional group properties were lower in the seed of Calotropis procera.

Table 2 FIR analysis of compounds.

Sr.no	Name of compounds	Molecular formula	Molecular weight	% Peaks	Structure
1	P-hydroxy-benzoic	C7H6O3	138.12 g/mol	170
2	Stictic acid	C19H14O9	386.3 g/mol	90
3	Usnic acid	C18H16O7	344.315 g/mol	21
4	Epigallocatechingallate	–	458.372 g/mol	55
5	Quercetin	C15H10O7	302.236 g/mol	40
6	Epigallocatechin	C22H18O11	458.372 g/mol	180
7	Catechin	C15H14O6	290.26 g/mol	160
8	Morin	C15H10O7	302.2357 g/mol	130
9	Epicatechin	C15H14O6	290.27 g/mol	92
10	Naringenin	C15H12O5	: 272.25 g/mol	55

3.3 Structural and molecular response of bioactive compounds

In the present study, the FRIR analysis was conducted in both herbal plants to identify the active functional group, the extracted samples were analyzed through IR on base of its peak, the results indicated the presences of groups explained in Table 3. The ten essential compounds were found in both herbal plants with their structural, Molecular formula Molecular weight, and % peaks. All ten compounds have varies their molecular formula and weight were shown in Table 5. The structural formula also varies for all ten compounds with their peaks values.

3.4 Various methods of antioxidants potential determination of bioactive isolated in various parts of Adhatoda vasica and Calotropis procera

The antioxidant potentials of various bioactive compounds like Epigallocatechin gallate (EGCG), Quercetin (QR), Epigallocatechin (EGC), Catechin (CT), Morin (MR), Epicatechin (EC), Naringenin (NG), and Epicatechin gallate (ECG) isolated from the leaves of Adhatoda vasica were presented in Tables 4 and 5. Modern and updated methods for determination of antioxidant activity were used like Electron spin resonance spectrometry, ABTS Assay and Diphenyl picrylhydrazyl (DPPH) radical scavenging methods (see Fig. 3).

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Fig. 3

Spectra obtained from ESR method of antioxidants.

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The ABTS Assay showed the higher antioxidant potential of Epigallocatechin gallate (EGCG) (13 ± 0.01) and Morin (MR) 13 ± 0.13 measured in Adhatoda vasica leaves followed by Epicatechin (EC) 12 ± 0.49 > Epicatechin gallate (ECG)11 ± 0.45 > Epigallocatechin (EGC) 10 ± 0.42 > Quercetin (QR) 9 ± 0.021 and Naringenin (NG) 9 ± 0.41 > Catechin (CT)8 ± 0.12. Similarly higher antioxidant potential of bioactive compounds isolated from leaves of Calotropis procera is shown in Table 4. A similar trend was found in both the plants when bioactive compounds were analyzed through ABTS radical scavenging assay. Morin (MR), Epigallocatechingallate (EGCG), and Epicatechin gallate (ECG) showed higher free radical scavenging potential 14 ± 0.14,12 ± 0.02 and 12 ± 0.47 respectively in µ/mol followed by Epigallocatechin (EGC) 11 ± 0.43 and Epicatechin (EC) 11 ± 0.49 which were > Naringenin (NG) 10 ± 0.42, Quercetin (QR)10 ± 0.03 and Catechin (CT) 9 ± 0.13. Spectra obtained from ESR method of free radicals measurement of bioactive compounds obtained from Seeds, Twigs, and Flowers of Adhatoda vasica and Calotropis procera is shown in Fig. 4, and results are summarized in Tables 4 and 5. The data shows that bioactive compounds isolated from C. procera and A. vasica leave like Morin (MR), Epigallocatechingallate (EGCG), and Epicatechin (EC) exhibited higher radical scavenging capability values of 14 ± 0.02, 12 ± 0.40, 13 ± 0.03 and 14 ± 0.02,12 ± 0.40, 13 ± 0.03 µg/mol respectively. The following order was observed from strongest to the weakest antioxidant potential of isolated antioxidant compounds from leaves of both the plants based on values obtained from ESR experimentation Morin > Epicatechin (EC) > Epigallocatechingallate (EGCG) > Quercetin (QR) = Naringenin (NG) = Epicatechin gallate (ECG) > Epigallocatechin (EGC) > Catechin (CT). The maximum radical scavenging ability was showed by Morin and minimum by Catechin (CT) both natural antioxidants present abundantly in many plants.Fig 5.

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Fig. 4

Flow of Spectra obtained from ESR method of free radicals measurement of both herbal plants (a) Seed (b) Twigs (c) Flowers of Adhatoda vasica (d) Seeds (e) Twigs (f) Flowers of Calotropis procera.

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Fig. 5

Scatter plot of antioxdants response data versus concentrations with calibration lines with standards (a) Gallic acid (b) DPPH^• (c) BHT (d) Ascorbic acid.

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We proceeded to further analyze the antioxidant potential of isolated compounds by performing Diphenyl picrylhydrazyl (DPPH) radical scavenging assay, FRAP assay, Total radical-trapping antioxidant assay, Phycoerythyrin assay, and ORAC-FL Oxygen radical absorbance capacity assay. The values obtained in all these environments are summarized in Tables 4 and 5.

Micellar ORAC_FL values have the following high to the low antioxidant capacity trend of bioactive compounds from A.vesica leaves: Epicatechin (EC) = Epicatechin gallate (ECG) > Quercetin (QR) = Catechin (CT) = Naringenin (NG) > Epigallocatechingallate (EGCG) = Epigallocatechin (EGC) > Morin (MR). In the case of C. procera bioactive compounds antioxidant potential following trend was found: Epicatechin gallate (ECG) > Epigallocatechin (EGC = Morin (MR) = Naringenin (NG) > Epicatechin (EC) > Epigallocatechingallate (EGCG). To explain these results in heterogeneous media (specifically we used triton X-100 micelle) we calculated free fluorescein concentration [FL].

The capacity of the studied compounds to quench the DPPH radical reactivity using a non-catalytic method was also determined. Tables 4 and 5 summarizes the values expressed as a percentage of decrease concerning a blank solution. For the bioactive compounds of plant, A. vasica Quercetin (QR) showed the highest radical quenching potential which was 13 ± 0.41 followed by Morin (MR) and Epicatechin gallate (ECG) which was 12  ± 0.43 and 12 ± 0.012 respectively. The order of decreasing DPPH radical scavenging ability was found as Quercetin (QR) > Morin (MR) = Epicatechin gallate (ECG) > Epigallocatechin (EGC) = Epicatechin (EC) > Epigallocatechin gallate (EGCG) > Catechin (CT) > Naringenin (NG). Bioactive compounds of C.procera showed the following trend in percentage reduction of DPPH induced radical: Catechin (CT) = Epicatechin gallate (ECG) > Epigallocatechin (EGC) = Naringenin (NG) > Epicatechin (EC) = Quercetin (QR) > Epigallocatechin gallate (EGCG) = Morin (MR).

The Diphenyl picrylhydrazyl (DPPH) radical scavenging method showed higher activity of Quercetin, Epigallocatechin (EGC) and Catechins (CT) isolated from leaves extract of both the plants as compared to antioxidant potential measured by ABTS assay and electron spin resonance spectrometry. The electron spin resonance spectrometry showed a higher potential of Morin (MR) 14 ± 0.02 and Epicatechin (EC) in leaves extract of Adhatoda vasica and Calotropis procera as compared to other methods. Epicatechin gallate (ECG) isolated from the leaves of Adhatoda vasica and Calotropis procera showed a higher percentage of DPPH radical scavenging values. The Phycoerythrin assay showed higher contents of Epigallocate cingulate (EGCG) 9 ± 0.41 and Quercetin (QR) 12 ± 0.44 in leaves of Adhatoda vasica. Epicatechin gallate (ECG) showed higher antioxidant activity estimated through the method of ORAC-FL while lower activity was showed in Morin (MR) 6 ± 0.44.

The antioxidants activity of different bioactive compounds isolated from the leaves of Calotropis procera is presented in Table 5. The Epigallocatechin gallate (EGCG) 11 ± 0.40 showed higher antioxidant contents through the Phycoerythrin assay method then estimated total radical-trapping method while it showed lower activity in FRAP assay. Epigallocatechin (EGC), Catechin (CT), Morin (MR), Epicatechin (EC), Naringenin (NG), Epicatechin gallate (ECG) all these essential compounds showed higher antioxidant potential measured through the Phycoerythrin antioxidants assay method.

In this study, the antioxidants activity was measured by the Ferric thiocyanate (FTC) method and the concentration of total phenolics was measured by the Folin-Ciocalteau method. The total antioxidant potential and total phenolic contents of both plants C. procera and A. vasica showed in Table 5. The higher antioxidant activity and total phenolic 3085.14 ± 8.51 and 3025.14 ± 8.56 showed by the leaves of Adhatoda vasica followed by the flower and twigs while lower values 1285.14 ± 3.56 1188.14 ± 3.55 were noted in the seed of Calotropis procera as presented in Table 5.