Antioxidant and cytotoxic activities of Dendrobium moniliforme extracts and the detection of related compounds by GC-MS

The stems of Dendrobium moniliforme were collected from Daman of Makawanpur district in central Nepal. The plant was identified by Dr. Keshav Raj Rajbhandari. A voucher specimen of this plant is deposited in the Tribhuvan University Central Herbarium (TUCH) (voucher number – M02). The collected stems were air-dried and powdered. A Soxhlet extraction was used to prepare plant extracts with solvents of increasing polarity: hexane, chloroform, acetone, ethanol and methanol in the ratio of 1:10 (w/v) [32]. The solvents were then evaporated at room temperature to obtain dry extract and the extract was stored at 4 °C.

The total polyphenol content (TPC) in each extract was determined using the Folin-Ciocalteu’s reagent method [33]. The reaction mixture was prepared by mixing 0.5 ml of a stock solution of extract, 2.5 ml of 10% Folin-Ciocalteu’s reagent, and 2.5 ml of 7.5% NaHCO₃. In tandem, a blank solution was prepared from 0.5 ml of ethanol, 2.5 ml of 10% Folin-Ciocalteu’s reagent and 2.5 ml of 7.5% NaHCO₃. The reaction mixtures were then incubated at room temperature for 45 min and their absorbance was measured using a Genesys UV-visible spectrophotometer at 765 nm. The TPC in extract was expressed as micrograms of gallic acid equivalent (GAE) per milligrams of dry extract.

The total flavonoid content (TFC) in each extract was determined using the aluminium chloride method [33]. The reaction mixture was prepared by mixing 1 ml of a stock solution of extract and 1 ml of 2% AlCl₃. In tandem, a blank solution was prepared by mixing 1 ml of ethanol and 1 ml of 2% AlCl₃. The reaction mixtures were incubated for an hour at room temperature and then their absorbance was measured using a Genesys UV-visible spectrophotometer at 415 nm. The TFC in extract was expressed as micrograms of quercetin equivalent (QE) per milligrams of dry extract.

The in vitro antioxidant activity of the extracts was determined using a DPPH (2,2-diphenyl-1-picrylhydrazyl) free-radical scavenging assay according to the method described in previous publication [34]. The stock solution of extract was diluted to prepare a series of concentrations (50, 100, 200, 400 and 800 μg/ml) for the antioxidant assay. An aliquot of 1.5 ml of 0.25 mM DPPH solution in ethanol and 1.5 ml of extract at each of the various concentrations was mixed. The mixture was shaken vigorously and allowed to sit for 30 min reach a steady state at room temperature. The decolourization of DPPH was determined by measuring the absorbance at 517 nm using a Genesys UV-visible spectrophotometer. The percentage of DPPH free-radical scavenging activity of each extract was then calculated using the following equation:

Where; A₀ was the absorbance of the control (only DPPH, without extract), A₁ was the absorbance of the extract with DPPH, and A₂ was the absorbance of the extract without DPPH.

The antioxidant capacity of extract was expressed as the 50% inhibition of DPPH radicals (IC₅₀ μg/ml of extract). The IC₅₀ of extract was calculated using a polynomial regression equation in which the abscissa represents the series of concentrations of extract and the ordinate represents the mean of the triplicate percentage of antioxidant activity at each concentration.

The cytotoxic activity of the extracts was determined by using MTT assay [29, 35]. Human brain tumor cells (U251) and cervical cancer cells (HeLa) were cultured in RPMI 1640 medium and incubated under 5% CO₂ at 37 °C for 48 h to reach 80% confluence. The cells were harvested by gently scraping them with a cell scraper and re-suspended in a medium. From the suspension, 5 × 10³ cells in 100 μl of medium were dispensed into each well of a 96-well microtiter cell culture plate and incubated under the same conditions for 48 h to allow time for the adherence and growth of cells. The supernatant was gently aspirated, and 100 μl of extracts were added with a range of four cytotoxic concentrations (100, 200, 400 and 800 μg/ml) prepared in a medium and incubated for 24 h. Ten microliter of 5 mg/ml MTT was added to every well and the plate was re-incubated for another 4 h. The formazan crystals formed were dissolved in 100 μl of DMSO. The plate was then read on a microplate reader (iMark™, Bio-Rad) at 595 nm. The number of dead cells per well was calculated as a percentage of the control, thereby measuring cell death after extract exposure. A dose-response curve was plotted for each extract to calculate the 50% inhibition of cell growth (IC₅₀ μg/ml of extract). The cytotoxic capacities of extracts (IC₅₀) were calculated using a regression equation in which the abscissa represents the series of concentrations of extract and the ordinate represents the mean of the triplicate percentage of inhibition of cell growth.

All data are expressed as a mean of three analyses. A linear regression model was applied to obtain equations for standards, gallic acid and quercetin. The total polyphenol and flavonoid contents were calculated by using linear equation of standards. The Duncan multiple-range test was applied to compare the significance differences of the total polyphenol and flavonoid contents in the extracts at p ≤ 0.05. The IC₅₀ values of the antioxidant and cytotoxic activities of the extracts were calculated by using an appropriate linear or non-linear regression equation obtained from the percentage activity of each extract at various concentrations with the F-statistic at p ≤ 0.05. All the analysis was done using statistical software R version 3.1.2 [36].

Total polyphenol contents (TPCs) in the extracts were calculated using the linear equation for standard gallic acid (y = 0.0154 x – 0.3285; R ²  = 0.989; p = 0.005) and total flavonoid contents (TFCs) was calculated using the linear equation for standard quercetin hydrate (y = 0.0242 x – 0.1845; R ²  = 0.976; p = 0.012). The TPCs in the extracts ranged from 32.68 to 116.65 μg GAE/mg of extract and TFCs from 54.13 to 116.67 μg QE/mg of extract (Additional file 1). The TPC of DMC (116.65 μg GAE/mg of extract) was much greater than those of other extracts. The TPCs of DME and DMA were the next highest respectively. The TFC of DMA (116.67 μg QE/mg of extract) was much higher than it was in other extracts, though the TFC of DME was also high (Fig. 1). The values of TPC and TFC in all the extracts were statistically significantly different at p ≤ 0.05.

The antioxidant activity of different extracts was determined by DPPH free-radical scavenging assay. The mean percentage of DPPH free-radical scavenging activity at different concentrations of extracts is shown in Fig. 2. DMH had the highest percentage of DPPH free-radical scavenging activity (94.48%), followed by DME (94.45%), DMC (94.35%), and DMA (93.71%), and, considerably lower by DMM (86.92%) at their 800 μg/ml concentration. The percentage of DPPH free-radical scavenging activity was measured at a range of concentrations from 50 to 800 μg/ml (Additional file 2). In all cases, it increased as the concentration increased. The antioxidant capacity of each extract was measured as the IC₅₀ value of the extract, or in other words, the amount required to scavenge 50% DPPH free-radicals.The antioxidant capacities of DMC, DMA, DMH and DME had much lower IC₅₀ values – 42.39 μg/ml, 49.56 μg/ml, 52.68 μg/ml and 58.77 μg/ml respectively than did DMM (223.15 μg/ml). Except for DMM, the IC₅₀ values of other extracts were statistically similar to the ascorbic acid (AA), whose IC₅₀ value was 38.21 μg/ml (Fig. 2).

The cytotoxic activity of extracts of D. moniliforme against the HeLa and U251 cell lines was determined using MTT colorometric assay. The mean percentage of the cytotoxic activity of extracts of different concentrations is shown in Fig. 3 (for HeLa cells) and in Fig. 4 (for U251 cells). The percentage of cell growth inhibition of extract-treated cells is increased as the concentration of the extract increased (Additional file 3 for HeLa cells and Additional file 4 for U251 cells). DMM at the concentration of 800 μg/ml showed the highest inhibition percentage of HeLa cells growth (78.68%), while DME at the concentration of 800 μg/ml had the highest cell growth inhibition of U251 cells (51.95%). DME at 800 μg/ml showed moderate inhibition of the growth of HeLa cells (72.66%) and DMM at 800 μg/ml showed moderate inhibition of the growth of U251 cells (39.93%). The cytotoxic capacity of an extract was measured as its IC₅₀ value, or the amount of extract required to inhibit the 50% cell growth. The cytotoxic capacities of DMM and DME against HeLa cells were 155.80 μg/ml and 181.93 μg/ml of extract respectively (Fig. 3). Their cytotoxic capacities were statistically significantly different from those of other extracts. In terms of inhibiting the growth of U251 cells, the cytotoxic capacity of DME was 772.50 μg/ml of extract, but neither DMH, DMC nor DMA showed any such capacity (Fig. 4).

The bioactive compounds present in the hexane, chloroform, acetone, ethanol and methanol extracts obtained from D. moniliforme stems were detected using gas chromatography (GC) and identified through mass spectrometry (MS). The compounds detected and identified in DME and DMM are shown in Tables 1 and 2. The results of the GC-MS analysis of the other extracts are not shown here because those extracts showed either no or little cytotoxic activity. The elution time of compounds with their base mass-to-charge ratio (m/z) and their contents by percentage were also determined. Based on abundance, two major compounds dimethylsulfoxonium formylmethylide (37.99%) and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (21.41%) were detected and identified in DMM and two, 5-(hydroxymethyl)-2-furancarboxaldehyde (45.59%) and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (16.74%) in DME. Both DME and DMM had a number of other phenol derivatives too including tetrahydro-1,1-dioxide-thiophene-3-ol, γ-sitosterol, 2-methoxy-4-vinylphenol, 2,6-dimethoxy-phenol, 2,6-dimethoxy-4-(2-propenyl)-phenol, n-nonadecanol-1, 1-heptacosanol and stigmast-5-en-3-ol. The GC chromatogram of DMM and DME are presented in Additional files 5 and 6 respectively.