Antioxidant activity of polyphenolic compounds isolated from ethyl-acetate fraction of Acacia hydaspica R. Parker
© The Author(s) 2018
Received: 19 October 2017
Accepted: 8 January 2018
Published: 25 January 2018
Acacia hydaspica belongs to family leguminosae possess antioxidant, anti-inflammatory and anticancer activities. During our search for antioxidant compounds from A. hydaspica, we carried out bioassay guided fractionation and obtained antioxidant compounds with free radical scavenging activity.
Materials and methods
The polyphenol compounds in the plant extract of A. hydaspica were isolated by combination of different chromatographic techniques involving vacuum liquid chromatography and medium pressure liquid chromatography. The structural heterogeneity of isolated compounds was characterized by high pressure liquid chromatography, MS–ESI and NMR spectroscopic analyses. The antioxidant potential of isolated compounds has been investigated by 1,1-diphenyl-2-picrylhydrazyl (DPPH), nitric oxide scavenging potential, hydroxyl radical scavenging potential, ferric reducing/antioxidant power (FRAP) model systems and total antioxidant capacity measurement.
The isolated compounds show the predominance of signals representative of 7-O-galloyl catechins, catechins and methyl gallate. Flash chromatographic separation gives 750 mg of 7-O galloyl catechin, 400 mg of catechin and 150 mg of methyl gallate from 4 g loaded fraction on ISCO. Results revealed that C1 was the most potent compound against DPPH (EC50 1.60 ± 0.035 µM), nitric oxide radical (EC50 6 ± 0.346 µM), showed highest antioxidant index (1.710 ± 0.04) and FRAP [649.5 ± 1.5 µM Fe(II)/g] potency at 12.5 µM dose compared to C2, C3 and standard reference, whereas C3 showed lower EC50 values (4.33 ± 0.618 µM) in OH radical scavenging assay.
Present research reports for the first time the antioxidant activity of polyphenolic compounds of A. hydaspica. Result showed good resolution and separation from other constituents of extract and method was found to be simple and precise. The isolation of catechin from this new species could provide a varied opportunity to obtain large quantities of catechin and catechin isomers beside from green tea. Free radical scavenging properties of isolated catechin isomers from A. hydaspica merit further investigations for consumption of this plant in oxidative stress related disorders.
Natural products from medicinal plants, either as pure compounds or as standardized extracts, provide unlimited opportunities for new drug leads because of the unmatched availability of chemical diversity. Due to chemical diversity in screening programs, interest has now grown throughout the world for making therapeutic drugs from natural products . However, the isolation of compounds remains a challenging and a mammoth task. Conventionally, the isolation of bioactive compounds is preceded by the determination of the presence of such compounds within plant extracts through a number of bioassays . The phytochemicals have been found to act as antioxidants by scavenging free radicals, and many have therapeutic potential for the remedy of diseases resulting from oxidative stress . Within the antioxidant compounds, considerable attention has been devoted to plant derived flavonoids and phenolic. Due to the presence of the conjugated ring structures and hydroxyl groups, many phenolic compounds have the potential to function as antioxidants by scavenging or stabilizing free radicals involved in oxidative processes through hydrogenation or complexing with oxidizing species . Moreover, naturally occurring agents with high effectiveness and fewer side effects are desirable as substitutes for chemical therapeutics which have various and severe adverse effects . Plants comprising phenolic constituents, such as phenolic diterpenes, flavonoids, phenolic acids, tannins and coumarins are possible sources of natural antioxidants. Numerous studies have revealed that these natural antioxidants possess numerous pharmacological activities, including neuroprotective, anticancer, and anti-inflammatory activities, and that these activities may be related to properties of antioxidant compounds to prevent diseases by scavenging free radicals and delaying or preventing oxidation of biological molecules .
There are different methods to evaluate the in vitro antioxidant capacity of isolated compounds, mixtures of compounds, biological fluids and tissues which involve different mechanisms of determination of antioxidant activity, for example: chemical methods based on scavenging of ROS or RNS, such as nitric oxide (NO∙) radical, DPPH radical and the hydroxyl radical (OH∙) radical [5, 6]. Other assays to determine the total antioxidant power include techniques such as phosphomolybdenum assay (TAC) , the ferric reducing/antioxidant power method . Various reaction mechanisms are usually involved in measuring the antioxidant capacity of a complex samples and there is no single broad-spectrum system which can give an inclusive, precise and quantitative prediction of antioxidant efficacy and antiradical efficiency , hence, more than one technique is suggested to evaluate the antioxidant capacities .
Acacia is a diverse genus comprising range of bioactive constituent such as phenolic acids , alkaloids , terpenes , tannins  and flavonoids , which are responsible for various biological and pharmacological properties like hypoglycaemic, anti-inflammatory, antibacterial, antiplatelet, antihypertensive, analgesic, anticancer, and anti-atherosclerotic due to their strong antioxidant and free radical scavenging activities .
Acacia hydaspica R. Parker belongs to family “Fabaceae (Leguminosae)”. This species is reported to be common in Iran, India and Pakistan, commonly used as fodder, fuel and wood . The bark and seeds are the source of tannins. The plant is locally used as antiseptic. The traditional healers use various parts of the plant for the treatment of diarrhea; the leaves and the bark are useful in arresting secretion or bleeding. Acacia hydaspica possesses antioxidant, anticancer, anti-hemolytic, anti-inflammatory, antipyretic, analgesic and antidepressant potentials [16–18]. Anticancer activity of A. hydaspica polyphenols has been determined against breast and prostate cancer .
In present study we determined the antioxidant activity of purified compounds from A. hydaspica by using five in vitro methods based on different mechanisms of determination of the antioxidant capacity in comparison with reference compounds. The inter-relationships between these methods were also examined for all the tested compounds to check the linearity of activity against different oxidants. Compounds showed linear activity in different antioxidant assays.
Materials and methods
The aerial parts (bark, twigs, and leaves) of A. hydaspica were collected from Kirpa charah area Islamabad, Pakistan. Plant specimen was identified by Dr. Sumaira Sahreen (Curator at Herbarium of Pakistan, Museum of Natural History, Islamabad). A voucher specimen with Accession No. 0642531 was deposited at the Herbarium of Pakistan, Museum of Natural History, Islamabad for future reference.
Preparation and extraction of plant material
General procedure and reagents
Mass spectrometer with both ESI and APCI spectra were obtained using a TSQ Quantum Triple Quadrupole (Thermo Scientific) ion sources. TLC was conducted on pre-coated silica gel 6OF254 plates (MERCK) spots were visualized by UV detection at 254 and 365 nm and Vanillin-HCL reagent followed by heating Semi-preparative HPLC was carried out using a agilent 1260 affinity LC system UV array detection system using a semi-preparative column (Vision HT™ classic; 10 μm, 250 × 10 mm). Flash liquid chromatography was carried on Combi-flash Teledyn ISCO (using Redisep column 40 g silica, mobile phase was dichloromethane:methanol (DCM:MeOH), flow rate 15 ml/min) with an ISCO fraction collector. Silica gel (230–400 mesh; Davisil, W. R. Grace) was used for open-column chromatography or vacuum liquid chromatography (VLC). All pure chemicals were purchased from sigma chemicals. All organic solvents were of HPLC grade. Water was purified by a Milli-Q plus system from Millipore (Milford, MA).
Vacuum liquid chromatography
The ethyl-acetate acetate extract (AHE) was fractionated with DCM:MeOH of increasing gradient polarity starting with 100% DCM (dichloromethane) to 100% MeOH (methanol) using vacuum liquid chromatographic (VLC) separation. Briefly 10 g of ethyl-acetate extract was dissolve in DCM, mixed with neutral acid wash (super cell NF) and dried down completely with rotavap. Pack 3/4 volume of glass column used for VLC with silica gel and load dried extract sample over the silica layer. After VLC separation, ethyl acetate extract sample was fractionated into 12 fractions of DCM:MeOH in the following gradients; 1:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4.5:5.5, 4:6, 3.5:6.5, 3:7, 2:8, 1:9, 0:1 (v/v). The 7:3 to 5:5 (DCM:MeOH) eluents (VLC-AHE/F3–F4) were mingled according to their TLC and 1H-NMR spectra similarity subjected to flash chromatography for further purification of the target compounds.
Flash liquid chromatography
VLC-AHE/F4–F6 (4 g/mixed in acid wash/dried) was loaded on Combi-flash Teledyn ISCO. Specifications of run are as follow.
Redisep column: 40 g silica, flow rate: 15 ml/ml, solvent A: dichloromethane (DCM), solvent B: methanol (MeOH), wavelength 1 (red): 205 nm, wavelength 2 (purple): 254 nm all wavelength (orange 200–780 nm) was monitored at all wavelengths (200–780 nm) with Peak width 2 min, and Thresh hold 0.02 AU. Air purge was set at 1 min peak tube volume: 5 ml, nonpeak tube volume 15 ml and loading type solid. 146 fractions collected with ISCO were pooled into 27 fractions according to their TLC and ISCO chromatogram spectral peaks. 1HNMR fraction indicated the presence of three pure compounds (C1, C2 and C3).
High performance liquid chromatography
Chromatographic analysis was carried out to check the purity of isolated compounds by using HPLC–DAD (Agilent USA) attached with Grace Vision Ht C18 column (Agilent USA) analytical column. Compounds stock solutions were prepared in methanol, at a concentration of 0.5 mg/ml. Samples were filtered through 0.45 μm membrane filter. Briefly, mobile phase A was H2O (prepared by a Milli-Q water purification system (Millipore, MA, USA) and mobile phase B was acetonitrile. A gradient of time was set as; 0–5 min (isocratic run) for 85% A in 15% B, 5–25 min for 15–100% B, and then isocratic 100% B till 30 min was used. The flow rate was 1 ml/min and injection volume was 20 μl. All the samples were analyzed at 220, 254, 280, 330, and 360 nm wavelengths. Every time column was reconditioned for 10 min before the next analysis. All chromatographic operations were carried out at ambient temperature.
% content of isolated compounds
The total content of each isolated compound was expressed as a percentage by mass of the sample.
Nuclear magnetic resonance spectroscopy (NMR)
1H- and 13C-NMR spectrum for all compounds was recorded on a CDD NMR instrument: Varian 600 MHz (1H and 13C frequencies of 599.664 and 150.785 MHz, respectively) at 25 °C using triple resonance HCN probe: for 1-D proton spectra and proton-detected experiments such as COSY, NOESY, and HMQC. Probe signal-to-noise specifications: 1H 1257:1 and broadband switchable probe was used for 13C. Chemical shifts were given in δ value Spectra of all compounds were obtained in methanol-d4 and DMSO-d6, typically 3–10 mg in 0.4 ml. Conventional 1D and 2D Fourier transform techniques were employed as necessary to achieve unequivocal signal assignments and structure proof for all compounds independently. In addition to 2D shift-correlation experiments (H–H COSY with long-range connectivity’s; C–H correlation via 1 J CH), extensive use was made of 1H-coupled 13C spectra and selective 1H-decoupling to determine long range J CH coupling constants and to assign all quaternary carbons unambiguously (DEPTH). Where necessary, stereo-chemical assignments were made with 2D ROESY and NOESY experiments. Detailed analysis of resolution enhanced spectra (Peak picking, integration, multiplet analysis) was performed using ACD/NMR processor (Advanced Chemistry Development, Inc). 1H and 13C chemical shifts are reported in ppm relative to DMSO-d6 (δ 2.5 and δ 39.5 for 1H and 13C respectively), CD3OD (δ 3.31, 4.78 for 1H and δ 49.2 for 13C) or internal standard Me4Si (TMS, δ = 0.0). The NMR spectra and chemical shifts of isolated compounds are matched with published data.
Antioxidant capacity determination assays
An amount of 10 mM stock solution of each compound and positive controls [Ascorbic acid, butylated hydroxytoluene (BHT) and Gallic acid] were prepared in 1 ml of solvent according to the assay protocol. These solutions were further diluted to get (0–100 µM) concentration. Positive control varied according to assay requirement.
Radical scavenging activity
DPPH radical scavenging activity assay
Non site-specific hydroxyl radical scavenging activity
EC50 values, which represent the concentration of sample that caused 50% hydroxyl radical-scavenging activity, were calculated from the plot of inhibition percentage against sample concentration. BHT was used as a positive control.
Nitric oxide radical scavenging activity
Rutin was used as a positive control.
Determination of antioxidant activity
Total antioxidant capacity (TAC) (phosphomolybdate assay)
The total antioxidant capacity of compounds was investigated by phosphomolybdate method of Afsar et al. . An aliquot of 100 µl of each sample was mixed with 1 ml of reagent (0.6 M H2SO4, 0.028 M sodium phosphate, and 0.004 M ammonium molybdate) and incubated for 90 min at 95 °C in a water bath. Absorbance was recorded at 765 nm after the mixture cooled to room temperature.
Ascorbic acid served as positive control.
Ferric reducing antioxidant power (FRAP)
A slightly modified method of Benzei and Strain  was adopted to estimate the ferric reducing ability of compounds isolated from A. hydaspica. Ferric-TPTZ reagent (FRAP) was prepared by mixing 300 mM acetate buffer, pH 3.6, 10 mM TPTZ in 40 mM HCl and 20 mM FeCl3·6H2O at a ratio of 10:1:1 (v/v/v). Compounds or reference were allowed to react with FRAP reagent in the dark for 30 min. In order to calculate FRAP values (µM Fe(II)/g) for compounds, linear regression equation for standard (FeSO4·7H2O) was plotted. The standard curve was linear between 100 and 1000 µM FeSO4. Results are expressed as μM (Fe(II)/g) dry mass.
All values are mean of triplicates. The Graph Pad Prism was used for One-way ANOVA analysis to assess the difference between various groups and calculation of EC50 values. Difference at p < 0.05 were considered significant. In addition, simple regression analysis on Microsoft excel was performed to seek relationship between different tests.
Compound 1: 7-O-galloyl-(+)-catechin
Light green shine crystals (H2O), C22 H 18 O10. MS/ESI(−) m/z 441.0977 [M−H], 1H-NMR (600 MHz, DMSO-d6), δ 7.04 (H-7, s, galloyl), δ 6.17 (H-8, J = 2.2 Hz), δ 6.11 (H-6, d, J = 2.2 Hz), δ 4.61 (H-2, d, J = 7.6 Hz), δ 3.88–3.93 (H-3, m), δ 2.52 (H-4a, dd, J = 16.7 Hz, J = 7.9 Hz), δ 2.71 (H-4b, dd, J = 16.3 Hz, J = 5.3 Hz). 13C NMR (methanol-d4-150.79 MHz): δ 27.21 (t, C-4), δ 66.941 (d, C-3), δ 81.975 (d, C-2), δ 100.946, δ 104.52 (each d, C-6 and C-8), δ 105.957 (s, C-4a), δ 109.179 (d, galloyl C-2 and C-6), δ 113.832, δ 114.548 (each d, C-2′ and C-5′), δ 119.201 (s, galloyl C-1), δ 130.656 (s, C-1′), δ 138.88 (s, galloyl, C-4), δ 144.973 (s, galloyl, C3 and C-5), δ 150.343 (s, C-7), δ 155.354, δ 156.070 (each s, C-5 and C-8a), δ165.734 (s, COO–).
Compound 2: Catechin
Light yellow amorphous powder, (H2O) (C15H14O6). MS/ESI(−) m/z [M−H]. 1H-NMR (DMSO-d6, 600 MHz): δ 5.67 (H-8 d, J = 2.3 Hz), δ 5.87 (H-6, d, J = 1.8 Hz), δ 4.46 (H-2, d, J = 7.6 Hz), δ 3.76–3.82 (H-3, m), δ 2.33 (H-4α, dd, J = 16.1 Hz, J = 7.9 Hz), δ 2.64 (H-4β, dd, J = 16.4 Hz, J = 5.3 Hz), δ 6.7 (H-2′, d, J = 1.8 Hz), δ 6.66 (H-5′, d, = 8.2 Hz), δ 6.57 (H-6′, dd, J = 8.2 Hz, J = 1.8 Hz). 13C-NMR (DMSO-d6-150.79 MHz). δ 28.01 (C-4), δ 66.717 (C-3), δ 81.411 (C-2), δ 94.314 (C-8), δ 95.389 (C-6), δ 99.331 (C-4a), δ 114.026 (C-2′), δ 115.10 (C-5′), δ 118.685 (C-6′), δ 130.870 (C-1′), δ 145.206 (C-4′), δ 146.281 (C-3′), δ 156.317 (C-5), δ 156.317 (C-8a), δ 156.317 (C-7).
Compound 3: Methyl gallate
White needle crystals. (C8H8O5). MS/ESI(−) m/z 183.0534 [M−H]. 1H-NMR (acetone-D6, 600 MHz),: δ3.79 (3H, s, OCH3), δ 7.11 (2H, s, H-2, H-6); 13C NMR (acetone-D6, 150.80 MHz) δ 51.0 (OCH3), δ 108.90 (C-2, C-6), δ 120.91 (C-1), δ 137.76 (C-4), δ 145.12 (C-3, C5), δ 166.27 (C=O).
Results and discussion
1H-NMR data of polyphenols isolated from Acacia hydaspica (Coupling constant J in Hertz)
δ in ppm
δ in ppm
δ in ppm
4.61 (d, J = 7.0 Hz)
4.46 (d, J = 7.6 Hz)
3.79 (s, OCH3)
2.71 (dd, J = 16.3 Hz, J = 5.3 Hz)
2.45 (dd, J = 16.5, 7.9 Hz)
2.64 (dd, J = 16.4, 5.3 Hz)
2.33 (dd, J = 16.1, 7.9 Hz)
6.11 (d, J = 2.2 Hz)
5.67 (d, J = 2.3 Hz)
6.17 (d, J = 2.2 Hz
5.87 (d, J = 1.8 Hz)
6.72 (d, J = 1.5 Hz)
6.70 (d, J = 1.8)
6.68 (d, J = 8.1 Hz)
6.67 (d, J = 8.2 Hz)
6.60 (dd, J = 8.1 Hz, J = 1.5 Hz)
6.57 (dd, J = 8.2 Hz, J = 1.8 Hz)
5.01 (d, J = 5.1 Hz)
4.84 (d, J = 4.7 Hz)
13C NMR data of polyphenols isolated from Acacia hydaspica ethyl-acetate extract
7-O-galloyl-catechins δ in ppm
δ in ppm
δ in ppm
The 1HNMR spectrum of C1 was similar to 1HNMR of (+)-catechin except for the additional signal at δ 7.04 (2H, s) due to a galloyl group. The location of the galloyl group was initially deduced to be at either C-5′ OH or C-7′ OH, C-4′ OH, C-3′ OH but not 3 of the catechins moiety from the HMBC spectrum in methanol-d4. In order to determine unequivocally the position of the galloyl group the HMBC was re-perform with DMSO and NOESY data indicate that the stereochemistry of isolated compound as 7-O-galloyl-(+)-catechin and which was further authenticated by comparison of the physical data with those reported previously [24, 26]. Consequently, the structure of C1 was concluded to be 7-O-galloyl-(+) catechin.
The 1HNMR spectrum and 13C-NMR of C2 was similar to assignment of catechin signals of those reported in previous literature [27, 28]. Consequently, the structure of C2 was concluded to be (+) catechin.
The molecular formula was determined from the MS and 13C NMR. 8 Carbons and five protons attached to carbon were observed in the 13C and 1HNMR spectra. In order to determine the position and number of hydroxyl groups, the NMR solvent was shifted to DMSO-d6 as hydroxyl were not seen with acetone-d6. 1H-NMR (DMSO-d6, 600 MHz) clearly reveal the presence two hydroxyls at δ9.44 and one hydroxyl at δ9.11. Close examination of the 1H and 13C NMR spectrum showed a symmetrical molecule with two aromatic protons, δ 7.11 (2H, s, H-2, H-6), three hydroxyl, two hydroxyl at δ C 145.12 (C-3, C-5), and one hydroxyl at δ C 137.76 (C-4), a methyl δ3.79 (3H, s, OCH3) and a ester carbonyl δ 166.27 (C=O). It is consistent with-NMR data have been reported from the literature [14, 15]. The structure (C3) revealed to be methyl 3, 4, 5-trihydroxybenzoate or methyl gallate.
Extractable compound yield
Acacia hydaspica ethyl-acetate extract (AHE) yields 187.5 mg/g of C1, 100 mg/g of C2 and 37.5 mg/g of C3.
Determination of anti-radical activity
DPPH radical scavenging
EC50 values (concentration causing 50% inhibition) in various antioxidant assays and FRAP potential of Acacia hydaspica polyphenols
% (dry weight of AHE extract)
1.60 ± 0.035a
4.33 ± 0.618b
6 ± 0.346a
649.5 ± 1.511a
6.24 ± 0.254b
8.0 ± 0.635a
12.3 ± 0.376b
432.9 ± 0.94b
2.9 ± 0.318a
6.25 ± 0.577a
7.67 ± 0.577a
505.5 ± 2.512c
0.781 ± 0.115c
36.3 ± 0.569d
53 ± 1.155c
9.1 ± 0.421c
9.67 ± 0.577a,d
49.5 ± 2.211c
Hydroxyl radical-scavenging activity
ROS constitute a major pathological factor causing many serious diseases, including cancer and neurodegenerative disorders . The generally formed ROS are oxygen radicals, such as hydroxyl radicals and superoxide, and non-free radicals, such as hydrogen peroxide and singlet oxygen. The hydroxyl radical is the most reactive and induces severe damage to adjacent biological molecules . The hydroxyl radical scavenging assay is based on ability of antioxidant to inhibit the formation of the hydroxyl radicals, malondialdehyde (MDA) formation and to prevent the degradation of 2-deoxyribose. Result demonstrated that all tested compounds inhibit hydroxyl radical generation in a dose dependent fashion. The respective EC50 values for isolated compounds C1, C2 and C3 were 4.33 ± 0.635, 8.00 ± 0.577 and 6.25 ± 0.618 μM respectively, exhibited greater potency to scavenge hydroxyl radical then Gallic acid (EC50 9.67 ± 0.577 µM) (Fig. 3b, Table 3). However none of tested compound showed better scavenging potential than standard BHT (EC50 0.781 ± 0.115). To our knowledge, the abilities of the compounds C2, and C3 to showed similar potency to scavenge hydroxyl radical to reported in previous studies . From our results, it was also possible to make a number of correlations regarding the relationships between the structures of isolated compounds and their hydroxyl radical-scavenging activities. Methyl gallate (C3) seemed to augment the bioactivity of Gallic acid (Reference compound). It was found that the antioxidant activities of flavan-3-ols decreased in the following sequence: C2 > C1 (i.e., 3-OH, 5′-OH > 7-O-gallate, 5′-OH). This suggests that a galloyl group and O-dihydroxy (i.e., catechol) is essential, and 5′-OH is not an important group in antioxidant activity. Comparing the hydroxyl radical-scavenging activities of isolated compounds revealed that the bioactivity decreased in the following sequence: C1 > C3 > C2. The results suggest that carbonyl, O-dihydroxy and galloyl group increased the hydroxyl radical scavenging activity.
Inhibition of RNS derived from nitric oxide
Nitric oxide a potent oxidizing radical leads to tissue damage in a number of pathological conditions in humans and experimental animals . Herein, isolated compounds from A. hydaspica were examined for their ability to protect against NO-dependent oxidation. Thus, the NO radical-scavenging activities of these isolated compounds were investigated by examining the oxidation of sodium nitroprusside. Figure 3c shows that exposure of nitric oxide generated by sodium nitroprusside to oxygen in the presence of the polyphenols isolated from A. hydaspica resulted in a significant inhibition of nitrite ion formation in a dose-dependent manner. The relative EC50 values of compound C1, C2 and C3 against RNS derived from nitric oxide are summarized in Table 3, which ranged from 6 to 12.3 µM compared to that of rutin (53.00 ± 1.155 µM). The bioactivity decrease in the following order: GG > MG > C > rutin. The addition of polyphenols significantly inhibited nitric oxide formation even at lower concentrations. Compounds at 25 µM dose showed inhibitory activity, ranging from 85.817±, 83.023± to 72.864± % for MG, GC and C respectively compared to rutin at same dose (39.845 ± 1.48%) as positive control. At a concentration of 100 μM, the scavenging activity of GC, C, and MG reached 97.34 ± 0.982% (p < 0.001), 93.825 ± 1.5 (p < 0.001) and 96.823 ± 1.501% (p < 0.01) respectively indicating significant difference from standard reference rutin (83.163 ± 2.79). These results reveal that the presence of hydroxyl and-carbonyl group in the flavonoid skeleton resulted in high nitric oxide inhibition of compounds. From these results, it was also possible to make a number of correlations regarding the relationship between the structures of isolated compounds and their NO radical-scavenging activities. Methyl gallate (C3) appeared to have enhanced the bioactivity then Gallic acid. It appeared that as far as the antioxidant activity was concerned, a galloyl group was essential, while C3 showed greater bioactivity. It was found that the antioxidant activities of flavan-3-ols decreased in the following sequence: C1 > C2 (i.e., 7-O-gallate, 5′-OH > 3-OH, 5′-OH). It is well known that nitric oxide has an important role in various inflammatory processes. Sustained levels of production of this radical are directly toxic to tissues and contribute to the vascular collapse associated with septic shock, whereas chronic expression of nitric oxide radical is associated with various carcinomas and inflammatory conditions including juvenile diabetes, multiple sclerosis, arthritis, and ulcerative colitis . The present study showed that GC, C and MG have good nitric oxide scavenging activity then rutin and gallic acid.
Total antioxidant capacity (TAC)
Phosphomolybdenum assay principal follows the chemistry of conversion of Mo (VI) to Mo (V) by compounds having antioxidant potential and resulting in the formation of green phosphate/Mo (V) having absorption maxima at 695 nm at acidic PH. TAC assay was used to assess the capacity total antioxidant capacity of isolated compounds compared Gallic acid . Isolated compounds showed good antioxidant index. Total antioxidant capacity (TAC) of compounds increase with increasing concentration of compounds. TAC order of A. hydaspica compounds TAC values were in following order; C1 (1.71 ± 0.040 µM) > C3 (1.54 ± 0.025 µM) > Gallic acid (1.39 ± 0.004) ~ C2 (1.379 ± 0.021) at 12.5 µM dose (Fig. 3d). To the best of our knowledge literature is scarce about the total antioxidant activity of 7-O-galloyl catechin (C1) by phosphomolybedate method. C1 significantly reduce Mo (VI) to Mo (V) and form a green colored complex of Mo (v) that gives absorbance at 695 nm. Antioxidant index of C2 is shown to be comparable with Gallic acid (p > 0.05), Methyl ester in C3 might responsible for significant (p < 0.01) enhancement in TAC capacity as compared to standard Gallic acid. From these results, it was also possible to make a number of correlations regarding the relationship between the structures of isolated compounds, their antioxidant activities and antioxidant index. The transfer of electron or hydrogen depends on the structure of compounds. These results reveal that the presence of hydroxyl and-carbonyl group in the flavonoid skeleton resulted in enhancement of total antioxidant capacity and moreover antioxidant index tested polyphenol compounds isolated from A. hydaspica correlated with the number of aromatic hydroxyl groups in the antioxidant assays . It was found that the antioxidant index of isolated compounds decreased in the following sequence: C1 > C3 > C2 (i.e., 7-O-gallate, 5′-OH > 3-OH, 5′-OH). The present study showed that C1, C2 and C3 have good TAC comparable to Gallic acid.
FRAP assay, based on the reduction of ferric tripyridyltriazine complex to its ferrous colored form. The antioxidant activities were measured three times to test the reproducibility of the assays. The Frap assay which measures the ability of isolated compounds to reduce TPTZ-Fe(III) complex to TPTZ-Fe(II) was used to assess the total reducing power of antioxidants . when a Fe3+-TPTZ complex is reduced by electron donating antioxidants under acidic conditions, change of absorbance of colorless less Fe3+ to blue colored Fe2+ form was measured at 593 nm . A higher value indicates higher ferric reducing power. The addition of polyphenols significantly reduces ferric ions to ferrous ions. Tested compounds C1, C2 and C3 at 12.5 µM dose showed FRAP values of 649.50 ± 1.501, 432.90 ± 0.949 and 505.5 ± 2.500 (µM Fe(II)/g) (Table 3). Results showed that 7-O-galloyl catechin (C1) has more significant (p < 0.001) FRAP values than catechin (C2), methyl gallate (C3) and standard reference Gallic acid at same dose; indicating significant electron donating capacity of C1 in comparison to C2, C3 and Gallic acid. C2 was less potent then Gallic acid, whereas methyl gallate (C3) showed bioactivity slightly but non-significantly enhanced than Gallic acid. Methyl gallate (C3) showed bioactivity slightly enhanced than Gallic acid. From these results, it was also possible to make a number of correlations regarding the relationship between the structures of isolated compounds and their FRAP activity. These results reveal that the presence of hydroxyl and-carbonyl group in the flavonoid skeleton resulted in high FRAP potential and reducing ability was concerned the number of aromatic hydroxyl and galloyl group. It was found that the antioxidant activities of isolated compounds decreased in the following sequence: C1 > C3 > C2 (i.e., 7-O-gallate, 5′-OH > 3-OH, 5′-OH). The present study showed that 7-O-galloyl catechin (C1), catechin (C2) and methyl gallate (C3) have good FRAP reducing potential comparable to Gallic acid.
Relationship between different antioxidant variables
Relation between antioxidant activity measurements of 3 AH polyphenols using different methods to evaluate the antioxidant activities of isolated compounds from Acacia hydaspica
y = 1.3736x + 3.7351
R2 = 0.9993
y = 0.7411x + 3.5321
R2 = 0.9301
y = 0.5354x + 1.5528
R2 = 0.9165
y = − 0.0666x + 1.7807
R2 = 0.9264, p < 0.01
y = − 0.0481x + 1.9586
R2 = 0.9124
y = − 0.0901x + 2.0993
R2 = 0.9999
y = − 41.969x + 680.02
R2 = 0.8271
y = − 30.177x + 790.87
R2 = 0.8073
y = − 59.277x + 896.42
R2 = 0.9742
Although catechin and methyl gallate were evaluated previously for antioxidant potential by various methods . Nevertheless, the present work provides more information about these features, since five different antioxidant methods were used to analyze the antioxidant capacity of these compounds in comparison with standards i.e., Ascorbic acid, gallic acid, BHT and rutin. In A. hydaspica ethyl-acetate extract 7-O-galloyl catechin appears to be the major antioxidant compound both in term of yield and activity. These results are in good agreement with the previous report of Zhao et al. , which showed that galloyl catechins contributes to the main antioxidant capacity of tea.
Antioxidant screening of active compounds from unexplored species of Acacia genus pave the way for the possible development of natural essences to substitute synthetic ones. There for further investigation for the isolation of compounds from other fractions and their pharmacological evaluations are still required. Moreover the isolation of catechin this new species could provide a new opportunity to obtained catechin beside from green tea. Acacia hydaspica provide a source of natural, significantly potent antioxidant constituents that might leads to the prevention of ROS mediated diseases by scavenging free radicals or preventing the oxidation of biomolecules.
TA made significant contributions to conception, design, experimentation, acquisition and interpretation of data and writing of manuscript. SR and MS revised the manuscript for important intellectual content. MRK supervised the study and reviewed the manuscript. All authors read and approved the final manuscript.
We acknowledge Higher Education Commission (HEC) of Pakistan for awarding IRSP scholarship for PHD research to the first author. We acknowledge Dr. Christine Salomon, Assistant Professor and Assistant Director Center for Drug Design, University of Minnesota, Minneapolis, MN 55455 for their help in purification of compounds and NMR data interpretation for structure elucidation. The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University, KSA for its funding the research group no (RGP- 193).
The authors declare that they have no competing interests.
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The project was partially funded by the Higher Education Commission (HEC) of Pakistan by awarding indigenous scholarship to the first author. We are grateful to the Deanship of Scientific Research, College of Applied Medical Sciences Research Center at King Saud University.
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