Anti sickling potential and chemical profiling of traditionally used Woodfordia fruticosa (L.) Kurz leaves

2.4.1 Human blood samples

Blood from SCD patients was collected by visiting the Primary Health Centre, Amarkantak, District Anuppur, Madhya Pradesh (India). The ethical approval for the research work was obtained from the IGNTU-Institutional Human Ethics Committee (Ref: IEC No. 003/IGNTU-IEC/2019–01) dated 16–02-19.

2.4.2 Reverse sickling assay

The reverse sickling assay was performed at different concentrations of the W. fruticosa extracts (250, 500 and 1000 μg/mL). Freshly collected blood was washed twice in five volumes of phosphate buffer saline (PBS, pH 7.4) by centrifugation at 3000 revolutions per minute (RPM) for 5 min (min). Cell suspension (100 μL) was aliquoted in different tubes for control, standard and test samples. 100 μL of freshly prepared 2% sodium metabisulfite was added to these tubes containing 100 μL of washed blood cells. After mixing the tubes were incubated for 2 h (h) at 37 °C. 100 μL of PBS (control), standard HU and plant extract (250, 500 and 1000 μg/mL) were added to the individual tubes that were further incubated for 2 h at 37 °C. Reaction mixture (10 μL) was taken and used for visualization under the microscope (EVOS XL Cell imaging system, Thermo Fischer Scientific) at 100x magnification. Cells were counted with the help of haemocytometer (Nurain et al., 2017). The assay was performed in triplicate.

The percentage of sickled cells was calculated as follows:

Percent sickling = (Number of sickled cells/total number of counted cells) × 100

Percent reverse sickling = (100 − Percent sickling).

2.4.3 Polymerization inhibition assay

Freshly prepared 2% sodium metabisulfite was used for inducing the partially sickled RBC. of sodium metabisulfite (880 μL) was mixed with 20 μL of pre-washed blood followed by addition of 100 μL PBS (control) / standard (HU)/ plant extracts (250, 500, 1000 μg/mL). The absorbance was taken immediately at 700 nm at every 2 min time interval for 30 min (UV-1800 24 V Shimadzu, Japan). Assay was performed in triplicates as reported earlier (Nwaoguikpe et al., 2013; Nurain et al., 2017). The rate of polymerization was calculated using the following formula:

Rate of polymerization = [(Final absorbance − Initial absorbance)/ 30] × 100.

2.4.4 Osmotic Fragility assay

NaCl solution in varying concentrations (0–0.9%) was prepared. To 3 mL of NaCl solution taken individually, 50 μL of prewashed blood was added followed by addition of 100 μL of PBS (control)/ standard (HU)/ plant extract (1 mg/mL) to the different tubes. Mixture was incubated at 37 °C for 4 h. The tube without NaCl was used as control while the tube having 0.9 % NaCl was labeled as blank respectively. After incubation, the tubes were centrifuged at 3000 RPM for 5 min. The supernatant was transferred to the cuvette and the absorbance was taken at 540 nm (UV-1800 24 V Shimadzu, Japan). Assay was performed in triplicates (Pauline et al., 2013; Nurain et al., 2017).

Percent of hemolysis was calculated as: (Absorbance of the supernatant in test sample / Absorbance of the supernatant without NaCl) × 100

2.5.1 Membrane stabilization assay

A mixture of PBS (1 mL), 50 mM hypo saline (2 mL) was taken in five different tubes followed by addition of 0.5 mL of RBC suspension to respective tubes. To these tubes 1 mL of different plant extracts/standard (200, 400, 600, 800, 1000 μg/mL) were added and mixed. The tubes were incubated for 30 min at 37 °C. The tubes were centrifuged for 20 min at 3000 RPM. The supernatant was collected in different tubes and the absorbance was measured at 560 nm using UV spectrophotometer (UV-1800 24 V, Shimadzu, Japan) to estimate the hemoglobin content (Bolajoko et al., 2015). The percentage of hemolysis was estimated as:

Hemolysis (%) = (Absorbance of test /Absorbance of control) X 100

The percentage of human RBC membrane stabilization or protection was calculated by the formula: % Protection = (100 – % Hemolysis)

2.5.2 Lipoxygenase inhibition assay

For determination of lipoxygenase (LOX) inhibition activity, a lipoxygenase inhibitor screening kit (760700, Cayman Chemical Company, USA) was used as per the manufacturer’s protocol. All required material buffers and standard was provided in the kit. Different concentration of the plant extract were prepared 100, 200, 300, 400 μg/mL in dimethyl sulfoxide (DMSO). Absorbance was taken at 590 nm using an ELISA plate reader. The percent inhibition was calculated as follows: $$\mathit{Inhibition}\left(\%\right)=\frac{(AbsorbanceControl-AbsorbanceSample)\times 100}{\left(\mathit{Absorbance}Control\right)}$$

2.5.3 Xanthine oxidase inhibition assay

Xanthine oxidase (XO) inhibitory activity of the extracts with xanthine as substrate was performed as described. Varying dilutions of each extract (100, 200, 300, 400, 500 μg/mL were prepared in phosphate buffer from the stock solution. To the assay mixture that consisted of 1 mL sodium phosphate buffer (0.15 mM, pH 7.5) and 1 mL 0.15 mM xanthine (prepared in phosphate buffer), 100 μL plant extract/ standard were added followed by incubation of 10 min at 37 °C. After incubation 100 µL (0.1 U/mL in phosphate buffer) of XO solution (Himedia, RM7620) was added to the reaction mixture and the tubes were incubated for another 30 min at 37 °C. Allopurinol (prepared in DMSO) was used as a standard. The reaction was stopped by addition of 1 mL 1 N HCl. The absorbance was measured at 290 nm (UV-1800 24 V, Shimadzu, Japan). The percentage of XO inhibitory activity was evaluated by estimation of the uric acid formation in control as compared to that formed in the samples (Wahyuningsih et al., 2016). The XO inhibition percentage was calculated as: $$\mathit{XO}inhibition\left(\%\right)=\frac{(AbsorbanceControl-AbsorbanceSample)\times 100}{\left(\mathit{Absorbance}Control\right)}$$

2.6.1 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay

DPPH is purple colored, free and stable radical used to evaluate the scavenging activity. DPPH solution and test samples were prepared in methanol. To 100 μL of 0.01% DPPH solution, 100 μL of plant extract/ standard (5, 10, 50, 100 μg/mL) were added and the plates were incubated in the dark for 30 min at RT. Absorbance was read at 517 nm (Multiskan ELISA reader, Thermo Scientific). Gallic acid was used as standard and DPPH alone was used as blank (Chang et al., 2001; Boly et al., 2016). The IC₅₀ value (μg/mL) values were calculated using linear regression analysis. Percentage of the radical scavenging activity (RSA) was calculated as: $$RSA\left(\%\right)=\frac{(AbsorbanceControl--AbsorbanceSample)\times 100}{\left(\mathit{Absorbance}Control\right)}$$

2.6.2 Ferric reducing anti-oxidant power (FRAP) assay

FRAP reagent was prepared immediately before the start of the experiment by mixing acetate buffer, 2, 4, 6-tripyridyl-Striazine (TPTZ) and FeCl₃·6H₂O in 10:1:1 Reduction of ferric to ferrous ion was measured as reducing power of the plant extract. Ascorbic acid was used as standard. The electron donating capability of different plant extracts was measured using previous methods (Gan et al. 2010). FRAP assay utilizes antioxidants as reductants in a redox–linked colorimetric assay employing easily reduced oxidant, Fe III. Reduction of a ferric tripyridyltrazine to Ferrous II can be monitored by the measurement of the absorbance at 593 nm. To 2 mL of FRAP reagent, 1 mL of plant extracts were added at varying concentrations (100, 200, 300, 400, 500 μg/mL). The reaction mixture was incubated in dark for 30 min before taking the absorbance at 593 nm (UV-1800 24 V Shimadzu, Japan). Reducing power of plant extract and standard was calculated using regression analysis [x = (y-0.1522)/1.375] of the standard calibration curve of ferrous sulphate prepared with concentrations ranging from 0.2, 0.4, 0.6, 0.8, 1.0 mM (Al-Abd et al., 2015).

2.6.3 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay

ABTS solution (14 mM) was prepared and mixed with potassium per sulfate (4.95 mM) (1:1 v/v) and left for overnight incubation at RT in the dark to yield a dark colored solution containing ABTS radical cations. The prepared ABTS solution was diluted with distilled water till the absorbance reached 0.70 ± 0.02 at 745 nm at 30 °C. To 300 μL of standard (ascorbic acid)/ plant extracts (100, 200, 300, 400, 500 μg/mL) 3.0 mL of ABTS working standard were added individually and mixed. The decrease in absorbance of tested samples was measured at 745 nm after five min of incubation (Re R et al., 1999). The scavenging effect was calculated as: $$\frac{(AbsorbanceControl--AbsorbanceSample)\times 100}{\left(\mathit{Absorbance}Control\right)}$$

The antioxidant capacity of the plant extracts was expressed in terms of IC₅₀ (Inhibitory concentration) values.

2.6.4 Total phenolic content (TPC)

Folin-Ciocalteu (FC) method was utilized for the determination of total phenolic content of W. fruticosa extracts. To 100 μL of FC reagent 1.16 mL of distilled water was mixed followed by addition of 20 μL of standard gallic acid at different concentrations (100, 200, 300, 400, 500 μg/mL) in individual tubes. To the mixture 300 μL of 20 % Na₂CO₃ solution was added. For the test samples, 20 μL of different plant extracts (1 mg/mL) were added to the sample tubes. The reaction tubes were incubated for 30 min at 40 °C followed by measurement of the absorbance at 760 nm. Phenolic content of plant extracts sample was expressed as mg gallic acid equivalent/g dry material (Arabshahi-Delouee et al., 2007, El Jemli et al., 2016).

2.6.5 Total flavonoid content (TFC)

For determining the total flavonoid content in different extracts of W. fruticosa, the aluminum chloride colorimetric method was used (Quettier et al., 2000; Hossain et al., 2011). Quercetin at different concentrations (100, 200, 300, 400 and 500 μg/mL) was used for preparing the calibration curve. 0.6 mL of quercetin solutions or plant extracts were separately mixed with 0.6 mL of 2% aluminum chloride and mixed. The mixture was incubated for 15 min at RT. The absorbance of the reaction mixtures was measured against blank at 440 nm wavelength (UV–Vis spectrophotometer, Shimadzu). The concentration of total flavonoid content in the test samples was calculated from the calibration plot (y = 0.0043x + 0.1585; R² = 0.9972) and expressed as mg quercetin equivalent (QE)/g of dried plant material. All the experiments were carried out in triplicate.

2.6.6 Total tannin content (TTC)

Estimation of total tannin content in the different leaf extracts of W. fruticosa was performed as described previously. For preparing the standard curve, 100 µL of tannic acid at different concentrations (100, 200, 300, 400, 500 μg/mL) was used and the volume of tube was made upto 7 mL with distilled water. To these tubes 20 mM ferric chloride and 8 mM potassium ferric cyanide (prepared in 0.1 M HCl) were added and the contents were mixed. The absorbance of the samples was taken at 700 nm. In the similar fashion, 100 µL of different plant extracts (1 mg/mL) were taken instead of standard and the measurements were done. The tannin content was expressed as mg of tannin per gram dry weight of extract (Muthukumaran et al., 2016).

5.2.1 Reverse sickling assay

Blood obtained from SCD patients was incubated with W. fruticosa plant extracts at three different concentrations. Microscopic observation for physiological changes in terms of reverse sickling was captured (Supplementary Fig. 1). The study demonstrated that methanolic extract was capable of reverse sickling in a concentration dependent manner. Approximately 66 ± 1% of the RBC was found to be reversed sickled at 1000 μg/mL. The reverse sickling in aqueous and hexane extract was 47.5 ± 0.5% and 42.7 ± 0.7% respectively while in the standard drug HU it was found to be 49.5 ± 0.5%. Chloroform and acetone extract displayed least reversal in sickling (Table 2).

5.2.2 Polymerization inhibition assay

Point mutation in β-globin chain of Hb leads to hydrophobic binding amongst the modified Hb molecules in the hypoxic conditions. The rate of polymerization inhibition was calculated for different extracts. The results clearly depict the significant rate of polymerization inhibition in the methanolic extract (1.42 ± 0.03 at 1000 μg/mL; 1.38 ± 0.03 at 500 μg/mL and 1.16 ± 0.01 at 250 μg/mL) followed by hexane (0.85 ± 0.001), chloroform (0.22 ± 0.005) and acetone (0.12 ± 0.005) extracts at 1000 μg/mL (Fig. 2).

Close

Fig. 2

Polymerization Inhibition Assay. W. fruticosa leaf extracts (250, 500 and 1000 μg/mL) were evaluated for their polymerization inhibition potential. Each value is represented as mean ± SEM of three replicate experiments. (*** represent P < 0.05 signifying highly significant results compared to standard). WFH (Hexane extract), WFA (Acetone extract), WFC (Chloroform extract), WFM (Methanol extract), WFW (Aqueous extract).

Export to PPT

5.2.3 Osmotic fragility assay

The assay was performed to decipher the capability of different W. fruticosa leaf extracts in conferring resistance to the hemolysis of the RBC obtained from SCD patients. The result of the assay revealed that there was decrease in percent hemolysis in the RBC from SCD patient in the presence of W. fruticosa extracts. The decreasing percent of hemolysis was displayed in the presence of hexane, acetone and methanol extracts at varying concentration of NaCl solution. Complete osmotic fragility is usually observed at 0.4–0.3% NaCl concentration. The percent hemolysis observed at these NaCl percentages in the methanol and hexane extracts were 42.7 ± 0.6, 53.7 ± 0.6 and 41.2 ± 0.7, 48.2 ± 0.07 respectively. The percent hemolysis observed in all other tested extracts is appended in Fig. 3.

Close

Fig. 3

Osmotic Fragility Assay. Different W. fruticosa extracts were evaluated for their cytotoxic potential in terms of hemolysis (%). All the values have been expressed as mean value obtained in three replicate experiments. Each value is represented as mean ± SEM of three replicate experiments. (*** represent P < 0.05 signifying highly significant results compared to standard). WFH (Hexane extract), WFA (Acetone extract), WFC (Chloroform extract), WFM (Methanol extract), WFW (Aqueous extract).

Export to PPT

5.3.1 Membrane stabilization assay

Different extracts of W. fruticosa at different concentrations were tested for membrane stabilizing potential of RBC treated with hypo saline solution. Concentration dependent protection from hemolysis was observed in the methanol and aqueous extracts that was comparable to the standard drug indomethacin. The maximum protection at 1000 μg/mL in methanol (73.8%) and aqueous extracts (68.8%) was comparable to the protection (77.4%) provided by the standard drug indomethacin (Fig. 4).

Close

Fig. 4

Membrane stabilization potential of W. fruticosa extracts. Different W. fruticosa extracts were analyzed at 200, 400, 600, 800, 1000 μg/mL for analysis of RBC protection from hemolysis. The results are presented in terms of percent protection in hemolysis. Each value is represented as mean ± SEM of three replicate experiments. (*** represent P < 0.05 signifying highly significant results compared to standard). WFH (Hexane extract), WFA (Acetone extract), WFC (Chloroform extract), WFM (Methanol extract), WFW (Aqueous extract).

Export to PPT

5.3.2 Lipoxygenase inhibition assay

Lipoxygenase inhibition potential of W. fruticosa leaf extracts was displayed by acetone and methanol extracts. Concentration dependent inhibition activity was observed in both these extracts that was comparable to the standard (15- Lipoxygenase standard) provided in the kit (Fig. 5). Hexane, chloroform and aqueous extracts were not able to inhibit the lipoxygenase enzyme.

Close

Fig. 5

Lipoxygenase Inhibition Assay. Different W. fruticosa extract and standard drug were analyzed for inhibition of the lipoxygenase enzyme analyzed at 100, 200, 300, 400 μg/mL. Each value represents the mean value of the three replicate experiments. Each value is represented as mean ± SEM of three replicate experiments. (*** represent P < 0.05 signifying highly significant results compared to standard). WFH (Hexane extract), WFA (Acetone extract), WFC (Chloroform extract), WFM (Methanol extract), WFW (Aqueous extract).

Export to PPT

5.3.3 Xanthine oxidase inhibition

Concentration dependent XO inhibition activity was primarily observed in the hexane (57.0 ± 0.5%) and chloroform (56.3 ± 0.6%) extracts while the inhibition obtained in methanol (46.3 ± 0.8%), aqueous extract (35 ± 0.5%) and acetone (5.8 ± 0.2) were lower. The inhibition observed in the standard drug allopurinol was highest compared to all the extracts at all concentrations (Fig. 6).

Close

Fig. 6

Xanthine Oxidase Inhibition Assay. Different W. fruticosa extract and standard drug were analyzed for inhibition of the Xanthine oxidase at 100, 200, 300, 400, 500 μg/mL. Each value represents the mean value of the three replicate experiments. Each value is represented as mean ± SEM of three replicate experiments. (*** represent P < 0.05 signifying highly significant results compared to standard). WFH (Hexane extract), WFA (Acetone extract), WFC (Chloroform extract), WFM (Methanol extract), WFW (Aqueous extract).

Export to PPT

5.4.1 DPPH assay

DPPH basically measures the free radical scavenging capacity of antioxidants and is a commonly used method. The IC₅₀ values of different extracts have been appended in Table 3. The methanol extract displayed significant IC₅₀ values (8.1 ± 1.5 μg/mL) as compared to the standard gallic acid (39.0 ± 0.6 μg/mL).

5.4.2 ABTS assay

The IC₅₀ values for different extracts were evaluated (Table 3). The results conferred that the acetone extract was most potent scavenger of the free radical with IC₅₀ values of 215.8 ± 5.7 μg/mL. Hexane, chloroform, methanol and aqueous extract of W. fruticosa displayed lower scavenging activities as compared to those of standard ascorbic acid (IC₅₀: 284.7 ± 0.9 μg/mL).

5.4.3 FRAP assay

Ferrous Reducing antioxidant power of plant extracts of W. fruticosa was measured and a concentration dependent response was observed in hexane, aqueous, methanol and standard groups. FRAP value or reducing (Fe (III) to Fe (II)) capacity of Hexane extract was highest (1.5 ± 0.5 mM (Fe(II)/g dry weight) followed by methanol (1.45 ± 0.1 mM (Fe(II)/g dry weight) and aqueous (1.45 ± 0.05 mM (Fe(II)/g dry weight) extracts at the highest tested concentration (500 μg/mL). The FRAP values were comparable to those observed in the standard ascorbic acid (0.93 ± 0.01 mM (Fe (II)/g dry weight). Acetone and chloroform extracts displayed least FRAP values (Fig. 7).

Close

Fig. 7

FRAP Assay. FRAP value (mM (Fe(II)/g dry weight) of different W. fruticosa extracts were analyzed at 100, 200, 300, 400, 500 μg/mL. Each value is represented as mean ± SEM of three replicate experiments. (*** represent P < 0.05 signifying highly significant results compared to standard). WFH (Hexane extract), WFA (Acetone extract), WFC (Chloroform extract), WFM (Methanol extract), WFW (Aqueous extract).

Export to PPT

5.4.4 TPC

The results of the assay revealed that methanol extract of W. fruticosa displayed highest phenolic content (113.7 ± 0.7) followed by acetone (68.1 ± 5.6), chloroform (22.4 ± 0.7), hexane (15.9 ± 0.7) and aqueous (7.2 ± 0.7) extracts (Table 3). The total phenolic content has been reported in terms of mg gallic acid equivalents/g dry material.

5.4.5 TFC

As a basis quantitative determination, Total flavonoid content in the W. fruticosa extracts was determined using aluminium chloride colorimetric method. The results have been expressed in quercetin equivalents (QE) per gram dry extract weight (Table 3). The flavonoid content in methanol extract was found to be highest 156.9 mg QE/g, while lowest flavonoid content was present in the hexane extract (0.45 ± 0.1) representing a great variation among the different extracts (Table 3).

5.4.6 TTC

The acetone extract of W. fruticosa had maximum total tannin content (365 ± 2.4), followed by methanol (338.1 ± 7.0), aqueous (101.2 ± 1.2), chloroform (15.8 ± 4.3) and hexane extracts (12.8 ± 9.0) (Table 3) The total tannin content has been reported in terms tannic acid equivalents/g dry weight.

5.5.1 Chemical profiling by LC-MS

Methanol extract of W. fruticosa was most active in terms of anti-sickling, anti-oxidant and anti-inflammatory activities. It was therefore selected for LC-MS analysis for identifying bioactive compounds that might be responsible for observed activities. The LC-MS analysis is based on the comparative analysis of the mass of the currently known compounds obtained from the literature, databases or libraries. The results revealed a total of eighty two compounds from those we were able to identify thirty six compounds (Table 4, Fig. 8). LC-MS result demonstrated the presence of many compounds like kempferol, β sitosterol and quercetin that are previously reported to have anti-sickling properties. Numerous quercetin derivatives are present in the fraction such as quercetin 3-O-(6″-galloyl)-D galactopyranoside, quercetin 3-O-(6″-galloyl)-D galactopyranoside, quercetin, quercetin 3-O-D-galactoside, quercetin 3-L-arabinoside, quercetin 3-rhamnoside, quercetin 3-L-arabinoside, quercetin 3-rhamnoside. Out of the total 82 compounds obtained forty seven compounds are unknown that may play crucial role in anti-sickling, anti-oxidant and anti-inflammatory activities.

Close

Fig. 8

LC-MS chromatogram of W. fruticosa methanolic leaf extract. Chromatogram in positive ion mode (A) and Chromatogram in negative ion mode (B).

Export to PPT