In-vitro and in-silico antioxidant, α-glucosidase inhibitory potentials of abutilins C and D, new flavonoide glycosides from Abutilon pakistanicum

2.1 General

The column chromatography (CC) was carried out using silica gel (230–400 mesh, E. Merck, Darmstadt/Germany). For UV/IR spectra with UV-3200 (Hitachi)/302-A Jasco) spectrophotometer were used. EI-MS, FAB-MS performed on a MAT 312 MS (Finnigan) and JMS-HX-110 MS (JEOL) with glycerol as a matrix respectively. The AMX-500 spectrometer (Bruker) to get the ¹H–¹³C NMR spectra with solvent C5D5N contains tetramethylsilane (TMS) as internal standard. The 2D NMR (COSY, HMQC, HMBC, NOESY) spectra were recorded within same instrument. Thin layer chromatography (TLC) accomplished with silica gel (F₂₅₄, E. Merck, Germany) precoated plates and spots were visualized with UV light (254 and 366 nm). Reference compounds of flavonol glycosides and biological activity standards were purchased from Sigma-Aldrich, Germany.

2.2 Plant material

The Abutilon pakistanicum (Malvaceae), whole specimen was collected at Karachi, Pakistan in June 2016. Department of Botany UOK plant taxonomist Prof. S Khatoon identified the plant material as a taxonomist, where 697 KUH voucher number deposited in the herbarium.

2.3 Extraction and isolation

Powder of air-dried A. pakistanicum (8 kg) was soaked in MeOH thrice to get the extract, which was concentrated in vacuo to get blackish crude extract. This semi-solid crude was suspended in H₂O and by using solvent–solvent extraction procedure this was divided into different polarity organic solvents and water-soluble fractions. CH₂Cl₂ sub-portion was subjected to CC over silica gel eluting with organic solvents in increasing order of polarity. Currently, CH₂Cl₂ portion was subjected to silica gel CC and eluted with organic solvents with increasing polarity, which yielded six major sub-fractions (Fr. 1 to Fr. 6). Fraction 5 and 6 showing the presence of flavonoidal glycoside within TLC. Repeated CC by fraction 5 yielded new abutilin A and B reported in our paper previously (Ali et al., 2010). The fraction 6 was eluted with CH₂Cl₂-MeOH with increasing polarity to yield four sub-fractions (Fr. A to Fr. D). Fraction C was subjected to final purification with CC using silica gel (230–400 mesh size) as adsorbent with mobile phase DCM-MeOH (90:10) to yield abutilin C (12 mg) and fraction D (0.31 g) was subjected to CC with DCM-MeOH (85:15) to yield abutilin D (10 mg).

2.4 Total phenolic and flavonoid contents

The method used to determine the TPC employed by Folin-Ciocalteu colorimetric method. Briefly, for TPC extracts each prepared sample was incubated and absorbance at UV–Vis. spectrophotometer (760 nm) was recorded. This was calibrating against the standards as gallic acid and attained values were expressed as (mg) gallic acid equivalent per (g) extract i.e (mg of GAE/g) (Vats, 2016).

Alike, total flavonoid contents (TFC) have been calculated following previously standard calorimetric methods (Cenobio-Galindo et al., 2019). After incubation of each sample, its absorbance value was determined at 415 nm. The amount of total flavonoids-TFC were uttered as rutine quivalents (mg RE/g extract). For both TPC/TFC calculation, triplicate experiments readings were determined, and results were averaged.

2.5 Characterizations of abutilins C (1)

Yellow gum. UV (MeOH): 262 (3.1), 274 (2.7), 354 (2.5). IR (KBr): 3350–3745 (br. s), 1654, 1550, 1505, 1297, 1064, 630. ¹H- ¹³C NMR: see Table 2. FAB-MS (pos.):627 ([M+H]⁺), 463 ([M−H−hex]⁺), 300 ([M−2H−2hex]⁺). EI-MS: 301 (30), 300 (10), 285 (17), 160 (15), 151 (30), 150 (32), 125(10), 110 (35). HR-FAB-MS (pos.): 627.1472 ([M+H]⁺, C₂₇H₃₁O₁₇; calc. 627.1483).

2.6 Characterizations of abutilins D (2)

Yellow gum. UV (MeOH): 264 (3.0), 273 (2.8), 355 (2.5). IR (KBr): 3355–3730 (br. s), 1660, 1545, 1510, 1295, 1060, 637. ¹H- ¹³C NMR: see Table 2. FAB-MS (pos.): 707.1037 ([M+H]⁺), 544 ([M+H-hex]⁺), 464 ([M+H-SO₃hex]⁺), 302 ([M+2H-hex- SO₃hex]⁺). EI-MS: 300 (10), 285 (17), 160 (15), 151 (30), 150 (32), 125(10), 110 (35), 97 (20), 80 (15). HR-FAB-MS (pos.): 707.1037 ([M+H]⁺, C₂₇H₃₁O₂₀S; calc. 707.1051).

2.7 Antioxidant bioassay

Antioxidant bioassay was done by DPPH and NO scavenging assay based on the principle that a -H donor is an antioxidant. The potential of antioxidant is proportional to DPPH and NO radical disappearance within sample. The test sample was prepared according to standard procedure (Sharma and Cannoo, 2017) and absorbance of the each sample was determined at 518 nm by using ascorbic acid and quercetin as reference standards (positive controls). The % age DPPH scavenging activities of the dichloromethane extracts, abulitin C-D and ascorbic acid was calculated by using equation: $$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}(\%)=100-(\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l})\times 100.$$

2.8 α- glucosidase inhibition assay

It was measured by the standard method with slight modifications (Tanase et al., 2019). Enzyme α-glucosidase solution (0.2 U/ml), sample solution (60 μl) phosphate buffer 0.1 M having pH 6.9 (50 μl) incubated in 96 well plates for 10 min at 40 °C. The 50 μl of 5 mM p-nitrophenyl-α-D-glucopyranoside in pesence of same buffer solution added to each well and incubated for minute (10) at 40 °C. It was quenched by adding 0.2 M NaCO₃ (150 μl) within well that were analyzed, and its absorbance were recorded at 405 nm using ascarbose (+ive control). The % age enzyme inhibition was determined by using equation: $$\mathit{Inhibition}(\%)=100-(AbsoftestsampleAbsofcontrol\times 100)$$

EZ-Fit Enzyme Kinetics Software used for calculation of IC₅₀ values (concentration at which 50% enzyme catalyzed reaction occurs) of phytochemicals.

2.8.1

2.8.1 Optimization of assay conditions

Optimization of reaction conditions were done by OVAT (one variable at a time) technique. Different chemical parameters were investigated including buffers, solvents, enzyme concentration and substrate concentration. Concentration of α-glucosidase enzyme were studied in the range of 0.0–0.12 units/well keeping the substrate concentration constant at 10 μl/well of 0.5 mM. Enzyme concentration of 0.057 unit/well was found optimum for α-glucosidase owing to maximum response and strength of reading (as shown in Fig. 2). Keeping the enzyme concentrations constant, different concentrations of substrate (ρ-nitrophenyl-α-D-glucopyranoside) were investigated in the range of 0–20 μl/well of 0.5 mM of substrate. Out of these substrate concentrations 10 μl/well was found optimum α- glucosidase for studies and it was finalized for further studies (Fig). All the finalized parameters discussed above were used for temperature studies done in the range of 10–40 °C. Among these 30 °C was found the most suitable for α-glucosidase in our laboratory conditions.

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Fig. 2

a. Dose dependent curve of α-glucosidase b. Standard curve of substrate for α-glucosidase.

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2.9 Computational details

In order to probe the diverse properties of interests first-principles approach is an interesting tools (Anastassova et al., 2018; Irfan et al., 2019a, 2019b, 2019c; Rammohan et al., 2020; Wazzan and Irfan, 2019). The prediction of extensive electronic characteristics which may replicated the experimental results was measured with Density functional theory (DFT) (Irfan, 2019). Its authentic stategies for the measurement of different compounds ground state (S₀) geometries, optimization along with its electronic characteristics (Irfan et al., 2018; Irfan et al., 2020a, 2020b, 2020c). The B3LYP is sensible for the S₀ geometries of several phytochemicals (Preat et al., 2010). Shifting of literature revealed that after optimizing the geometries of phytochemicals with functional (B3LYP) with broad range of basis sets has no extraordinary effect on various geometrical parameters (Preat et al., 2009). Currently, optimizations were conducted by B3LYP functional with basis set as triple zeta with polarization function (TZP) (Irfan et al., 2013). The detailed computations were executed with ADF. Additionaly, for docking purposes Autodock version 4.2 was employed with H₂O molecule elimination succeeded by adding polar hydrogen. Autodock 4.2 endorsed using Autodock tools, MGL tools. To find location of binding site among native ligand Autogrid utilized with organizing the grid X, Y, and Z-axis coordinates. The Autodock 4.2 along with Pymol version 1.7.4.5 Edu softare employed for docking computations.

3.1 Total phenolic content TPC and TFC

Total phenolic and flavonoid contents of crude methanol and its soluble organic fractions like hexane, CH₂Cl₂, EtOAc, n-butanol and H₂O extracts of A. pakistanicum presented in Table 1. According to our results, phenolic content of hexane, CH₂Cl₂, EtOAc, n-butanol and H₂O extracts were 45.15, 77.51, 54.89, 37.81 and 46.03 respectively. The TPC of Abutilon pakistanicum was evaluated Folin-Ciocalteu assay using standard gallic acid (GA) by plotting the relationship between absorbance vs concentration. The TPC of Abutilon pakistanicum result revealed that the phenolic compounds were best extracted dichloromethane for extraction having more amount of interesting phytometabolites.

Total flavonoids content was measured using a standard procedure (Genovese et al., 2020) using Rutin as positive control (standard). The TFC of the analyzed samples (Table 1) were expressed as mg rutine/g each extract. Each result represented the mean ± S.D. of three experimental determinations. According to our flavonoid contents results hexane, CH₂Cl₂, EtOAc, n-butanol and H₂O extracts were 44.91, 71.47, 56.06, 38.11 and 40.18 respectively, revealed the presence of high flavonoids within dichloromethane extract.

3.2 Abutilin C (1)

Bioguided directed isolation of dichloromethane soluble portions of A. pakistanicum using repeated column chromatography affoarded four new flavonoids namely Abutilin A-D. The spectral information of Abutilin A-B was reported by our previous study (Ali et al., 2010) and herin structural elucidation and antioxidants and α-glucosidase activity of Abutilin C-D was explained. Abutilin C (1) was obtained as a yellow gum from dichloromethane soluble fractions. The HRFABMS (+ive mode) of 1 showed a quasi-molecular-ion [M+H]⁺ peak at m/z 627.1472, consistent with the M.F. C₂₇H₃₁O₁₇. The prominent peak in EI-MS exhibited at m/z 300 ([M − 2 × sugar]⁺), which indicate the loss of hexose moieties. The UV spectrum was characteristic of a flavonol, which exhibited maximum absorption at 262, 274 and 354 nm. The 40 nm bathochromic shift was seen with addition of AlCl₃/HCl, revealed chelated —OH presence at C-5 of a flavonoid (Ali et al., 2010). The IR absorption band were evident of a —OH (3350–3745 cm⁻¹), carbonyl conjugated (1654 cm⁻¹) and aromatic moieties (1550 and 1505 cm⁻¹) and ether groups (1297, 1064, 630 cm⁻¹). The UV spectrum, IR peaks, ¹H NMR spectrum in pyridine‑d₅ indicated that 1 was a quercetin derivative. The ¹³C NMR spectra exhibited 27 carbon signals corresponding to two methylene, fifteen methin and ten quaternary C-atoms (Table 2). The resonances of ring B were characteristic ABX spin system having resonances at δ_H 6.85 (H-5′, d, J = 8.4), 7.42 (H-2′, d, J = 2.1) and 7.60 (H-6′, dd, J = 2.1, 8.4). The two meta-coupled doublets in the ¹HNMR spectrum were exhibited at δ_H 6.20 (d, J = 2.0) and 6.37 (d, J = 2.0) corresponded to H-6 and H-8, were regarded to quercetin moiety. Instead of quercetin signals, the two sugar residues were revealed by the corresponding anomeric protons signals having doublets at δ_H 4.80 (d, J = 5.2) and 5.30 (d, J = 7.5).

The hexose units have larger coupling constant values endorsed for β-configuration assignment for hexoses moieties (Ali et al., 2012). Further signals of oxymethines were observed between δ_H 4.21–4.43 and 3.91–4.20 and oxymethylene at δ_H 4.25–4.27, 4.36–4.39, 4.46–4.48 and 4.50–4.53 for the sugar moities. The ¹³CNMR signals at δ_C 157.2, 134.1, 178.7, 157.5 and 105.2 were typical of a flavonol moiety. The signals of benzene ring moiety were resonated at δ_C 115.7, 116.2, 121.8, 121.9, 145.3 and 148.8 forming an ABX system. The anomeric carbons were observed at δ 107.2 and 100.9 along with further signals of the hexose moieties. The ¹H -¹³C NMR signal revealed towards skeleton as flavonoid class of quercetin type (Halabalaki et al., 2011). The acid hydrolysis of abutilin C provided quercetin and glycones binary mixture identified as D -glucose and D-mannose by TLC with standard as well as by gas chromatography (GC) by comprising of retention times of their tetramethylsilane (TMS) ethers with corresponding standards (Miyazawa and Hisama, 2003). The 2D-HMBC experiments, position of D-glucose moiety was established in which the anomeric proton at δ_H 5.30 exhibited ³J correlation with C-3 at δ_C 134.1 and also anomeric proton of D-mannose at δ_H 4.80 showed ³J correlation with C-7 at δ_C 162.9. Thus above evidences support to allocate the D-glucose moiety at C-3 followed by D-mannose at C-7. Further, the HMBC correlations illustrated in Table 2 are in complete support with the assigned structure of abutilin C as quercetin-3-O-[β-D-glucopyranosyl]-7-O-β-D-mannopyranoside (Fig. 1).

3.3 Abutilin D (2)

It was obtained as a gummy yellow material from dichloromethane soluble extract which shows similar characteristics and tests as those of 1. The HR-FAB-MS (positive mode) of abutilin D 2 gives a quasi-molecular-ion [M+H]⁺ peak at m/z 707.1037, consistent with the molecular formula C₂₇H₃₁O₂₀S. The EI-MS exhibited prominent peak at m/z 300 ([M - hexose - SO₃hexose]⁺), which indicate the loss of hexose moieties. The further ions fragment observed at m/z 97 and 80, confirmed the presence of sulfohexose moiety. Also the UV/IR spectra were alike to those of 1. The ¹³C NMR spectra (BB and DEPT) showed twenty-seven well-resolved carbon peaks corresponding to two CH₂, fifteen CH groups, and ten quaternary C-atoms (Table 3). Similar to compound 1, meta-coupled doublets of H-6, 8 were resonated at δ_H 6.24 and 6.45 (J = 2.3 Hz). The resonances of ring B were characteristic ABX spin system having resonances at δ_H 6.84 (d, J = 8.5, H-5′), 7.43 (d, J = 2.5, H-2′) and 7.70 (dd, J = 2.5, 8.5, H-6′). The anomeric protons shows doublets at δ_H 4.85 (d, J = 5.0) and 5.32 (d, J = 7.5) in the spectrum. These coupling constant value larger, allowed β-configuration for both hexose units. The sugar moieties further showed signals for oxymethines between δ_H 4.20–4.43 and 3.83–4.54 and oxymethylene at δ_H 4.23 (dd, J = 5.1, 5.2), 4.27–4.29, 4.44–4.47 and 4.50–4.54. Further the double doublet appeared at δ_H 5.10 (dd, J = 5.0, 5.3, H-2″) confirm the sulfate group in the sugar moiety.

The ¹³CNMR signals resonated at δ_C 157.3, 134.0, 178.8, 157.4 and 105.1 were characteristic of a flavonol moiety. The signals of benzene ring moiety were resonated at δ_C 115.4, 116.2, 121.5, 121.8, 145.5 and 149.1 forming an ABX system. The anomeric carbons signal observed at δ_C 106.6 and 100.5 along with further signals of the hexose moieties. Thus compound 2 was assigned as an analogue 1 (Safder et al., 2012), except slight differences in the chemical shifts of the hexose unit.

The structure of abutilin D was further confirmed by acid hydrolysis, gives quercetin and binary mixture of glycones,that identified as D -glucose and D-mannose by TLC with standard as well as by gas chromatography (GC) by comprising of retention times of their tetramethylsilane (TMS) ethers with corresponding standards. The sulphate moiety attached to D-mannose was resonated at δ_H 5.10 (dd, J = 5.0, 5.3) and was assigned to C(2″) (δ_C 82.4) through HMBC correlations (Fig. 1). The HMBC correlations showed within Table 3 allowed to assigned the structure abutilin D (2), as quercetin-3-O-(β-D-glucopyranosyl)-7-(2‘‘-O-sulfo-β-D-mannopyranoside).

3.4 In-vitro antioxidant and α-glucosidase inhibition activity

The antioxidant activities of the dichloromethane extracts and pure constituents 1–2 of A. pakistanicum were determined by employing 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH) and nitric oxide (NO) scavenging assay. The CH₂Cl₂ extracts of A. pakistanicum exhibited the antiradical activity against DPPH and NO with radical scavenging activities (RSA) of 92.75and 89.25% with IC₅₀ values 20.35and 39.41 µg/mL respectively. In present work the CH₂Cl₂ extracts showed good abilities to quench both DPPH and NO. The ability of the A. pakistanicum CH₂Cl₂ extracts to quench and it was related with its observed phenol content as higher. Among pure constituents abutilins C showed significant inhibition, with IC₅₀ values 41.66 (DPPH), 39.04 (NOS) µg/mL, with +ive control ascorbic acid (IC₅₀, 7.06 µg/mL) and quercetin (IC₅₀, 14.47 µg/mL) respectively, shown in Table 4.

The inhibitory activity of a-glucosidase was measured with modified method of Kim et al. It was shown that the Abutilon pakistanicum CH₂Cl₂ extracts exhibited excellent inhibitory activity against α-glucosidase, (extract IC₅₀: 10.85 μg/mL) as compared to standard ascarbose (IC₅₀: 5.92 μg/mL). Inhibitory effects of flavonoids 1–2 against enzyme α-glucosidase were also investigated and abutilins C-D showed activity (Abutilin C, IC₅₀ 8.27 µg/mL; Abutilin D, IC₅₀: 52.43 μg/mL) compared to +ive control as exhibited in Table 5. Optimization of assay conditions were maintained by standardizing the enzyme assay as mentioned above within experimental portion. The α-glucosidase inhobition was done by using standard protocol established after stanadadizing the assay. Phytochemical screening of the active components of Abutilon pakistanicum have been revealed that several types of total phenolic, flavonoids contents including, new abutilins C-D (1–2), flavonoid glycosides, are responsible for antioxidant activities as well as to inhibit the α-glucosidase activities of isolated pure constituents. These compounds were isolated and showed inhibitory effects on α-glucosidase with IC₅₀ values close to that of acarbose as the standard. The results showed that compound 1 showed more potent inhibitory activity, and may be used clinically as antidiabetic agents to control blood glucose (Nguyen et al., 2019). It was observed that the A. pakistanicum dichloromethane extracts and and abutilins C exhibited excellent activity against antioxidant (DPPH and NO) scavenging assay and α-glucosidase inhibiton.

3.5 Electronic properties and molecular descriptors

The FMOs, i.e., highest occupied molecular orbitals (HOMOs, HOMOs-1) and lowest unoccupied molecular orbitals (LUMOs, LUMOs + 1) of abutilin_C, abutilin_D and reference compounds, i.e., ascorbic acid and quercetin at level-B3LYP/TZ2P are shown in Fig. 2. In gallic acid, spatial distribution of HOMO-1 is on dihydroxyethyl, HOMO and LUMO at dihydroxyfuran-2(5H)-one while the LUMO + 1 at hydroxyl groups of furan moiety. The intra-molecular charge transfer (ICT) can be established from dihydroxyethyl (HOMO-1) to dihydroxyfuran-2(5H)-one (LUMO) and hydroxyl groups (LUMO + 1). The ICT was also observed from dihydroxyfuran-2(5H)-one (HOMO) to hydroxyl groups (LUMO + 1). In quercetin, spatial distribution of HOMO-1 can be seen on dihydroxyphenyl and benzene of the chromene, HOMO is on caqz of the chromene moiety, LUMO at the entire molecule while LUMO + 1 at the chromene unit. The ICT can established from benzene of the chromene (HOMO-1) to pyran (LUMO) and dihydroxyphenyl (HOMO-1) to chromene moiety (LUMO + 1). The ICT was found from dihydroxyphenyl (HOMO) to chromene (LUMO and LUMO + 1). In abutilin_C, spatial distribution of charge is almost similar to that of the reference compound quercetin, i.e., spatial distribution of HOMO-1 can be seen on dihydroxyphenyl and benzene of the chromene, HOMO is on dihydroxyphenyl and pyran of the chromene moiety, LUMO at the entire molecule while LUMO + 1 at the chromene unit. The ICT can be found from benzene of the chromene (HOMO-1) to pyran (LUMO) and dihydroxyphenyl (HOMO-1) to chromene moiety (LUMO + 1). The ICT was found from dihydroxyphenyl (HOMO) to chromene (LUMO and LUMO + 1). In abutilin_D, HOMO-1 is on dihydroxyphenyl ring, HOMO at dihydroxyphenyl and pyran, LUMO and LUMO + 1 at chromen unit. The ICT can be found from dihydroxyphenyl (HOMO-1 and HOMO) to chromene moiety (LUMO and LUMO + 1). The ICT can be seen from HOMO-1 → LUMO, HOMO-1 → LUMO + 1, HOMO → LUMO, HOMO → LUMO + 1 in abutilin_C, abutilin_D and reference compounds. The antioxidant ability of phytochemicals is also firmly associated with HOMO orbital spatial distribution, revealed the sites thats are most plausible within studied phytochemicals that may attacked by reactive agents and free radicals. In abutilin_C, HOMOs are overlapped on main core substantially disclosing remarkably reactive nature of the studied phytochemicals toward antioxidant and biological activity (see Fig. 3).

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Fig. 3

Ground state charge density of FMOs of studied compounds (contour value = 0.035).

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The energies of FMOs, i.e., HOMO-1 (E_HOMO-1), HOMO (E_HOMO), LUMO (E_LUMO), LUMO + 1 (E_LUMO+1), HOMO-LUMO energy gaps (ΔE_{HOMO –LUMO}) and HOMO-1 – LUMO + 1 energy gaps (ΔE_{HOMO–1−LUMO+1}) are important parameters to explore the materials electronic properties. The E_HOMO-1, E_HOMO, E_LUMO, E_LUMO+1, ΔE_{HOMO –LUMO} and ΔE_{HOMO–1−LUMO+1} of reference compounds as well as abutilin_C and abutilin_D at B3LYP/TZ2P level at S₀ in eV are exhibited within Table 2. The E_HOMO-1 at S₀ reduce as: quercetin (-6.54) > abutilin_D (-6.68) > abutilin_C (-6.80) > ascorbic acid (-8.03). The tendency in the E_HOMO is as: quercetin (-5.78) > abutilin_D (-6.09) > abutilin_C (-6.34) > ascorbic acid (-6.71). The trend in the E_LUMO is as: ascorbic acid (-1.16) > quercetin (-1.99) > abutilin_C (-2.25) > abutilin_D (-2.38). The tendency in the E_LUMO+1 is as: ascorbic acid (-0.45) > quercetin (-0.91) > abutilin_C (-1.27) > abutilin_D (-1.47). The ΔE_HOMO–LUMO increases in the following order: abutilin_D (3.71) < quercetin (3.79) < abutilin_C (4.64) < ascorbic acid (5.55). The ΔE_{HOMO–1−LUMO+1} increases in the following order: abutilin_D (5.21) < abutilin_C (5.53) < quercetin (5.63) < ascorbic acid (7.58).

The work functions (W) of Au and Al are 5.10 and 4.08 eV, respectively . The hole/e injection energies (HIE/EIE) of abutilin_C, abutilin_D, and reference compounds to Au and Al electrodes are probed. For Al, (EIE =  − E_LUMO − (W of Al) has been estimated as: ascorbic acid (2.92 eV = -1.16 − (−4.08)) > quercetin (2.09 eV = -1.99 − (−4.08)) > abutilin_C (1.83 eV = -2.25 − (−4.08)) > abutilin_D (1.70 eV = -2.38 − (−4.08)). In case of Au, (EIE =  − E_LUMO − (W of Au) has been estimated as: ascorbic acid (3.94 eV = -1.16 − (−5.10)) > quercetin (3.11 eV = -1.99 − (−5.10)) > abutilin_C (2.85 eV = -2.25 − (−5.10)) > abutilin_D (2.72 eV = -2.38 − (−5.10)). The HIE were observed as: ascorbic acid (2.63 eV =  − 4.08 − (-6.71) > abutilin_C (2.26 eV =  − 4.08 − (-6.34) > abutilin_D (2.01 eV =  − 4.08 − (-6.09) > quercetin (1.70 eV =  − 4.08 − (-5.78) by considering Al electrode. In case of Au, HIE were observed as: gallic acid ascorbic acid (1.61 eV =  − 5.10 − (-6.71) > abutilin_C (1.24 eV =  − 5.10 − (-6.34) > abutilin_D (0.99 eV =  − 5.10 − (-6.09) > quercetin (0.68 eV =  − 5.10 − (-5.78). It was recognized that for better e/hole injection ability, Al/Au would be more suitable electrode.

Global chemical reactivity descriptors (GCRD) are important parameters to realize the reactivity and structure stability. Here, we estimated various GCRD parameters like chemical hardness-η, chemical potential-µ, electronegativity-χ, softness-S and electrophilicity index-ω of isoflavones using HOMO and LUMO energy values, see Table 6 (computational details can be found in SI). The η value of phytochemical is aromaticity interrelated (Geerlings et al., 2003; Vektariene et al., 2009). The μ value express its electron tendency to rush out with its electronic cloud. The η value also symbolizes electronic cloud obstruction extent deformation and ω signifies the stabilization energy when the systenm is saturated by es from the external environment.

The Molecular electrostatic potential (MEP) maps are important to envision the charged region of phytochemicals. In Fig. 4, the MEP mapped for pure constituents and reference compounds are displayed in color visual images. The red, blue shades within figure identifies the sophisticated -ive and +ive potential regions which would be favorable for electrophilic/nucleophilic attack, respectively. The concentrated negative electrostatic potential can be seen on the oxygen atoms of –OH while +ive potential is determined on hydrogen atoms of –OH.

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Fig. 4

Molecular electrostatic potential surfaces views of studied compounds.

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The Hirshfeld charges of the abutilin_C, abutilin_D and reference compounds are demonstrated within Fig. S1. The -ive charge can be located on the -O atoms while +ive charge on -H atoms. These results showed that studied compounds have favorable sites for electrophilic/nucleophilic attacks would bring about greater antioxidant potential.

3.6 Molecular docking

As far as we know, no theoretical studies for Abtulin_C and Abtulin_D compounds were conducted especially resistance with NADPH (PDB ID-2CDU) enzyme. We investigated the interactions of the studied phytochemicals with NADPH crystal structure in order to attain better understanding on the potency and SAR studies. Molecular docking was employed by inserting isolated phytochemicals (Abtulin_C and Abtulin_D) into the binding site of NADPH crystal structure. The determination of the 2CDU NADPH enzyme structure exceuted with Worldwide Protein Data Bank. The 2CDU NADPH enzyme crystal structure without H₂O and inhibitors are displayed within Fig. 5. The binding energies values between phytochemicals and 2CDU NADPH enzyme (active sites in the phytochemicals and enzyme) are displayed within Table 7 and Fig. 6. The hydrogen bonding between keto oxygen of Abtulin_C and hydrogen of ARG308 was observed with the distance 1.7 Angstrom. Another hydrogen bonding was found between hydrogen atom of Abtulin_C and oxygen of GLN18 was observed with the distance 1.8 Angstrom. The hydrogen bonding between hydrogen of Abtulin_D and keto oxygen of GLY254 was observed with the distance 2.3 and 2.9 Angstrom. Another hydrogen bonding was found between hydrogen atom of Abtulin_D and oxygen of LYS255 was observed with the distance 2.2 Angstrom. One more hydrogen bonding was found between oxygen atom of Abtulin_D and hydrogen of SER273 was observed with the distance 2.8 Angstrom. The +ive binding energy of Abtulin_D mean that affinity is too low. As a results of association of phytochemicals with a target drug binding energy is released, which leads to its overall energy reduction. This binding energy decompress also counterbalance mpensates for any phytochemical transformation therefrom energy minimal to its confined conformation along with the relevant protein. As a consequences of phytochemical binding with protein more energy released resulted enhanced tendency of association of phytochemical among with relevant protein. Based on reduction within binding energy made more consistent complex, i.e., phytochemicals having −1.05 Kcal/mol binding energy, is more stable than ligand having 1.91 Kcal/mol binding energy. The vale of Gibbs Free Energy –ive is preferable for its better binding. The obtained docking results for Abtulin_C were found better that is illuminating that this compound would be better anti-oxidant than the Abtulin_D which is in good agreement with our experimental studies.

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Fig. 5

Crystal structure of the NADPH (PDB ID-2CDU) enzymes (water molecules and inhibitors are removed for clarity).

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Fig. 6

Docking simulation of the interaction between isolated compounds and NADPH enzyme.

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