An antioxidant is a compound that inhibits or delays the oxidation of substrates even if the compound is present in a significantly lower concentration than the oxidized substrate. The scavenging of Reactive Oxygen Species (ROS) is one of possible mechanism of action. Others include the prevention of ROS formation by metal binding or enzyme inhibition. The antioxidant compounds can be recycled in the cell or are irreversibly damaged, but their oxidation products are less harmful or can be further converted to harmless substances (Halliwell, 1995; Halliwell and Gutteridge, 2007). The antioxidant testing can reveal various mechanisms of action, depending on features of the particular assay. Simple methods include free radical scavenging with use of colored, artificial stable free radicals such as 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS used in the TEAC assay-Trolox equivalent antioxidant capacity) (Re et al., 1999) and DPPH (1,1-diphenyl-2-picrylhydrazyl free radical) (Molyneux, 2004), as well as transition metal reduction that can be monitored by colorimetry. The metal based methods include the reduction of ferric ions: FRAP-(ferric reducing ability of plasma) and ferric thiocyanate assays (Halliwell, 1995; Aruoma, 2003) or molybdenum ion-phosphomolybdenum (P-Mo) assay (Prieto et al., 1999). These tests are easy and affordable and can be used in high throughput screening.

The genus Rubus is very diverse, includes over 750 species in 12 sub genera and is found on all continents except Antarctica (Finn, 2008). R. ellipticus is a weedy raspberry is well established in disturbed wet forests, 1800-5580 ft elevation and thrives in sunny open pastures as well as deep rain forests. R. niveus is native from Indian to southeastern Asia, the Philippines and Indonesia (Gerrish et al., 1992). They contain a range of biologically active substance including polyphenolics, flavanols, alkanols, anthocyaninis, lignans and tannins. In the present study we are following the quantification assays such as total phenolics, tannins and flavonoids, in vitro antioxidant assays such as DPPH, ABTS, FRAP, P-Mo, nitric oxide, super oxide and hydroxyl radical scavenging, the analysis of in vivo antioxidant molecule and enzymes in blood and liver such as Glutathione, Glutathione Peroxidase, Glutathione Reductase, Glutathione-S-Transferase, Superoxide Dismutase and Catalase for the comparative evaluation of antioxidant potential of the selected Rubus species. A survey of literature revealed that the antioxidant potentials of these plants have not yet been fully evaluated. Keeping this in view, in the present investigation we have investigated the antioxidant potential of R. ellipticus and R. niveus in vitro as well as in vivo and compared with the known natural and synthetic antioxidants to put forward a scope to develop an effective natural antioxidant supplement to fight against the free radical generated diseases.

MATERIALS AND METHODS

Chemicals: 2,2-diphenyl-1-picryl-hydrazyl (DPPH), potassium persulfate, 2,2’-azinobis- (3-ethylbenzothiozoline-6-sulfonic acid) diammonium salt (ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 3-(2-pyridyl)-5,6-bis (4-phenyl-sulfonic acid)-1,2,4-triazine (Ferrozine), ferrous ammonium sulfate, ethylene diamine tetra acetic acid (EDTA) disodium salt, sodium nitroprusside, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene (CDNB), GSH (γ-glutamyl cysteinyl glycine), 5,5’-Dithio-Bis (2-Nitrobenzoic Acid) (DTNB), Glutathione disulfide (GSSG) and Nitro blue tetrazolium (NBT) were obtained from Himedia, Merck or Sigma. All other reagents used were of analytical grade.

Plant material: The fresh plants of R. ellipticus and R. niveus were collected during the month of August from Mannavan Shola forest of Marayoor, Idukki dist, Kerala, India. The whole plants were separated in to leaves, stem, root etc., and washed in running tap water to remove dirt and other foreign materials and then shade dried.

Preparation of extracts: The dried, powdered plant samples were successively extracted in Soxhlet with Petroleum ether, Chloroform, Acetone and Methanol. Each time before extracting with the next solvent, the material was dried in hot air oven at 40°C. The different solvent extracts were concentrated by rotary vacuum evaporator (Yamato BO410, Japan) and then dried. The dry extract obtained with each solvent was weighed to determine the yield of soluble components. The percentage yields were expressed in terms of the air dried weight of sample material.

The extracts thus obtained were used directly to assess the antioxidant potential.

Determination of total phenolics and tannins: The total phenol content was determined according to the method described by Siddhuraju and Becker (2003) and Siddhuraju (2007). The analysis was performed in triplicates and the results were expressed in Gallic Acid Equivalents (GAE). The same extract can be used to estimate the tannin content by treating with polyvinyl polypyrrolidone (PVPP) (Siddhuraju, 2007). From the above results, the tannin content of the sample was calculated as follows:

Estimation of total flavonoids: The flavonoid contents of the extracts were quantified according to Zhishen et al. (1999). The pink colour developed due to the presence of flavonoids was read spectrophotometrically at 510 nm. The results were expressed in Rutin Equivalents (RE).

Determination of non-enzymatic antioxidant activities
DPPH radical scavenging activity: The antioxidant activity of the extract was determined in terms of hydrogen donating or radical scavenging ability using the stable radical DPPH according to Blois (1958). The absorbance was measured at 517 nm against the blank (methanol). Radical scavenging activity of the samples was expressed as IC₅₀ which is the concentration of the sample required to inhibit 50% of DPPH^• concentration.

Trolox equivalent antioxidant capacity (TEAC) assay: The total antioxidant activity of the samples was measured by ABTS radical cation decolourization assay according to Re et al. (1999). Triplicate determinations were made at each dilution of the standard and the percentage inhibition was calculated against the blank (ethanol) absorbance at 734 nm and then plotted as a function of Trolox concentration. The unit of Total Antioxidant Activity (TAA) is defined as the concentration of Trolox having equivalent antioxidant activity expressed as μM g^-1 sample extracts.

Ferric reducing antioxidant power (FRAP) assay: The antioxidant capacities of different extracts of samples were estimated according to Pulido et al. (2000). Equivalent concentration was calculated as the concentration of antioxidant giving an absorbance increase in the FRAP assay equivalent to the theoretical absorbance value of a 1 mM concentration of Fe (II) solution.

Phosphomolybdenum assay: The antioxidant activity of samples was evaluated by the green phosphomolybdenum complex formation according to the method of Prieto et al. (1999). The results were expressed in milligrams of ascorbic acid equivalents per gram extract.

Nitric oxide radical scavenging activity: The procedure is based on the method of Sreejayan and Rao (1997), where sodium nitroprusside in aqueous solution at physiological pH, spontaneously generates nitric oxide which interacts with oxygen to produce nitrite ions that can be estimated using Greiss reagent. Scavengers of nitric oxide compete with oxygen leading to reduced production of nitrite ions. The inhibition percentage was calculated using the equation:

Superoxide radical scavenging activity: The assay was based on the capacity of various extracts to inhibit formazan formation by scavenging the superoxide radicals generated in riboflavin light NBT system (Beauchamp and Fridovich, 1971). The scavenging activity on superoxide anion generation was calculated as:

Hydroxyl radical scavenging activity: The scavenging activity of acetone, methanol and water extracts of the plant samples along with the reference standard tannic acid and quercetin on hydroxyl radical was measured according to the method of Klein et al. (1991). The analysis was performed in triplicate. The scavenging activity on hydroxyl radical generation was calculated as:

Determination of in vivo antioxidant activity of RELM and RNRA extracts: Thirty six Swiss albino male mice were divided into 6 groups of 6 animals and they were treated orally with RELM and RNRA extracts dissolved in Carboxy Methyl Cellulose (CMC) (0.1%) at different doses for 30 days:

At the end of the experiment, animals were sacrificed and blood was collected by heart puncture and liver was excised and washed in ice-cold Tris-HCl buffer (0.1 M, pH 7.4) and cytosolic samples of liver homogenate were prepared by centrifugation at 10,000 rpm for 30 min at 4°C. Estimation of the total protein was carried out by the method of Lowry, Rosenbrough, Farr and Randall (Lowry et al., 1951). The following parameters were assayed in both blood and liver to assess the oxidative stress.

Superoxide dismutase (SOD): The SOD activity was measured by the NBT reduction method of McCord and Fridovich (1969).

Catalase activity (CAT): CAT activity was estimated by the method of Aebi (1974) by measuring the rate of decomposition of hydrogen peroxide at 240 nm.

Glutathione (GSH): GSH (γ-glutamyl cysteinyl glycine) activity was assayed by the method of Moron et al. (1979), based on the reaction with DTNB.

Glutathione peroxidase (GPX): The assay of glutathione peroxidase was carried out by the method of Hafeman et al. (1974) based on the degradation of Hydrogen peroxide in the presence of GSH.

Glutathione reductase (GR): Glutathione reductase activity was measured by the method of Racker (1955), where the amount of reduced form of NADP consumed during the conversion of GSSG to GSH was estimated.

Glutathione-S-transferase (GST): The method of Habig (1974) was followed to assay the activity of glutathione-S-transferase (GST) based on the rate of increase in conjugate formation between GSH and 1-chloro-2, 4-dinitrobenzene (CDNB).

Statistical analysis: All the values are expressed as Mean±SEM. The values are analyzed using one-way ANOVA and the significance of the difference between means was determined by Duncan’s, Tukey Kramer multiple comparisons or Dunnet multiple range test using SPSS software version 17 and GraphPad InStat3 software.

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RESULTS AND DISCUSSION

Total phenolics, tannins and flavonoids contents of R. ellipticus and R. niveus extracts: The non-enzymatic antioxidants like total phenolics, tannins and flavonoids were estimated and presented in Table 1. The amount of total phenolics, tannins and flavonoid contents in the tested extracts were determined and expressed as Gallic acid equivalents (GAE) and Rutin equivalents. Highest total phenolics and tannin contents were identified in R. ellipticus and R. niveus root chloroform extract, 80.28 and 66.2 g GAE/100 g extract, respectively. The total flavonoid contents recorded maximum in the R. ellipticus and R. niveus root petroleum ether extracts (308.89 and 264.76 mg g^-1 extract) expressed in terms of Rutin Equivalents (RE).

The total phenolic content of methanol and aqueous extracts Rubus chingii Hu. Fruit was found to be 4.54 and 4.02 g/100 g DW and the major types of phenolic compounds such as; Phenolic acids (gallic acid) and tannins (ellagic acid) were also reported by Cai et al. (2004). The total phenolic and flavonoid contents of R. sanctus reported that 4.52 mg g^-1 plant extract in gallic acid equivalent and 4.66 mg g^-1 plant extract in Rutin equivalent (Motamed and Naghibi, 2010). Similar to the previous reports our study also showed significant phenolic, flavonoid and tannin contents. The root extracts of the tested Rubus species showed higher levels of total phenol, flavonoid and tannin contents.

In vitro antioxidant activities of R. ellipticus and R. niveus extracts: The stable free radicals such as DPPH and ABTS were effectively scavenged by R. ellipticus and R. niveus extracts. DPPH radical scavenging activity was expressed as IC₅₀ values compared to the standard antioxidants BHT, BHA, Quercetin and Rutin (Fig. 1 and 2) and ABTS by Trolox Equalents (Table 2). Acetone and methanol extracts of both the plants showed comparable activity with standards by scavenging DPPH radical.

However, stem acetone extract of R. ellipticus and root acetone extract of R. niveus showed significant activity. Similar activities were observed in R. sanctus plant extracts which were found to scavenge the DPPH free radical by 83.27% when compared with Vitamin C and BHT (97.15 and 96.47%) (Motamed and Naghibi, 2010). In case of ABTS assay the methanol extract of leaf and acetone extract of root showed significant activity for R.ellipticus, where as for R. niveus, the acetone and methanol extract of root showed significant activity. Cai et al. (2004) reported the total antioxidant capacity TEAC values of the methanolic and aqueous extracts of R. chingii Hu fruits which were found to be 946.1 and 817.0 (μmol Trolox/100 g DW). The DPPH and ABTS radical scavenging activities of R. ulmifolius (TEAC 3.8±0.3 mM Trolox and DPPH 5.10±0.5 μg mL^-1) were reported by Dall’Acqua et al. (2008). Raspberry (R. idaeus) leaves, collected in different locations of Lithuania were extracted with ethanol and were tested for their antioxidant activity by using ABTS and DPPH scavenging methods. All extracts were active, with radical scavenging capacity at the used concentrations from 20.5-82.5% in DPPH reaction system and from 8.0-42.7% in ABTS reaction (Venskutonis et al., 2007).

Antioxidant potential of R. ellipticus and R. niveus was estimated from their ability to reduce TPTZ-Fe (III) complex to TPTZ-Fe (II) and are given in Table 2. Among the different extracts methanol and acetone extracts showed comparable ferric reducing ability.

The root acetone extract of R. niveus showed higher activity (8024.44 mM Fe (II)/mg extract), followed by stem acetone and leaf methanol extracts (7548.88, 7524.44 mM Fe (II)/mg extract). The ferric reducing antioxidant power assay measures the antioxidant effect of any substance in the reaction medium as reducing ability. The total antioxidant capacity observed for the extracts of R. ellipticus and R. niveus can be correlated with its free radical scavenging activity equivalent to that of natural antioxidant ascorbic acid. The total antioxidant capacities of different extracts are shown in Table 2. R. niveus root acetone extract showed superior activity (89.68 mg AAE/g extract) followed by R. niveus stem acetone, R. ellipticus leaf methanol and acetone extracts (78. 19 and 75. 60 mg AAE/g extract). The acetone extracts of all the parts showed better radical scavenging capacity.

FRAP values in blueberries were highly correlated with phenol content (r = 0.981), implying that the antioxidant activity of blueberries is largely due to presence of phenolic compounds (Koca and Karadeniz, 2009). Similar results have been reported by other researchers (Wang and Lin, 2000), who found a linear correlation between total antioxidant activity and phenol content in blackberries (r = 0.961). In addition, Deighton et al. (2000) reported that there were apparent linear relationships between antioxidant activity (assessed by FRAP) and total phenols (r = 0.965), whereas anthocyanin content had a minor influence on antioxidant activity (r = 0.588) of Rubus juices. The leaf methanol extracts of R. ellipticus showed better results for nitric oxide (71.37%), superoxide (68.71%) and hydroxyl radical (62.37%) scavenging activities. Whereas, in case of R. niveus, stem acetone showed best results for nitric oxide (60.10%), root pet ether (63.14%) for super oxide and stem pet ether (65.03%) for hydroxyl radical scavenging activities (Table 3).

Effect of RELM and RNRA extracts on antioxidant enzymes and Glutathione: RELM and RNRA extracts showed commendable in vitro antioxidant activities. The effect of RELM and RNRA extract on in vivo antioxidant enzymes and Glutathione in the blood and serum of mice after a period of thirty days is shown in Table 4. The Catalase and SOD were found to be increased significantly in animals treated with 250 mg kg^-1 b.wt. of RELM, 250 and 100 mg kg^-1 b.wt. of RNRA extract (p<0.01).

GR in the serum was found to be significantly increased only in 250 mg kg^-1 b.wt. of RNRA (p<0.05). Similarly GSH level was found to be significantly elevated (p<0.05) in the blood of animals treated with 250 mg kg^-1 b.wt. of RELM and RNRA extracts.

The effect of RELM and RNRA extracts on the antioxidant enzymes in mice liver after treatment for 30 days is given in Table 5. Catalase was found to increase significantly in 250 mg kg^-1 b.wt. of RELM and RNRA extracts (p<0.05). SOD was found to be increased significantly only in 250 mg kg^-1 b.wt. RNRA extracts treated group. GPX depicted an increase in 250 mg kg^-1 b.wt. RNRA (p<0.001), 250 mg kg^-1 b.wt. of RELM (p<0.01), 100 mg kg^-1 b.wt. of RELM and RNRA groups (p<0.05). GR activity was also increased in 250 mg kg^-1 b.wt. of RELM and RNRA extract (p<0.01) treated groups. Significantly increase in GST level was found in both 100 and 250 mg kg^-1 b.wt. of RNRA treated groups (p<0.01). GSH level was found to be increased significantly in both 100 and 250 mg kg^-1 b.wt. of RNRA groups (p<0.01 and 0.001), 100 and 250 mg kg^-1 b.wt. of RELM groups (p<0.05 and 0.01).

The human body has several mechanisms to counteract damage by free radicals and other reactive oxygen species. These act on different oxidants as well as in different cellular compartments. One important line of defence is a system of antioxidant enzymes including SOD, catalase, GPX, GST and GR. SOD is a metalloprotein, converts two superoxide radicals into hydrogen peroxide and O₂. To eliminate H₂O₂, before the Fenton reaction which can create highly reactive hydroxyl radicals, organisms use catalase-a homotetrameric ferri heme containing enzyme and/or GPX-a selenium dependent enzyme. The Km value for GPX is lower than that for catalase and hence GPX is considered most important in physiological conditions. GSH abundant in most cells is an important substrate for GPX and Glutathione S-Transferase (GST) and also act by quenching free radicals. GST is GPX like enzyme and its function is to eliminate various hydroperoxides. Glutathione Reductase (GR) is a member of the disulphide oxidoreductase family, catalyses the NADPH-dependent reduction of glutathione disulphide (GSSG) to GSH. In plants the role of glutathione was reported as free radical scavenger and membrane stabilizer, keeping this in view the high levels of GSH and GPX in the RELM and RNRA extracts plays an important role in the lipid peroxidation (Firdous et al., 2010). The survey of literature showed that related species of Rubus possess promising antioxidant and pharmacological activities; the anti-inflammatory, analgesic and antipyretic properties of R. niveus root acetone extract and R. ellipticus leaf methanol extract have been reported (George et al., 2013a, b). Therefore R. ellipticus and R. niveus may also have the potentiality to treat other free radical generated disorders since it contains significant antioxidant activities.

CONCLUSION

The present study confirms the promising antioxidant activities of R. ellipticus and R. niveus. This is the first report available in the comparative in vitro and in vivo antioxidant studies on these two Rubus species. Four different extracts from the leaf, stem and root of these plants were screened for its in vitro antioxidant potentials and two which gave maximum activity (RELM and RNRA) from both the plants were selected for testing the in vivo antioxidant potentials. Among the 24 different extracts tested for seven in vitro antioxidant assays and three quantification tests, RELM and RNRA extracts showed superior activities in five and six assays such as RELM showed superior activities in ABTS, Phosphomolybdenum, NO, SO, OH etc., and whereas RNRA stood first in quantification of phenolics and tannins, ABTS, FRAP, DPPH, Phosphomolybdenum etc. The in vivo results indicated that RELM and RNRA 100 and 250 mg kg^-1 extract administration significantly increased the levels of SOD, CAT, GSH, GPX, GST and GR in both blood and liver of the 30 days extract treated mice. This antioxidant effect could be due to the phenolic/flavonoid constituents. The increased exposure to free radicals or the impaired efficiency of the protective enzymes and molecules may lead to many free radicals generated diseases including cancer. Further studies are warranted in order to characterize the exact compound responsible for the antioxidant activity. In summary, R. ellipticus and R. niveus can act as a natural antioxidant to fight against free radical related diseases.