Experimental
Plant collection
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
Partial purification or separation of crude methanol extract was done by solvent–solvent extraction. Briefly 12 g of crude methanol extract was suspended in 500 ml distilled water in separator funnel (1000 ml) and successively partitioned with n-hexane, ethyl-acetate, chloroform and n-butanol. Each extraction process was repeated three times with 500 ml of each solvent same process was repeated to get enough mass of each fraction to use for chromatographic separation. These solvents with varying polarities theoretically partitioned different plant constituents. The filtrate was concentrated using rotary evaporator (Buchi, R114, Switzerland) and weigh to determine the resultant mass. After this initial partitioning we got four soluble extracts beside crude methanol extract and remaining aqueous extract. The ethyl-acetate (AHE) and butanol (AHB) fractions revealed significant antioxidant potential in various in vitro antioxidant enzyme assays. Estimation of total phenolic content (TPC) and total flavonoid content (TFC) indicate that these AHE and AHB possess high TPC (120.3 ± 1.15,129 ± 2.98 mg Gallic acid equivalent/g dry sample) and TFC (89 ± 1.32, 119 ± 1.04 mg rutin equivalent/g dry sample) respectively [18]. These results prompted us to choose these two extracts for further fractionation and purification of active compounds. Here we report only the isolation and fractionation of ethyl-acetate extract. The scheme of fractionation is summarized in Fig. 1.
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 1JCH), extensive use was made of 1H-coupled 13C spectra and selective 1H-decoupling to determine long range JCH 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
The DPPH assay was done according to the method previously describe with slight modifications [20]. The stock solution was prepared by dissolving 24 mg DPPH with 100 ml methanol (80%) and then stored at 20 °C until needed. The working solution was obtained by diluting DPPH solution with methanol to obtain an absorbance of about 0.751 ± 0.02 at 517 nm using the spectrophotometer. An aliquot of 1 ml aliquot of this solution was mixed with 100 μl of the samples at varying concentrations (0–100 µM). The mixture was mixed vigorously and allowed to stand at room temperature in the dark for 10 min. The absorbance of the solution was measured at 517 nm using a UV-1601 spectrophotometer (Shimadzu, Kyoto, Japan). Ascorbic acid was used as a reference compound. The decrease in absorbance was correlated with the radical scavenging potential of test samples. The percentage of inhibition was calculated as follow
$${\text{DPPH scavenging }}\left( \% \right) = \left[ {\frac{{{\text{A}}0 - ({\text{A}}1 - {\text{As}})}}{{{\text{A}}0}}} \right] \times 100.$$
where 0 is the absorbance of the DPPH solution, 1 is the absorbance of the test compound in the presence of DPPH solution, and is the absorbance of the compound solution without DPPH. Each sample was analyzed in triplicate. The EC50 value was calculated by a graphical method as the effective concentration that results in 50% inhibition of radical formation [35].
Non site-specific hydroxyl radical scavenging activity
The hydroxyl radical-scavenging activity was monitored using 2-deoxyribose method of Halliwell et al. [21]. Phosphate buffer saline (0.2 M, PH 7.4) was used as a solvent in this assay. Sample solution (0–100 µM) was mixed with assay mixture containing 2.8 mM 2-deoxyribose, 20 mM ferrous ammonium sulphate solution, 100 µM EDTA in a total volume of 1 ml of solvent buffer (0.2 M phosphate buffer saline, PH 7.4). Ferrous ion solution and EDTA were premixed before adding to the assay mixture. The reaction was initiated by the addition of 100 µl of 20 mM H2O2 and 100 µl of 2 mM Ascorbic acid and incubated at 37 °C for 15 min. Then, thiobarbituric acid solution (1 ml, 1%, w/v) and trichloroacetic acid solution (1 ml, 2%, w/v) were added. The mixture was boiled in water bath for 15 min and cooled in ice, and its absorbance was measured at 532 nm. All experiments involving these samples were triplicated. The scavenging activity were calculated by following formula.
$${\text{Radical} - \text{scavenging capacity }}\left( \% \right) = \left[ {\frac{{{\text{Control absorbance}} - {\text{sample absorbance}}}}{\text{control absorbance}}} \right] \times 100$$
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
The interaction of isolated compounds with nitric oxide was accessed by nitrite detection method as previously describe [22]. Nitric oxide was generated with Sodium-nitroprusside previously bubbled with and measured by the Greiss reaction. 0.25 ml of sodium-nitroprusside (10 mM) in phosphate buffer saline was mixed with 0.25 ml of different concentrations (0–100 µM) of compounds and incubated at 30 °C in dark for 3 h. After incubation 0.25 ml of Greiss reagent A (1% sulphanilamide in 5% phosphoric acid) was added and kept at 30 °C for 10 min. After incubation, 0.25 ml of Greiss reagent B (0.1% N 1-naphthylethylenediamine di-hydrochloride) was added mixed and incubated for 20 min. The absorbance of chromophore form during the diazotization of nitrite with sulphanilamide and subsequent coupling with naphthyl-ethylenediamine was read at 546 nm. The same reaction mixture without extract was served as control
$$\% {\text{ inhibition}} = \left[ {1 - \frac{\text{sample absorbance}}{\text{control absorbance }}} \right] \times 100.$$
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. [18]. 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 [7] 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.
Statistical analysis
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.
Chemistry
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).