A Study of DNA Protective Ability of Peels of Different Citrus Species

The aim of the present study was to assess the DNA protection ability, free radicals scavenging activity, and phytochemical constituents of peel extracts of various species of citrus. Results showed the presence of carbohydrates, amino acids, glycosides, tannins, steroids and alkaloids. DNA protection assay showed maximum protection with methanolic extracts of peels of C. limon , C. reticulata , C. aurantium and C. sinensis as compared to C. limetta which showed less protection. DPPH assay used to assess the antioxidants showed the highest activity in the methanolic extract of peels of C. reticulata and C. aurantium with 75% and 86% respectively at 10 mg/ml. It is therefore concluded that C. limon , C. reticulata , C. aurantium and C. sinensis in terms of DNA protection and C. reticulata and C. aurantium in terms of free radicals scavenging activity are the best ones and these are the potential candidates to be used for further studies.


INTRODUCTION
Nature has provided each cell with protective mechanisms against all kinds of effects caused by free radicals, which help in curing many degenerative and chronic diseases such as heart disease, diabetes mellitus, cancer, Alzheimer and aging (Lele et al., 2004). Intake of antioxidants by eating fruits and vegetables in the diet should protect cells against the reactive oxygen species and also help in decreasing the DNA damage caused by oxidation (Moller et al., 2003). Various plant phytochemicals such as tannins, phenolic compounds and flavonoids are reported to have important antioxidant activity against the wide variation of free radicals (Koleckar et al., 2008;Kirmizibekmez et al., 2009;Choudhary & Swarnkar, 2011).
Oranges are a member of Rutaceae, which is commonly known as the rue or citrus family. In recent research studies, it has been proved that the phytonutrients compound of citrus has healing properties. Perez et al. (2010) studies showed Citrus limetta has healing effects in antihypertensive treatment. Kefford et al. (1960) found that citrus fruit consists of soluble solids content, ascorbic contents and acidity. The screening activity of citrus fruits identified the composition as carbohydrates in citrus juice, in citrus peels and pulps, ascorbic acid, vitamins, etc. Citrus is used for curing various illnesses, and reducing vomiting or diarrhea. So, the main objective of the current study is to determine the phytochemical constituents, DNA protective ability and scavenging activity of peel extracts of various citrus species.

MATERIAL AND METHODS
The fresh oranges of different varieties (Citrus limetta, Citrus aurantium, Citrus sinensis, Citrus reticulata and Citrus limon) were bought from a local market and their peels were air dried under shade. The peels were ground into fine powdered. Crude methanolic extracts (CME) of each plant were prepared by dissolving 5g of powdered sample into 50ml of methanol. Then, it was kept at room temperature by shaking it after every 2 hours for one day. Thereafter, it was filtered using Whattmann filter paper No. 1. The crude methanolic extracts were used for the phytochemical analysis by using procedures described by Harborne, 1973 andSofowora, 1993. DNA protection assay was conducted by following the protocol described by Tian and Hua (2005). The antioxidant activity of all six samples was done by following the protocol described by Kulisic et al. (2004).

RESULTS AND DISCUSSION
For the estimation of antioxidant and DNA protection ability of different species of citrus: phytochemical screening, plasmid DNA protection assay and DPPH free radicals scavenging assay was performed in this study. Qualitative analysis of phytochemicals present in peels extract of citrus species is summarized in Table 1.
-----Positive sign (+) indicated presence of compound and negative sign (-) indicated absence of compound. Lalitha et al. (2009) also evaluated preliminary phytochemical screening of Citrus limon and confirmed the presence of cardiac glycosides and steroids, which is similar to our results. Further, a study of phytochemical screening conducted by Kumar et al. (2011) showed the presences of flavonoids, saponins, steroids, terpenoids, tannins and alkaloids. This study partially supports our study with the presence of steroids, tannins and alkaloids while saponins were absent in the peel extracts. This difference may be due to the difference of fruits used.
DNA protective ability of the methanolic extract of Citrus species was determined by using a DNA protection assay, which is based on photolysing H2O2 and FeSO4. In this assay, plasmid pBR322 was used with four dilutions, i.e. 1000 ppm, 100 ppm, 10 ppm and 1 ppm of different samples. After one hour of incubation, samples were loaded, along with the positive and negative controls, on agarose gel electrophoresis. Normal pBR322 of the control series showed 2 bands in agarose gel electrophoresis. The faster moving band represented the native form of supercoiled circular DNA (scDNA) and the slower moving band corresponded to the open circular form (ocDNA). The obtained results are given in Figure 1. DPPH assay performed for the evaluation of the antioxidant activity of the peel extracts of different citrus fruit samples showed C. limon and C. sinensis have 50% and C. limetta have 17% antioxidant activity, while C. reticulata and C. aurantium showed more than 75% activity at 10 mg/ml. Since C. reticulata and C. aurantium showed more than 75% antioxidant activity further dilutions were made as 5 mg/ml, 2.5 mg/ml, and 1.25 mg/ml. Barreca et al. (2011), in their study, carried out DPPH radical quenching and FRAP assays which demonstrated that C. limetta juice showed remarkable antioxidant activity. Their results do not support our study. This may be due to their use of C. limetta juice, while in the current study the peels extract was used. Four citrus species were characterized in relation to their contents of minerals, ascorbic acid, total polyphenols and the antioxidant capacity of pulps and peels by Barros et al. (2012). They concluded that aside from citrus pulps, the peels are also good sources of bioactive compounds and minerals, and can play a considerable role in food products. This study supports our study in terms of the use of peels. The results of antioxidant activity of citrus species are mentioned in Table 2.

CONCLUSION
Citrus species used in the current study consists of various phytochemicals such as carbohydrates, amino acids, glycosides, tannins, steroids and alkaloids. They also have the capability for DNA protection, which was shown by the methanolic extracts of peels of C. limon, C. reticulata, C. aurantium and C. sinensis. C. limetta showed less protection as compared to other species. DPPH assay used to assess the

Samples
Dilutions 10 mg/mL 5 mg/mL 2.5 mg/mL 1.25 mg/mL antioxidants showed the highest activity in the methanolic extract of peels of C. reticulata and C. aurantium with 75% and 86% respectively at 10 mg/ml. It is therefore concluded that C. limon, C. reticulata, C. aurantium and C. sinensis, in terms of DNA protection, and C. reticulata and C. aurantium, in terms of free radicals scavenging activity, are the best ones and these are the potential candidates which can be used for further studies. Barreca