Phytochemical studies, antioxidant activities and identification of active compounds using GC–MS of Dryopteris cochleata leaves

2.1 Chemicals

Folin–Ciocalteu’s reagent, 1,1-diphenyl-2-picrylhydrazyl (DPPH), gallic acid, (±)-catechin, rutin, α-tocopherol, ascorbic acid, bovine serum albumin, glucose, tannic acid, solvents and other reagents used were of analytical grade purchased from Sigma Chemical Co. (St. Louis, MO, USA).

2.2 Plant collection and extraction

Healthy D. cochleata leaves were collected from Kolli Hills, Namakkal District, Tamil Nadu, identified and authenticated by Botanist, St. Xavier’s College, Tirunelveli, Tamil Nadu, India.

The plant materials were washed with tap water, prior to distilled water, shade dried and powdered. The powdered plant materials were subjected to successive extraction with petroleum ether, chloroform, ethylacetate, acetone, methanol and water using Soxhlet extractor. The extracts were dried in vacuum pump at 40 °C. The dried crude extracts were stored in freezer at 0 °C for future use.

2.3 Phytochemical screening

The preliminary phytochemical screening tests were carried out to identify the useful constituents by standard methods (Onwukeame et al., 2007).

2.4 Determination of total phenolic contents

The total phenolics in the extracts were estimated by spectrophotometric assay (Barreira et al., 2008). One milliliter of sample (concentration 1 mg/mL) was mixed with 1 mL of Folin and Ciocalteu’s phenol reagent. After 3 min, 1 mL of saturated sodium carbonate solution was added to the mixture and made upto 10 mL with distilled water. The reaction was kept in the dark for 90 min, after which the absorbance was read at 725 nm. Gallic acid was used for constructing the standard curve (20–100 μg/mL, Y = 0.025x + 0.0378, R² = 0.997) and the results were expressed as μg of gallic acid equivalents/mg of extract (GAEs).

2.5 Determination of total flavonoid contents

Flavonoid contents in the extracts were determined by spectrophotometric method (Barreira et al., 2008). The (250 μL) extract (concentration 1 mg/mL) was mixed with 1.25 mL of distilled water and 75 μL of a 5% NaNO₂ solution. After 5 min, 150 μL of 10% AlCl₃ solution was added. After 6 min, 500 μL of 1 M NaOH and 275 μL of distilled water were added to prepare the mixture. The solution was mixed well and the absorbance was read at 510 nm. (±)-catechin was used to calculate the standard curve (20–160 μg/mL, Y = 0.0006x + 0.0603, R² = 0.9997) and the results were expressed as μg of (±)-catechin equivalents (CEs) per mg of extract.

2.6 Estimation of total flavonol contents

One millilitre of leaf extract (concentration 1 mg/mL) was mixed with 1 mL aluminium trichloride (5 mg/mL) and 3 mL sodium acetate (25 mg/mL). The absorbance read at 440 nm was read after 2.5 h. The absorption of standard rutin solution (0.5 mg/mL) in methanol was measured under the same conditions (Grubesic et al., 2005). All determinations were carried out in duplicates. The amount of flavonols in plant extracts in rutin equivalents (RE) were calculated by the following formula. X = (A.m₀)/(A₀.m), where X is the flavonol content, mg/mg plant extract in RE, A is the absorption of plant extract solution, A₀ is the absorption of standard rutin solution, m is the weight of plant extract (mg), and m₀ is the weight of rutin in the solution (mg).

2.7 Estimation of tannin contents

Tannin content of the extracts was measured by Folin–Denis method (Oyaizu, 1986). The various extracts (50 μL) were made up to 7.5 mL by adding double distilled water. Then 0.5 mL Folin–Denis reagent and 1 mL of Na₂CO₃ were mixed with it. Again the volume was made up to 10 mL by double distilled water. Absorption was recorded at 700 nm. Tannic acid was used to calculate the standard curve (20–120 μg/mL, Y = 0.069x + 0.0091, R² = 0.9985) and the results were expressed as μg of tannic acid equivalents per mg of extract.

2.8 DPPH radical scavenging activity

Various concentrations of D. cochleata leaf extracts (0.3 mL) were mixed with 2.7 mL of methanol solution containing DPPH radicals (6 × 10⁻⁵ mol/L). The mixture was shaken vigorously and allowed to stand for 60 min in the dark. The reduction of the DPPH radical was determined by reading the absorbance at 517 nm (Barros et al., 2007). The radical-scavenging activity (RSA) was calculated as a percentage of DPPH discoloration, using the equation: %RSA = [(A_DPPH−A_S)/A_DPPH] × 100, where, A_S is the absorbance of the solution when the sample extract is added at a particular level and A_DPPH is the absorbance of the DPPH solution. The extract concentration providing 50% of radical-scavenging activity (IC₅₀) was calculated from the graph of RSA percentage against extract concentration. Ascorbic acid and α-tocopherol were used as standards.

2.9 Reducing power

The reducing power of D. cochleata leaf extracts was determined (Oyaizu, 1986). Various concentrations of different solvent extract (1 mL), phosphate buffer (1 mL, 0.2 M, pH 6.6) and potassium ferricyanide (1 mL, 10 mg/mL) were mixed together and incubated at 50 °C for 20 min. Trichloroacetic acid (1 mL, 100 mg/mL) was added to the mixture and centrifuged at 8000 rpm for 5 min. The supernatant (1 mL) was mixed with distilled water (1 mL) and ferric chloride (0.1 mL, 1 mg/mL) and then the absorbance was measured at 700 nm.

2.10 Estimation of carbohydrate content

Total carbohydrate contents were estimated by Anthrone method (Hedge et al., 1962). Glucose was used to calculate the standard curve (20–120 μg/mL, Y = 20x, R² = 1) and the results were expressed as μg of glucose equivalents per mg of extract.

2.11 Estimation of protein content

Total proteins were estimated by Lowry’s method (Lowry et al., 1951). Bovine serum albumin was used to calculate the standard curve (20–160 μg/mL, Y = 0.0004x + 0.032, R² = 0.9569) and the results were expressed as μg of bovine serum albumin equivalents per mg of extract.

2.12 Estimation of ascorbic acid content

One mg of various extracts was treated with 4.0 mL of 10% trichloro acetic acid and centrifuged for 20 min at 3500 rpm and 0.5 mL of supernatant was then, mixed with 0.1 mL DTC reagent (2,4-dinitrophenylhydrazine-thiourea-copper sulfate reagent). The tubes were incubated at 37 °C for 3 h. Ice cold 65% H₂SO₄ (0.75 mL) was added and the tubes were allowed to stand at room temperature for an additional 30 min. The color developed was read at 520 nm (Omaye et al., 1962). Ascorbic acid was used to calculate the standard curve (20–160 μg/mL, Y = 0.0043x + 0.03682, R² = 0.9652) and the results were expressed as μg of ascorbic acid equivalents per mg of extract.

2.13 GC–MS analysis

For the GC–MS analysis a 30 × 0.25 mm × 0.25 μm df a 5% diphenyl/95% dimethyl poly siloxane column; was used in Clarus 500 Perkin–Elmer gas chromatograph with a Turbo mass gold-Perkin–Elmer detector. For GC–MS detection, an electron ionization system with ionization energy of 70 eV was used. Helium gas (99.999%) was used as the carrier gas at a constant flow rate of 1 mL/min. and an injection volume of 3 mL was employed (split ratio of 10:1). Injector temperature was 250 °C; ion-source temperature was 280 °C. The oven temperature was programed from 110 °C (isothermal for 2 min.), with an increase of 10 °C/min to 200 °C (no hold), then from 5 °C/min to 280 °C, ending with a 9 min. isothermal at 280 °C. Mass spectra were taken at 70 eV; a scan interval of 0.5 s and fragments from 45 to 450 Da. Total GC running time was 36 min. The relative percentage amount of each component was calculated by comparing its average peak area to the total areas. Software adopted to handle mass spectra and chromatograms was a Turbomass version 5.2.0.

2.14 Statistical analysis

Statistical analyses were conducted using SPPS software (16.0 version). Analysis of variance (ANOVA) in a completely randomized design and Turkey’s multiple range tests were used to compare any significant differences between samples. Values were expressed as means ± standard deviations. All determinations were done at least in triplicate and all were averaged. The confident limits used in this study were based on 95% (p < 0.05).

3.1 Phytochemical screening

The quantitative screening of the crude leaf extracts had revealed the presence of numerous phytoconstituents. The presence of them from copious, slightly to moderately present in all the test extracts has been listed in Table 1.

3.2 Total phenolic content

The acetone extract recorded the highest content of about 90.45 μg/mL. The next highest was observed in ethylacetate extract. Chloroform and methanol extracts closely ranged in their contents and the least observed extract was water of about 28.64 μg/mL.

3.3 Total flavonoid content

The acetone extract had recorded the highest content for flavonoid too, then stood the ethylacetate (145.78 μg/mL) and chloroform (122.56 μg/mL) extracts. The expected methanol extract recorded a low content of about 77.71 μg/mL. Least value was obtained for water and petroleum ether extract.

3.4 Total flavonol content

Both acetone and ethylacetate extract recorded a significant flavonol content ranging around 47 μg/mL. Then thenext higher content of about 46.35 μg/mL was recorded by methanol extract. Unlike the other extracts chloroform recorded 26.40 μg/mL.

3.5 DPPH radical scavenging activity

Fig. 1 illustrates a significant decrease in the concentration of DPPH free radical with various concentrations of the plant extracts, due to their scavenging ability against the standard ascorbic acid and α-tocopherol. IC₅₀ values have been calculated that represents the concentration of an inhibitor that is required for 50% inhibition of its target (i.e.) DPPH radicals. The scavenging effects of the extracts were in the order: ascorbic acid > acetone > ethylacetate > α-tocopherol > chloroform > methanol > water > petroleum ether.

Close

Figure 1

DPPH radical scavenging activities of Dryopteris cochleata leaves extracts in different concentrations. Each value represents a mean ± S.D. Symbols represent statistical significance from control (^*p < 0.05).

Export to PPT

3.6 Reducing power

Reducing power was seen to increase with increase in the concentration of the extracts as shown in the Fig. 2. EC₅₀ was calculated from the graph to determine the concentration of extracts that provided 0.5 absorbance at 700 nm. The reducing ability of the extracts was in the order: ascorbic acid > acetone > ethylacetate > α-tocopherol > chloroform > methanol > water > petroleum ether, unlike the order seen for DPPH scavenging activity.

Close

Figure 2

Reducing power activity of Dryopteris cochleata leaves extracts in different concentrations. Each value represents a mean ± S.D. Symbols represent statistical significance from control (^*p < 0.05).

Export to PPT

3.7 Estimation of carbohydrates

In the case of carbohydrates, acetone and ethylacetate extracts exhibited higher contents while a moderate content for water and chloroform extracts were observed. A lesser content (38.10 μg/mL) was observed for methanol extract.

3.8 Estimation of tannins

The highest value of about 102.02 μg/mL was recorded by the acetone extract. About one-half of the content was only recorded by the ethylacetate extract and then the methanol extract. Chloroform and water extracts showed lesser contents.

3.9 Estimation of protein

The order obtained in the protein content of extracts were acetone > ethylacetate > methanol > chloroform > water > petroleum ether. Protein content of about 174.02 μg/mL was recorded for the acetone extract.

3.10 Estimation of vitamin C

The acetone and ethylacetate extracts exhibited higher vitamin content of about 157.37 and 156.04 μg/mL, respectively followed by methanol (152.19 μg/mL) extract. While the remaining extracts showed insignificant amounts of vitamin-C.

3.11 GC–MS analysis

Interpretation on mass spectrum GC–MS was conducted using the database of National Institute Standard and Technology (NIST) having more than 62,000 patterns. The spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST library. Six compounds were identified in D. cochleata leaf extract by GC–MS analysis was shown in Fig. 3. The active principles with their retention time (RT), molecular formula, molecular weight (MW) and concentration (%) were taken into account.

Close

Figure 3

Chromatogram obtained from the GC–MS with the extract of Dryopteris cochleata leaves.

Export to PPT

For almost all the contents and activities thereby, the petroleum ether extract exhibited lower effects, which may be due its less polarity. All the contents and the estimations are neatly portrayed in Table 2 for the readers to get the fruit of this labor.