1. INTRODUCTION:

The free radicals are produced as byproducts of normal cellular metabolic processes and scavenged by enzymatic and non-enzymatic antioxidative defense system of body. In healthy organisms, production and scavenging of free radicals is balanced by antioxidant defense system of body and any imbalance in the same leads to a condition called oxidative stress (Galle, 2001). This stress leads to definite deleterious effects which are associated with many life threatening anomalies such as malignant cancers, cardiovascular diseases and inflammatory diseases (Valko et al, 2007).

To surmount these problems, use of plant derived antioxidants for dietary and remedial intent is a very hopeful and a novel way in recent times (Laguerre et al, 2007). Antioxidants protect us from a variety of diseases by scavenging the free radicals. Antioxidants bring to bear their mode of action by suppressing the construction of free radicals either by inhibition of enzymes or by chelating trace elements (Subhashini et al, 2011). Major dilemma is to extend tools to determine the antioxidant capacity of plants with precision. There are many rapid methods to determine the antioxidant capacities which differ in terms of their determination principles, aims and experimental conditions.

In this research, the antioxidant activity of oil extract derived from a gymnosperm, namely, Araucaria cunninghamii Aiton ex D.Don has been investigated. To measure the antioxidant activity, DPPH radical scavenging activity, reducing power capacity, nitric oxide scavenging activity and total antioxidant capacity were determined. Total phenolic, flavonoid and tannin contents were also estimated. Keeping the immense significance of these compounds in mind, it was decided that the experimental work should revolve around the determination of antioxidant capacity and identification of these classes of antioxidants with HPLC analysis.

2. MATERIALS AND METHODS:

2.1 Study material:

The study material was the leaves of Araucaria cunninghamii Aiton Ex D.Don which were collected from Botanical garden of Guru Nanak Dev University, Amritsar and authenticated at department of botanical and environmental sciences Guru Nanak Dev University, Amritsar.

2.2 Oil extraction:

An oil is a dense fluid containing volatile fragrance compounds from plants. To extract the oil from the leaves of Araucaria cunninghamii Aiton Ex D.Don, 150 g of freshly ground, air-dried leaves were distillated in sohxlet apparatus for 48 h. Petroleum ether (1500 mL) was used as the extraction solvent. The oil was stored at 4°C until investigated for antioxidant properties.

2.3 Evaluation of antioxidant activity:

2.3.1 DPPH radical scavenging activity:

Hydrogen donating ability of the oil extract of Araucaria cunninghamii Aiton Ex D.Don was measured through the DPPH assay (Blois, 1958). Different concentrations of the oil extract were mixed with DPPH (0.1mM) in methanol solution. After 20 min incubation at room temperature the absorbance was read at 517 nm. The inhibitory percentage of DPPH was calculated according to the following equation:

% Inhibition = Ac-As x100

Ac = Absorbance of control

As = Absorbance of sample

2.3.2 Reducing power assay:

1 ml of different concentrations (0-1000 µg/µl) of oil extract was mixed with 2.5 ml phosphate buffer and 2.5 ml of 1% potassium ferricyanide and incubated at 50ºC for 20 minutes. 2.5 ml of 10% TCA was added to 2.5 ml of supernatant. Then 2.5 ml distilled water and 0.5 ml FeCl₃was added. The absorbance was noted at 700 nm (Oyaizu, 1986). Rutin was used as standard.

2.3.3 Nitric Oxide scavenging assay:

Different concentrations of the sample were mixed with 8.1 ml (10 mM) sodium nitropruside in 0.5 M phosphate buffer (pH 7.4) and incubated at 25^оC for 15 min. Greiss reagent (1% sulphanilamide in 5% H₃PO₄ and 0.1% N-(1-napthyl) ethylenediamine dihydrochloride in water) was added to the above solution and the absorbance was noted at 546 nm (Acharyya et al, 2010). Ascorbic acid was used as standard.

2.4 Phytochemical analysis:

2.4.1 Determination of total phenolic compounds:

100 µl of oil extract was taken in a test tube and diluted with 900 µl distilled water. Then 500 µl FC reagent and 1.5 ml of 20% Na₂CO₃was added to this solution and the volume was raised to 5 ml with distilled water. The mixture was incubated at room temperature for 2 hours and the absorbance of the solution was taken at 765 nm on UV-VIS spectrophotometer (Yu et al, 2002). Total phenol content was estimated from standard curve. Gallic acid was used as standard and total phenolic content was expressed as gallic acid equivalents (GAE) in mg/g of dry sample.

2.4.2 Determination of total flavonoid content:

4 ml of distilled water was added to 1 ml oil extract of 100µg/ml concentration. Then, added 0.3 ml NaNO₂ and 0.3 ml AlCl₃. The mixture was incubated for 5 minutes at room temperature. 2 ml of NaOH and 2.4 ml distilled water was added to the incubated solution and the absorbance was taken at 510 nm using UV-VIS spectrophotometer (Kim et al, 2003). Total flavonoid content was estimated from standard curve. Rutin was used as standard and total flavonoid content was expressed as rutin equivalents (RE) in mg/g of dry sample.

2.4.3 Determination of total tannin content:

100 µl sample solution was mixed with 7.5 ml distilled water, 500 µl FC reagent (1:1), 1 ml of 35% Na₂CO₃ and the volume was raised to 10 ml with distilled water. The mixture was incubated for 30 min. at room temperature. The absorbance was observed at 725 nm (Polshettiwar et al, 2007).

Phytochemical testing was performed on the oil using standard procedures (Anyasor et al, 2010) to identify the presence and absence of chemical constituents like anthraquinones, cardenolides, cardiac glycosides and saponin.

2.4.4. Test for anthraquinones:

0.5 g of the extract was shaken with 10 ml of benzene and filtered. 10% of ammonia solution was added to filtrate and the mixture was shaken. The formation of a pink, red or violet colour on the ammoniac phase indicates the presence of anthraquinones.

2.4.5. Test for cardenolides:

1 ml of the sample extract was added to 2 ml of benzene. The formation of a turbid brown colour is a sign of the presence of cardenolides.

2.4.6. Test for cardiac glycosides:

2 ml glacial acetic acid was mixed with 1 drop of ferric chloride solution and 0.5 g of the extract was dissolved in it. Above solution was under layered with 2 ml of concentrated sulphuric acid. A brown ring formation at the inter phase indicates the presence of deoxy sugar characteristics of cardiac glycosides.

2.4.7. Test for saponins:

1 ml of the extract was mixed with one ml of distilled water and shaken briskly. A stable continuous lather specifies the presence of saponins.

2.5 Phytochemical investigation with HPLC:

The investigation of phenolic compounds was carried out using HPLC. The chromatography was carried out at room temperature with a flow rate of 1ml/min at λ 280 nm. A 150x4.6 mm C-18 column with a pore size of 5µm was used. Injection volume was 5 µL. The solvent system comprised of solvent A (0.01% acetic acid in water) and solvent B (methanol). The gradient runs as 70% A and 30% B, reaching 45% B at 12 min, 75% B at 13.5 min, 75% B at 15 min, 50% B at 16.6 min, 25% B at 18 min, 25% B at 20 min, 30% B at 21 min, and stopped at 22 min with elution of 4 min. The mixture of 11 standards compounds namely, Gallic acid (C₇H₆O₅), Catechin (C₁₅H₁₄O₆), Chlorogenic acid (C₁₆H₁₈O₉), Epicatechin (C₁₅H₁₄O₆), Caffeic acid (C₉H₈O₄), Umbelliferone (C₉H₆O₃), Coumaric acid (C₉H₈O₃), Rutin (C₂₇H₃₀O₁₆), Ellagic acid (C₁₄H₆O₈), Quercetin (C₁₅H₁₀O₇) and Kaempferol (C₁₅H₁₀O₆) was diluted with methanol at different concentrations by serial dilutions for quantitative analysis. The calibration curves were generated for plotting concentrations versus peak areas. The detection of every compound was based on a combination of retention time and spectral similarity. The detection and quantification limit for all detected compounds were calculated on the basis of signal-to-noise ratio (S/N) of 3 and 10 with corresponding standard solution, respectively.

2.6 Statistical Analysis:

All experimental measurements were carried out in triplicate. The data was analyzed statistically by one-way analysis of variance (ANOVA) and comparisons with P-values ≤ 0.05 were considered significantly different (Bailey, 1995). Mean value, standard deviation and standard error were also calculated and data was presented as ± SE

3. RESULTS:

3.1 DPPH assay:

In the present study, the DPPH radical scavenging activity of oil extract increased with an increase in concentration (Fig. 1).

Fig. 1: The hydrogen donating ability of oil extract in DPPH assay

3.2 Reducing power assay:

With the increasing concentration of extract, the reducing power increased but as compared to the standard (Rutin), very less reducing power was shown by the oil extract of Araucaria cunninghamii Aiton ex D.Don. (Fig. 2).

Fig. 2: The effect of oil extract in reducing power assay

3.3 Nitric oxide scavenging assay:

Oil extract of Araucaria cunninghamii Aiton ex D.Don. exhibited a good effect in prevention of nitric oxide radical and the effect increased with increase in concentration (Fig. 3).

Fig. 3: The effect of oil extract in nitric oxide scavenging assay

3.4 Phytochemical investigation:

Anthraquinones, cardenolides, Saponins and cardiac glycosides were absent in the oil.

3.5 Phenol, flavonoid and tannin content:

The amount of phenolic compounds in the oil extract was 0.111 mg gallic acid equivalents. The flavonoid content was 2.5 mg rutin equivalents and the tannin content was 0.030 mg tannic acid equivalents.

3.6 HPLC analysis:

HPLC analysis documented the presence of Gallic acid (C₇H₆O₅), Catechin (C₁₅H₁₄O₆), Chlorogenic acid (C₁₆H₁₈O₉), Epicatechin (C₁₅H₁₄O₆), Caffeic acid (C₉H₈O₄), Umbelliferone (C₉H₆O₃), Rutin (C₂₇H₃₀O₁₆), Ellagic acid (C₁₄H₆O₈), Quercetin (C₁₅H₁₀O₇) and Kaempferol (C₁₅H₁₀O₆) in the oil extract of Araucaria cunninghamii Aiton ex D.Don. Fig. 4 is the chromatogram for the standards and Fig. 5 is the chromatogram for compounds in oil extract of Araucaria cunninghamii Aiton ex D.Don. The results of HPLC analysis are shown in Table 1 which depicts the present phytochemicals in the oil extract of Araucaria cunninghamii Aiton ex D.Don. along with their concentrations and retention time.

4. DISCUSSION:

Free radicals are responsible for the induction of oxidative disintegration of lipids, which leads to harmful effects on health (Stohs and Bagghi, 1995). Oxidative stress is countered intrinsically in all living systems by the scavenging and protective role played by antioxidants against free radicals (Narayanaswamy and Balakrishnan, 2011). It is very important to find out natural sources of antioxidants. In present study, the oil extract of Araucaria cunninghamii Aiton ex D. Don was investigated for its antioxidant potential. The oil performed differently in different assays used for the experimentation in this study. When compared to the standards, the antioxidant activity of oil was very less. However, in all the assays, the oil extract showed the antioxidant activity which increased with the increase in the concentration. The phenolic portion of plant extracts has been associated to their antioxidant capacity (Ali et al, 2008). Flavonoids show their antioxidant action by means of scavenging or chelating process (Xu et al, 2009). Tannins are potential biological antioxidants (Hagerman et al, 1998). Therefore, total phenolic, flavonoid and tannin contents (mg/g) in oil extract of Araucaria cunninghamii Aiton Ex D. Don were determined by using regression equation of calibration curve. Total phenols were expressed as Gallic acid equivalents (GAE), flavonoids were expressed as rutin equivalents (RE) and tannins were expressed as tannic acid equivalents (TAE). Our results show that the leaf oil of Araucaria cunninghamii Aiton Ex D. Don has tannins, flavonoids and phenolic compounds. On the whole, the types of phenolic compounds present in the oil may be responsible for the antioxidant activity. To identify the phytochemicals present in the oil extract, the HPLC study was carried out. HPLC investigation recognized the occurrence of 10 polyphenolic compounds namely, gallic acid, catechin, chlorogenic acid, epicatechin, caffeic acid, umbelliferone, rutin, ellagic acid, quercetin and kaempferol in the oil extract of Araucaria cunninghamii Aiton ex D.Don.

Fig. 4:Chromatogram for standard polyphenols

Fig. 5:Chromatogram for polyphenols in oil extract of Araucaria cunninghamii Aiton ex D.Don.

Table. 1: Phytochemicals with retention time and concentrations detected in Oil extract of Araucaria cunninghamii Aiton ex D.Don by HPLC.

Sr. No.	Name	Peak No.	Retention Time (min.)	Concentration (µg/10mg dry sample)
1	Gallic acid	1	2.665	216.028
2	Catechin	3	4.064	91.200
3	Chlorogenic acid	4	5.048	22.515
4	Epicatechin	5	6.218	31.461
5	Caffeic acid	6	7.228	28.624
6	Umbelliferone	7	9.517	59.481
7	Rutin	8	15.181	72.285
8	Ellagic acid	10	15.621	17.161
9	Quercetin	14	16.494	524.230
10	Kaempferol	16	17.226	617.323

5. CONCLUSION:

Results of the present study disclose the antioxidant potential of Araucaria cunninghamii Aiton ex D.Don. This is apparent that the antioxidant activity of oil extract is attributable to the polyphenolic compounds recognized by the HPLC analysis. In future, the oil extract of Araucaria cunninghamii Aiton ex D.Don may serve as the compound for crafting the medicine to take care of a variety of diseases linked with free radicals. As per our knowledge, this is the first study reporting the antioxidant activity of oil extract of Araucaria cunninghamii Aiton ex D.Don.

6. ACKNOWLEDGEMENTS:

The authors thankfully acknowledge the financial assistance by Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar, India for carrying out this work.

7. CONFLICT OF INTERESTS:

The authors affirm no conflicts of interests with respect to the authorship and/or publication of this article.

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Received on 28.04.2016 Modified on 28.05.2016

Research J. Pharm. and Tech. 2016; 9(7):875-879.

DOI: 10.5958/0974-360X.2016.00166.9