Potent anticancer, antioxidant and antibacterial activities of isolated flavonoids from Asplenium nidus

2.1 Chemicals and reagents

All chemicals used were of the highest purity (⩾99.0%) and were purchased from Sigma Aldrich and Merck Malaysia, as follows: NaCl, HCl, FeCl_2, ethanol, methanol, acetone, H₂SO_4, ferric chloride, KCl, Na₂HPO₄, chloroform, conc. H₂SO₄, acetic anhydride, KH₂PO₄, NaNO₂, AlCl_3, NaOH, H₂O₂, Na₂CO₃, ferrozine, FeSO₄ and glacial acetic acid. Bacterial strains were bought by University Malaya (UM), Malaysia. The GC and GC/MS techniques were used on Hewlett-Packard (HP) 5973 with HP-6890 gas chromatograph having HP5 column of 30 m × 0.25 mm i.d with 0.25 μm of film thickness. Strains were cultivated in nutrient broth. The human carcinoma HeLa cells were provided by the University Malaya (UM) research laboratory. The Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma Chemical Company, MO, Saint Louis, USA) with 10% heat-inactivated fetal bovine serum in a humidified atmosphere with 5% CO₂ at 37 °C were used for cell maintenance.

2.2 Preparation of plant extract

The fresh fern/plant material (whole plant) was washed under running tap water, air-dried and homogenized into fine powder. The powered preparations were stored in air-tight glass vials. For preparing tissue extracts, 1.0 g of the homogenized powder was placed in 10 ml of methanol in a conical flask, covered with cotton cap and kept on a rotary shaker at 200 rpm for 48 h. The extract was filtered through Whatmann filter paper No.1, the resultant filtrate of which was centrifuged at 10,000 rpm at 4 °C for 15 min. The supernatant was collected and completely evaporated at room temperature. The left-over methanolic fern extract (MFE) residue was dissolved in 0.05 M Phosphate buffered saline (PBS), pH 7.2 to reach a final concentration and was sterilized by filtration (0.22 μm Millipore filter). The filter-sterilized preparation of MFEs thus obtained was stored in a freezer at −70° C in air-tight vials for further studies.

2.3 Flavonoid assays

The flavonoid assays were performed according to the reported procedure (Belmekki and Bendimerad, 2012). The determination of flavonoids was performed according to the colorimetric assay. Diluted ammonia (5 ml) solution was mixed with plant extract, followed by slow addition of concentrated H₂SO_4. A yellow coloration in the extract which diminished on standing indicated the presence of flavonoids.

2.4 Quantitative assay for flavonoids

The flavonoid assay was done according to the reported procedure. The aluminum chloride (AlCl₃) colorimetric assay was used to determine the total flavonoid content in MFE (Marinova et al., 2005). 0.5 ml of sample (2000 μg/ml) was added to 2.8 ml of distilled water, 0.1 ml of 1 M potassium acetate, and 0.1 ml of 10% aluminum chloride. The content was mixed using a vortex and incubated at 37 °C for half an hour. The absorbance was measured at 510 nm with a micro plate reader (Molecular Devices).

2.5 Fractionation of crude extracts – using column chromatography

A column of 40 cm was filled up to 31 cm height having 2.5 cm diameter with slurry of silica gel 60 (Merck) (0.063–0.2 mm being the particle size 70–230 mesh). After equilibration of the column with 100% methanol for 30 min, 5 g of the plant extract along with 10 g gel was mixed in concentrated methanol slurry. Air-dried mixture was then decanted to the top of the packed column up to 7.5 cm in height. Firstly the column was eluted by methanol at 40 drops per minute followed by solvent combination of ethyl acetate/methanol/water (EMW) in the ratio 20:2.7:2.5 (Masoko et al., 2008). Each fraction was collected with a standard eluent volume of 200 mL. A total of 15 fractions thus collected were subjected to a rotary evaporator, concentrated until dryness and subsequently transferred to glass Petri dishes. Residual solvents were than gathered after air-drying the dishes for 48 Hours. To determine the number of phytocomponents in each fraction, primary phytochemical methods were used (Harborne, 1984; Kim et al., 2003).

2.6 Compound identification through gas chromatography & mass spectrometry (GC/MS)

A Hewlett-Packard (HP) mass spectrometer interfaced with an HP-6890 gas chromatograph with an HP5 column was used for GC and GC/MS analysis. In split mode, sample was added through the injector along with helium (carrier gas), with initial flow rate 0.7 ml/minute and a 32 cm/sec average linear velocity. The settings of the temperature were as described here in: injector temperature 220 °C; oven temperature 70 °C (initial), a 30 mL/min purge flow and 0.2 minute purge time, 5 °C/minute ramp, final temperature 240 °C, pressure of 8.20 psi. Mass spectra with pervious literature (Adams, 1995) were used as identifier for compounds. Peak areas of the total fraction composition were observed to record the percentage composition of every compound.

2.7 Anti-oxidant activity assay

Antioxidants react with 1, 1-diphenyl-2-picrylhydrazyl (DPPH) stable free radicals to produce its reduced form (DPPH-H). The DPPH-H formation in the reaction mixture is assayed (Xiao et al., 2011) at A517. The reduction of the DPPH radical to the DPPH-H form is indicated by the formation of a yellow color. A lower A517 of the reaction mixture indicated a higher free radical scavenging activity. DPPH solution (0.1 mM) in methanol was prepared and 3.0 ml of purified flavonoids at different concentrations was added to 1.0 ml of this solution and 30 minutes later the A517 values were recorded. A blank was prepared without adding flavonoids to serve as a control. Ascorbic acid (1–18 μg/ml) was used as a standard. The capability to scavenge the DPPH radicals was determined as follows: $$\text{DPPH}\text{scavenged}(\%)=\frac{A517\text{control}-A517\text{test}}{A517\text{control}}\times 100$$

2.8 Determination of antibacterial activity by inhibitory concentration (MIC₅₀) of fractions

Micro-broth dilution method performed in 96-well plates to determine the MIC₅₀ of test fractions. Test fractions were prepared at a concentration of 5.0 mg/mL, while gliricidin-7-O-hexoside and quercetin 7-O-rutinoside were prepared at a concentration of 2 μg/mL and 200 μg/mL respectively. For nutrient broth two-fold dilutions of every fraction were prepared in test wells. For such fractions, the ranges of the final concentration were 2–2500 μg/ml, 0.001–2 μg/ml and 0.003–200 μg/ml, gliricidin-7-O-hexoside and quercetin-7-O-rutinoside, respectively. Then, 20 μL of an 18-h-old broth culture of Gram-negative multidrug resistant suspension was mixed with 100 μL of the fraction having the culture medium. The preparation of control was done with the bacterial suspension, culture medium and broth only. Alongside each batch of tests at a concentration range of 0.0002–2 μg/mL, positive-control antibiotic was amoxicillin. To check absorbance of plates after and before three days of incubation at room temperature observed under micro-aerophilic situations, an automatic enzyme-linked immunosorbent assay (ELISA) microplate reader adjusted to 610 nm was used. To determine increase and decrease in bacterial growth, absorbencies were compared and were represented graphically against concentration. Least concentration of fraction resulting in inhibition of 50% bacterial growth was calculated as MIC₅₀.

2.9 Cytotoxicity assay

Uniform volumes of HepG2 and HeLa cell suspension (200 μl/well; ∼2 × 10⁴cell/ml) were poured into the selected wells of a 96-wells sterile tissue culture plate. The filter-sterilized fern extract (200 μg/ml stock) was dispensed into the wells of the microtiter plate to achieve final concentrations of 0–600 μg/ml in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma Chemical Company, MO, Saint Louis, USA). The cells treated with gliricidin-7-O-hexoside and quercetin-7-O-rutinoside (the flavonoids isolated in greatest quantities) were incubated in a CO₂ incubator with 95% humidity at 37 °C for 16–18 h. The DMEM (100 μl) was removed from each of the wells and 20 μl of freshly prepared MTT (5 mg/ml) was mixed in these wells followed by incubation for 1 h at 37 °C in a CO₂ incubator. Thereafter, the DMEM containing MTT was completely removed from the wells using a multi-channel auto-pipette and the purple colored product by the cells in each of the wells was extracted in DMSO (100 μl/well) and A570 values were recorded in an ELISA reader. Each of the MFE concentrations was tested in quadruplicate and mean values ± standard deviation (SD) were calculated after the MTT assay. The appropriate controls (containing an appropriate volume of methanol alone) were also considered as a negative control to determine the effect of methanol on the viability of the proliferating cells cultured in vitro. Each analysis was performed in triplicate and resulted as reproducible within experimental errors (RSD < 5.0%), as was nearly comparable to standard amoxicillin.

2.10 Statistical analysis

Results values were taken as mean ± S.D. of three independent and different experiments and were checked by a one-way analysis of variance (ANOVA). Remarkable differences were spotted at p < 0.05.

Gliricidin-7-O-hexoside, being the most abundant flavonoids (3.87%), followed by quercetin-7-O-rutinoside, (3.09%), keampferol-3-O-rutinoside and myricetin-3-O-rhamnoside were used against multidrug-resistant pathogens, Proteus mirabilis, Proteus vulgaris and Pseudomonas aeruginosa.

3.1 Chemical composition and percentage composition of flavonoids in A.nidus

EAMW fractions 5 and 6 with the least number of components were based on GC/MS analysis. 12 flavonoids were recognized from present fractions, 3 were unknown flavonoids. EAMW two of the fractions (fractions 1 and 3) revealed the presence of compounds 3 and 12, respectively, representing 13.12% and 2.61% and the total composition was 15.12%. The flavonoids detected were gliricidin-7-O-hexoside (3.87%) and quercetin-7-O-rutinoside (3.09%) followed by keampferol-3-O-rutinoside (0.9%) and myricetin-3-O-rhamnoside (1.10%). Further information on the present phytochemical compound composition is outlined in Table 1.

3.2 Minimum inhibitory concentration (MIC₅₀) of fractions

Out of 13 fractions collected, 12 showed remarkable anti-bacterial activity. The MIC₅₀ values of the fractions ranged from 310 μg/ml to 2500 μg/ml; the ranges of amoxicillin, gliricidin-7-O-hexoside and quercetin-7-O-rutinoside were 0.0003–0.06 μg/ml, 0.004–0.06 μg/ml and 0.005–6.0 μg/mL, respectively, as shown in Table 2. Inhibition values for gliricidin-7-O-hexoside and quercetin-7-O-rutinoside were found to be comparable standard (amoxicillin) (p > 0.05).

3.3 DPPH assay

The flavonoids from A. nidus exhibited notable DPPH radical scavenging activity, which increased with increasing concentrations. The scavenging activity of gliricidin-7-O-hexoside (78.1%) was higher than that of quercetin-7-O-rutinoside (69.2%) as presented in Fig. 2. The results indicate that the maximum yield extracted flavonoids by A. nidus had a remarkable DPPH radical-scavenging activity, underscoring the potential clinical utility of these flavonoids.

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Figure 2

Anti-oxidant activity of flavonoids (gliricidin7-O-hexoside, and quercetin 7-O-rutinoside, from Asplenium nidus assessed by the DPPH radical-scavenging assay. Data represent means ± SD of three independent experiments. ■ – Gliricidin-7-O-hexoside. ▴ – Quercetin-7-O-rutinoside.

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3.4 Cytotoxicity assay

The observed results of MTT assay indicated that both potent extracted flavonoids (gliricidin-7-O-hexoside and quercetin-7-O-rutinoside) at all the selected concentrations (240 to 600 μg/ml) exhibited marked cytotoxicity or inhibitory effect on the growth of HepG2 and HeLa cells (Table 3). The maximum viability of the mammalian cells was observed at 600 μg/ml of gliricidin-7-O-hexoside (HepaG2 76.15 ± 0.04) and HeLa (66.91 ± 1.93). At enhanced concentration of gliricidin-7-O-hexoside (600 μg/ml), a marked decline in the viability of the cells was noticed indicating the inhibitory effect of A.indus. These results indicate that flavonoids extracted by A. indus had a remarkable cytotoxic activity, with an IC₅₀ value of 507 μg/ml. Sylvestre et al. (2007) showed that the bioactive compounds did not affect cell viability at concentrations <100 μg/mL, thus validating its safety within this range. It is interesting to note that in the current study, the compound was active at concentrations <1 μg/mL. The high IC50 of the two compounds indicate that they may be safe for human consumption.