AntioxidAnt StudieS uSing Sunflower oil AS oxidAtive SubStrAte And dnA Protective ASSAy by Antirrhinum majus

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* For correspondence.

Oxidation Communications 36, No 2, 542–552 (2013)

Biological systems – antioxidants

AntioxidAnt StudieS uSing Sunflower oil AS oxidAtive SubStrAte And dnA Protective ASSAy by Antirrhinum majus

M. Riaza, N. Rasoola*, i. H. BukHaRia, M. zuBaiRa, M. sHaHidb, T. H. BokHaRia, Y. Gulla, k. RizwaNa, M. iqBalb, M. zia-ul-Haqc

aDepartment of Chemistry, Government College University, 38 000 Faisalabad, Pakistan bDepartment of Chemistry and Biochemistry, University of Agriculture, 38 040 Faisalabad, Pakistan cDepartment of Pharmacognosy, Research Institute of Pharmaceutical Sciences, University of Karachi, 75 270 Karachi, Pakistan E-mail: [email protected]

aBsTRaCT

in this work we evaluated the antioxidant activity of absolute methanol extract and its fractions from the snapdragon (Antirrhinum majus) plant. The presence of total phenolics content, iC50 and the % inhibition in linoleic acid oxidation were evalu-ated. The antioxidant activity of plant extract and fractions was also studied using sunflower oil as an oxidative substrate. Peroxide value (PV), free fatty acids (FFA), conjugated dienes (Cd), conjugated trienes (CT) and para-anisidine values were also determined by stabilising the sunflower oil as oxidation substrate. Moreover, it was observed to provide a protective effect in H2o2-induced oxidative damage in plasmid pBR322 dNa, indicating that the plant has antioxidant properties. The results of present study revealed that snapdragon plant may be considered as a good source of natural antioxidants.

Keywords: snapdragon, antioxidant activity, sunflower oil, oxidative substrate.

aiMs aNd BaCkGRouNd

Phytochemicals from plant sources play a pivotal role in human health1. in fact, the pro-duction of free radicals during metabolism and other activities beyond the antioxidant ability of a biological system leads to oxidative stress2 and foods or medicinal plants rich in natural antioxidants such as phenolics were able to reduce this risk. Today, the

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use of synthetic antioxidants in food products is mostly discouraged because of their adverse effects. on the other hand, the use of natural antioxidants in foods is gaining a considerable attention due to their prospective health benefits and pharmacological properties3–6. The oxidation of food causes the development of undesirable smell, creates toxicity and greatly affects the shelf-life of food products7. Natural extracts with a pleasant smell combined with a preservative action have properties to avoid lipid deterioration and spoilage by microorganisms. The use of natural sources as functional ingredients in foods, drinks and cosmetics is gaining greater recognition, as the synthetic additives used as antioxidants are potentially harmful8. Food-borne diseases are major problem in developing countries, moreover 80% of the population in developing countries rely on traditional plant-based medicines for their primary health care needs9. Even in developed nations there is a demand for safe preventives such as natural antioxidants10,11.

snapdragon (Antirrhinum majus) belongs to scrophulariaceae family and tradi-tionally has been used as a tonic against diseases and also as aperient, emmenagogue and antipyretic. Its leaves and flowers were claimed to be antiinflammatory and were also used to cure hemorrhoids12. The plant was also used as a diuretic, in the treatment of scurvy, ulcer and liver disorders and as an antitumoral13.

Previous phytochemical investigations revealed the presence of flavonoids12–15 and alkaloids16 in plants of this family. Fatty acids in seed oil have also been charac-terised17. Various authors have evaluated the nutritional, antioxidant and biological studies of various plants in Pakistan18–25.

according to our knowledge no literature is available on the stabilisation of oil and antioxidant activity of the extract and various fractions of Antirrhinum majus.

EXPERIMENTAL

Plant material and extraction. The whole plant was collected from Faisalabad, Pakistan. The plant was authenticated by Assistant Prof. Mansoor Hameed Botany, department of Botany, university of agriculture, Faisalabad. after collection, the plant material was washed, shade dried and grinded. The whole plant was extracted thrice with absolute methanol by dipping for 7 days. The extracts were mixed and concentrated to dryness using rotary evaporator. The absolute methanol extract was further fractioned by using solvents of increasing polarity: n-hexane, chloroform, ethyl acetate and n-butanol14–16. after fractionation, samples were concentrated to dryness and stored in a refrigerator at 4°C, until used for analysis.

Chemicals. The refined, bleached and deodourised (RBD) sunflower oil, without addition of any antioxidant was used. DPPH (1,1-diphenyl-2-picrylhydrazyl) and other reference chemicals were purchased from sigma Chemical Co. (st. louis, Mo, usa). all other chemicals used were of analytical grade and purchased from Merck (darmstadt, Germany).

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Determination of total phenolic contents. The total phenolic contents (TPC) were determined using the Folin–Ciocalteau method26,27. The results were expressed as gallic acid equivalent (GAE) for TPC. The experiments were performed in triplicate and the mean values were reported.

ANTIOXIDANT ACTIVITY

DPPH free radical scavenging assay. DPPH free radical scavenging activity was determined by methods previously described in literature28,29. The iC50 was calculated from the plot of concentration against percentage inhibition. Three replicates were recorded for each sample.

Percent inhibition of linoleic acid oxidation. The antioxidant activities of the extract and fractions were also calculated in terms of measurement of percentage inhibition of the peroxidation in the linoleic acid system, using a method previously described28,30. a control was performed with linoleic acid, without extract and fractions. The syn-thetic antioxidant butylated hydroxy toluene (BHT), was used as a positive control at 200 mg/l concentration. The maximum inhibition in peroxidation level was noted at 360 h (15 days). The absorbance of the samples was measured at 500 nm using a spectrophotometer (u-2001 Hitachi, Japan). The sample that contained no antioxidant component was used as a blank.

DNA protection assay. The antioxidant activity was also evaluated by deoxyribonucleic acid (dNa) protection assay by the method described by kalapna and coworkers31 with some modifications. For this assay pBR 322 DNA 0.5 µg/µl was diluted up to 2 folds (0.5 µg/3 µl using 50 mM sodium phosphate buffer pH 7.4). A sample of 3 µl of diluted pBR 322 DNA was treated with a 5-µl test sample. After this, 4 µl of 30% H2O2 were added in the presence and absence of 1000 µg/ml concentration of Antir-rhinum majus absolute methanol extract and its fractions. The volume was made up to 15 µl with sodium phosphate buffer (pH 7.4). The relative difference in migration between the native and oxidised dNa (ensured on 1% agarose gel electrophoresis) was observed. The gels were documented by Gel doc, Bio Rad instrument (Gene-genius sYNGENE, England).

Evaluation of antioxidant activity by stabilisation of sunflower oil. The Antirrhinum majus extract and fractions were separately added at a concentration of 500 ppm to refined, bleached and deodourised (RBD) sunflower oil heated at 50oC to establish a homogeneous distribution. The standard BHT at a concentration of 200 ppm was used for comparison of antioxidant activity for stabilisation of sunflower oil; the sunflower oil alone was used as a control. The treated and untreated sunflower oil samples (100 ml), were transferred to brown glass bottles, stored in an electric oven at 60oC and were analysed after an interval of 10 days. The oxidation of the oils was determined by the determination of the free fatty acid (FFA), peroxide value (PV), conjugated trienes (CT), conjugated dienes (Cd) and para-anisidine values. The PV

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and FFA of the stabilised and control sunflower oil samples were measured follow-ing the AOCS Official Method Cd 8-53 and F 9a-44, respectively32. The conjugated dienes and trienes were analysed by following the IUPAC Method II.D.23 (Ref. 32). The absorbance was noted at 232 and 268 nm, respectively. para-anisidine value was determined following the IUPAC method II.D.26 (Ref. 33).

sTaTisTiCal aNalYsis

all the experiments were performed in triplicate and statistical analysis of the data was carried out by analysis of variance, using Costat 6.3 software. a probability value of difference p ≤ 0.05 was considered to denote a statistical significance. All data were presented as the mean values ± standard deviation (sd).

REsulTs aNd disCussioN

Percent of yield, total phenolics contents and antioxidant activity. The percents of yield and total phenolics content (TPC) in the absolute methanol extract and its fractions are presented in Table 1. The amounts ranged within 1.92–15.25 GaE (mg/g) of dry extract. Absolute methanol extract showed the highest TPC value (15.25), while its chloroform fraction showed the lowest (2.61) mg/g of dry extract. Table 1 also shows the percent inhibition of linoleic acid oxidation caused by the Antirrhinum majus whole plant extract and fractions; BHT was used as a positive control. The percent inhibition ranged from 33.12% (n-hexane fraction) to 70.25% (absolute methanol extract) whereas BHT showed 90.69% inhibition. The free radical scavenging activ-ity was measured by DPPH assay. The IC50 resulted for BHT (9.96 ± 0.08 µg/ml),

table 1. antioxidant potential by Antirrhinum majus absolute methanol extract and its fractions by different assays

assays absolute methanol

n-Hexane Chloroform Ethyl ac-etate

n-Butanol BHT

Extract yield (g/100 g)

16.2±0.12a 1.57±0.03e 3.59±0.03c 4.51±0.31b 3.25±0.51d –

TPC (mg/g of dry extract)

15.3±0.12a 1.92±0.03e 2.61±0.02d 5.01±0.04b 3.57±0.03c –

DPPH assay (iC50 µg/ml)

19.15±0.15e 92.12±0.82a 46.27±0.35c 39. 10±0.26d 49.51±0.32b 9.96±0.08f

Percent inhibi-tion of linoleic acid oxidation

70.25±0.79b 33.12±0.21f 49.17±0.39d 55.09±0.41c 41.35±0.51e 90.69±1.02a

The values are the mean ± sd of 3 different runs (n = 2 × 3), (p ≤ 0.05); the alphabets in superscript represent the significant differences among the extract and its fractions.

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absolute methanol (19.15 ± µg/ml), ethyl acetate (39.10 ± 0.26 µg/ml), chloroform (46.27 ± 0.35 µg/ml) and n-butanol (49.51 ± 0.32 µg/ml) and n-hexane (92.12 ± 0.82 µg/ml), respectively. The observed antioxidant activity of Antirrhinum majus absolute methanol extract might be due to the presence of considerable amounts of phyto-chemical constituents. a large group of naturally occurring compounds in plant such as phenolic compounds and alkloids possessed numerous pharmacological activities and biological activities29,34.

DNA protection assay. The antioxidant activity was also analysed by the protective effect on plasmid pBR322 dNa by H2o2-induced damage exerted by the absolute methanol extract and its fractions at a concentration of 1000 µg/ml of Antirrhinum majus (Fig. 1). In the first lane plasmid pBR322 DNA constituted the control (without treatment) and it may be in super-coiled form. when the plasmid was exposed to H2o2, the dNa damage was evident, this might be with the conversion of the super-coiled form of pBR322 dNa into open linear form (2nd lane).

fig. 1. dNa protective effects exerted by Antirrhinum majus absolute methanol extract and its fractions in H2o2-induced oxidative damage on pBR322 dNaLane 1 – plasmid pBR322 DNA without treatment (super-coiled); lane 2 – plasmid pBR322 DNA; treated with H2o2 (open circular or damaged); lane 3 – plasmid pBR322 DNA treated with absolute methanol extract + H2o2; lane 4 – plasmid pBR322 DNA treated with n-butanol fraction + H2o2; lane 5 0 1 Kb DNA ladder; lane 6 – plasmid pBR322 DNA treated with chloroform fraction + H2o2; lane 7 – plasmid pBR322 dNa treated with ethyl acetate fraction + H2o2; lane 8 – plasmid pBR322 DNA treated with n-hexane extract fraction + H2o2

when compared with other lanes, the data showed the protective effects of the absolute methanol extract (3rd lane), the band almost equal to the pure dNa band. The n-hexane fraction (8th lane) showed a less protective effect on dNa (the band of dNa was near to the damaged dNa, 2nd lane, treated with H2o2). similarly, other fractions showed protective effectiveness on dNa (4th to 7th lane) but the protection was less than the absolute methanol extract and higher than the n-hexane fraction when compared with the other lanes (Fig. 1). Therefore, it may be concluded that the most effective dNa protection was showed by absolute methanol extract, the lowest by the n-hexane fraction. The protective effect observed by absolute methanol extract was probably linked to its antioxidant activity, as the protective effects of plant extract on

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dNa can also be explained by its ability to scavenge reactive oxygen species (Ros) (Ref. 35).

Peroxide value (PV). The antioxidant activity of plant extract and fractions was also evaluated by using sunflower oil as an oxidative substrate. The peroxide value (PV) was usually used to evaluate the extent of primary oxidation products in oil36. Figure 2 shows the peroxide values of sunflower oil untreated (control) and treated with absolute methanol extract of Antirrhinum majus and its fractions. at the end of the storage period of 90 days, the control showed the highest level of PV (relative to the initial value), indicating a higher extent of primary oxidation. The samples of sun-flower oil stabilised with absolute methanol extract showed the maximum antioxidant activity. The variations in peroxide value of stabilised sunflower oil were found to be significant (p < 0.05) for the incubation period and PV values among the extract and fractions used.

fig. 2. Peroxide value (meq/kg) of sunflower oil stabilised with Antirrhinum majus absolute methanol extract and its fractions

Free fatty acids (FFA). The free fatty acids (FFA) might be considered as a significant measure of deterioration of food products. The free fatty acids were formed by the hydrolysis of triglycerides and may increased by reaction of oil with oxygen37. The free fatty acid formation increased in the oil with an increase of storage period. The control sample of the oil (without antioxidant) showed the higher FFa value, while the oil sample with absolute methanol extract and BHT exhibited the lowest FFa values (Fig. 3). The variations in free fatty acid constituents of stabilised sunflower oil were found to be significant (p ≤ 0.05) for incubation period and FFA values among the extract and fractions used.

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fig. 3. Free fatty acid (%) of sunflower oil stabilised with Antirrhinum majus absolute methanol extract and its fractions

Conjugated dienes (CD) and conjugated trienes (CT). The estimation of conjugated dienes (Cd) and conjugated trienes (CT) were valuable parameters for the early stages of peroxidation in the study of oils38. After 90 days, the untreated sample of sunflower oil (control) showed higher levels of these oxidation products. The sample treated with absolute methanol extract showed the lowest increase in peroxide value. during the period of the experiment, the contents of CD and CT in oils increased; however these levels were considerably lower for samples treated with Antirrhinum majus extract and fractions. The control exhibited higher levels of these oxidation products predicting that it had undergone extensive oxidative deterioration. among the tested samples, n-hexane fraction, showed higher values of CD and CT reflecting its lower antioxidant activity for stabilisation of sunflower oil (Figs 4 and 5). The results were found to be statistically significant (p < 0.05).

fig. 4. Conjugated dienes of sunflower oil treated with Antirrhinum majus absolute methanol extract and its fractions

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fig. 5. Conjugated trienes of sunflower oil treated with Antirrhinum majus absolute methanol extract and its fractions

Para-anisidine value (PAV). The results of para-anisidine assay, which typically de-scribed the amount of aldehydic secondary oxidation products in the oils36,39 as shown in Fig. 6. The control sunflower oil showed the maximum increase in para-anisidine value indicating a higher rate for the formation of secondary oxidation products. The smallest increase in para-anisidine value of the oils was observed with absolute methanol extract, while the maximum increased with n-hexane fraction. There was a significant difference (p ≤ 0.05) among the para-anisidine values among the extract and fractions used.

fig. 6. para-Anisidine value (PAV) of sunflower oil treated with Antirrhinum majus absolute methanol extract and its fractions

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CoNClusioNs

Antirrhinum majus whole plant showed good antioxidant potential as indicated by its total phenol content (TPC), and its antioxidant properties. The absolute methanol extract proved to be the most active in terms of antioxidant activity between the tested extract and fractions. The protective effect of absolute methanol extract by H2o2-induced oxidative damage in plasmid pBR322 dNa was also demonstrated and it is possible that it protected the dNa due to its antioxidant activity. in conclusion, Antir-rhinum majus whole plant can be considered a good source of natural antioxidants.

aCkNowlEdGEMENTs

The financial support by Higher Education Commission (HEC), Islamabad, Pakistan, for providing scholarship to Muhammad Riaz by indigenous scholarship scheme for purchase chemicals and other research material is gratefully acknowledged.

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Received 8 November 2012 Revised 2 Jnauary 2013