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Phenolic profile, antioxidant potential and DNA damage protecting activity of sugarcane (Saccharum officinarum)
Food Chemistry 147 (2014) 10–16
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Phenolic profile, antioxidant potential and DNA damage protecting activity of sugarcane (Saccharum officinarum) Syed Rizwan Abbas a, Syed Mubashar Sabir b,⇑, Syed Dilnawaz Ahmad c, Aline Augusti Boligon d, Margareth Linde Athayde d a
Department of Plant Breeding and Molecular Genetics, Faculty of Agriculture, University of Poonch Rawalakot, Azad Kashmir, Pakistan Department of Chemistry, University of Poonch Rawalakot, Azad Kashmir, Pakistan University of Azad Jammu and Kashmir, Muzaffarabad, Azad Kashmir, Pakistan d Phytochemical Research Laboratory, Department of Industrial Pharmacy, Federal University of Santa Maria, Build 26, Room 1115, Santa Maria CEP 97105-900, Brazil b c
a r t i c l e
i n f o
Article history: Received 8 July 2013 Received in revised form 16 September 2013 Accepted 19 September 2013 Available online 29 September 2013 Keywords: Sugarcane DNA damage Antioxidant activity HPLC analysis Hydroxyl radical
a b s t r a c t The present study investigated the antioxidant and phenolic composition of sugarcane. The leaves and juices of thirteen varieties of sugarcane were studied for their antioxidant activity and protective effect on DNA damage. 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH) assay was used to determine the radical scavenging activities in leaves and juices. Different varieties of sugarcane showed good antioxidant properties, IC50 values ranged from 20.82 to 27.47 lg/ml for leaves and from 63.95 to higher than 200 lg/ml for juice. The leaves and juice possess strong ability to protect against DNA damage induced by hydroxyl radical generated in Fenton reaction. The major phenolic acids, some flavonoid aglycone and glycosides were identified in leaves by high performance liquid chromatography. Ferulic acid (14.63 ± 0.03 mg/g), cumaric acid (11.65 ± 0.03 mg/g), quercetrin (10.96 ± 0.02 mg/g), caffeic acid (9.16 ± 0.01 mg/g) and ellagic acid (9.03 ± 0.02 mg/g) were prédominant in infusion of sugarcane. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction In the last several decades, there has been a huge interest in identifying free radical scavengers or antioxidants that can inhibit or retard the oxidative DNA damage. These antioxidants supplied by diets, include vitamin C, vitamin E, carotenoids (b-carotene, acarotene, b-cryptoxanthin, lutein, zeaxanthin and lycopene) to several polyphenolic compounds including flavo noids (catechins, flavonols, flavones and isoflavonoids) can impede carcinogenesis by scavenging free radicals or interfering with the binding of carcinogens to DNA (Craig, 1997; Stoner & Mukhtar, 1995). Accordingly, antioxidants, abundant in foods, have received the great attention and have been studied extensively, since they can reduce risks for cardiovascular disease or several types of cancers (Hertog, Feskens, Hollman, Katan, & Kromhout, 1993; Kromhout et al., 1995). Sugarcane is an important cash crop of Pakistan. It is mainly grown for sugar production. It is an important source of income and employment for the farming community of the country. It also forms essential item for industries like sugar, chip board, paper, barrages, confectionery, uses in chemicals, plastics, paints, synthetics, fiber, insecticides and detergents (Aslam & Khan, 2001). Sugar⇑ Corresponding author. Tel.: +92 3455171876; fax: +92 05824960007. E-mail address: [email protected] (S.M. Sabir). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.09.113
cane, popularly known as noble cane, due to its high sucrose content and low fiber content is one of the important industrial crops of the world. The sugarcane juice contains flavonoids such as apigenin, luteolin and tricin derivatives and among phenolics, hydroxycinnamic, caffeic and sinapic acid, representing a total content of around 160 mg/L (Joaquim, Maurício, Adyary, Franco, & Maria, 2006) whereas sugarcane leaves contains luteolin-8-C(rhamnosylglucoside) as major compound with radical scavenging activity (Fabiana, Renata, Tatiana, & Janete, 2008). It is principal raw material for the sugar industry as 70% of the world’s sugar comes from sugarcane. When the sugar is produced, the sugarcane leaf is removed prior to a series of manufacturing procedures. The leaves are by products of agro-industries and are wasted or used as feedstuff and fertilizer. Sugarcane leaves are commonly discarded or burned. However, the disposal has become a matter of serious concern. Hence, more rational uses of sugarcane leaves are necessary. Besides sugar production, large number of population in the tropics and subtropics relishes its juice and consume raw cane. In the Pakistan chewing raw sugarcane is recommended for sound and healthy body. Both the roots and stems of sugarcane are used in medicine to treat skin and urinary tract infections, as well as for bronchitis, heart conditions, loss of milk production, cough, anemia, constipation as well as general debility. It is also used for jaundice and lowering blood pressure (Mira et al., 2011). The DPPH radical assay has been widely used to test the ability of compounds
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as free-radical scavengers or hydrogen donors and to evaluate the antioxidative activity of plant extracts and foods (Porto, Calligaris, Celloti, & Nicoli, 2000; Seow, Amru, & Chandran, 2012). In view of the potential role of sugarcane as a dietary source of flavonoids as well as its possible use as functional food, this study was aimed to evaluate the antioxidant activity of thirteen varieties of sugarcane, their phenolic and flavonoid contents and to examine their protective effect on hydroxyl radical mediated DNA damage. To our knowledge this is first report on the antioxidant and DNA protecting ability of these sugarcane cultivars.
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aqueous extract of sugarcane. The mixture was shaken vigorously and left to stand for 30 min in the dark, and the absorbance was measured at 517 nm. The capacity to scavenge the DPPH radical was calculated using the following equation: (%) = [(Ao A1)/ Ao)] 100, Where, Ao is the absorbance of the control reaction and A1 is the absorbance of the sample itself. The IC50 values (Extract concentration that cause 50% scavenging) were determined from the graph of scavenging effect percentage against the extract concentration. All determinations were carried out in triplicate. 2.5. Total antioxidant activity assay
2. Material and methods 2.1. Chemicals and reagents 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH Sigma, 90.0%), 1,1,1-tris (hydroxymethyl) ethane (Tris) and Low melting agarose were purchased from Sigma and Aldrich Chemicals (St, Louis, MO, USA). Ethanol (95%), iron sulphate, phosphate buffer, hydrogen peroxide, sodium acetate and sodium carbonate were purchased from Bio-Chemical (Lahore). Methanol, acetic acid, gallic acid, chlorogenic acid, caffeic acid, ferulic acid, cumaric acid and ellagic acid were purchased from Merck (Darmstadt, Germany). Quercetin, quercitrin, isoquercitrin, rutin, catechin and epicatechin were acquired from Sigma Chemical Co. (St. Louis, MO, USA). All the chemicals and reagents were of analytical grade. 2.2. Sugarcane samples Studies were conducted on thirteen genotypes of sugarcane which were collected from different growing areas of Pakistan. Genotypes selected for experiments were NSG-45, CPF-198, Lho 83-153, CPF-246, CSSG-668, S-2003-US-718, Rb-72, S-2002-US160, CO-1148, NSG-555, S-2003-US-694, S-2003-US-633 and CPF237. These genotypes were chosen for their frequent cultivation in the area and use by local communities. All genotypes were grown under glass house conditions in Faculty of agriculture, University of Poonch Rawalakot Pakistan. Mature sugarcane leaves (4 months old) yellowish-green in color were harvested from aerial parts of plants. Three samples of leaves were chosen from each plant.
The assay was based on the reduction of molybdenum, Mo(VI)– Mo(V) by the extract and subsequent formation of a green phosphate/Mo(V) complex at acidic pH (Prieto, Pineda, & Aiguel, 1999). The extract (0.1 mg/ml) was mixed with 3 ml of the reagent solution (0.6 M H2SO4, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were incubated at 95 °C for 90 min. The mixture was cooled to room temperature and the absorbance of the solution was measured at 695 nm. 2.6. DNA extraction DNA was extracted from leaves of sugarcane with new modified method (Li & Midmore, 1999). DNA of all sample were stored at 20 °C. The presence of DNA was confirmed by gel electrophoresis (1.5%). 2.7. Site-specific hydroxyl radical-mediated DNA strand breaks The site specific hydroxyl radical-mediated DNA strand breaks were measured by the procedure described by Yeung et al. (2002) with some minor modifications. Briefly, 0.5 lg DNA was incubated with 1 ll of 1 mM FeSO4, 1 ll of 10% H2O2, 3 ll of 100 lg/ml of aqueous extracts of sugarcane, and the final volume was made up to 15 ll with 50 mM phosphate buffer (pH 7.0). The mixture was incubated in water bath at 37 °C for 30 min. After the incu bation, the sample was immediately loaded in a 1.5% agarose gel along with 3 ll ethidim bromide, containing 40 mM Tris, 20 mM sodium acetate and 2 mM EDTA, and electrophoresed in a horizontal slab apparatus in Tris/boric/EDTA gel buffer. The gel was then photographed under UV light.
2.3. Preparation of sugarcane extracts
2.8. HPLC–DAD- analysis of phenolic compounds
The leaves were air dried in order to remove the moisture. The leaves of sugarcane (5 g) were ground and soaked in boiling water (250 ml) for 15 min, allowed to cool and filtered using whatman filter paper. The highly significant total phenolic content and antioxidant activity has been recorded in plant sample extracted with distilled boiling water (Ponamari, Sathishkumar, & Lakshmi, 2011). The obtained residue was further extracted twice and finally the whole extract was concentrated. The extract weight and percentage yield were found to be 0.5–1.5%, respectively. The serial dilution of the extract was made to obtain the desired concentration of plant for experiment. Sugarcane juice was extracted from the genotypes and filtered to obtain the juices.
High performance liquid chromatography (HPLC–DAD) was performed with a Shimadzu Prominence Auto Sampler (SIL-20A) HPLC system (Shimadzu, Kyoto, Japan), equipped with Shimadzu LC-20AT reciprocating pumps connected to a DGU 20A5 degasser with a CBM 20A integrator, SPD-M20A diode array detector and LC solution 1.22 SP1 software. Reverse phase chromatographic analyses were carried out under gradient conditions using C18 column (4.6 mm 250 mm) packed with 5 lm diameter particles; the mobile phase was water containing 2% acetic acid (A) and methanol (B), and the composition gradient was: 5% (B) for 2 min; 25% (B) until 10 min; 40%, 50%, 60%, 70% and 80% (B) every 10 min; following the method described by Boligon et al. (2012) with slight modifications. Saccharum officinarum (sugarcane) infusion leaves and mobile phase were filtered through 0.45 lm membrane filter (Millipore) and then degassed by ultrasonic bath prior to use, the extracts of sugarcane were analyzed at a concentration of 30 mg/ml. The flow rate was 0.7 ml/min and the injection volume was 50 ll. The sample and mobile phase were filtered through 0.45 lm membrane filter (Millipore) and then degassed by ultrasonic bath prior to use. Stock solutions of standards references were prepared in the HPLC mobile phase at a concentration range
2.4. Antioxidant activity by DPPH radical scavenging The antioxidant activities of the sugarcane extracts were measured using the stable DPPH radical according to the method of Hatano, Kagawa, Yasuhara, and Okuda (1998). Briefly 0.25 mM solution of DPPH radical (0.5 ml) was added to the sample solution in ethanol (1 ml) at different concentrations (25–300 lg/ml) of
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of 0.025–0.300 mg/ml catechin, epicatechin, quercetin, quercitrin, isoquercitrin and rutin, and 0.045–0.500 mg/ml for gallic, chlorogenic, caffeic, ferulic, cumaric and ellagic acids. Quantification was carried out by integration of the peaks using the external standard method, at 257 nm for gallic acid, 280 nm for catechin and epicatechin, 325 nm for ellagic acid, chlorogenic acid, caffeic acid, ferulic acid and cumaric acid, and 365 for quercetin, quercitrin, isoquercitrin and rutin. The chromatography peaks were confirmed by comparing its retention time with those of reference standards and by DAD spectra (200–600 nm). All chromatography operations were carried out at ambient temperature and in triplicate.
2.10. Statistical analysis The results were expressed as means ± standard deviation. The data was analyzed by one way AOVA and different group means were compared by Duncan’s multiple range (DMR) test where necessary. P < 0.05 was considered significant in all cases. The software Package Statistica was used for analysis of data.
3. Results and discussion 3.1. DPPH radical scavenging activity
2.9. Limit of detection (LOD) and limit of quantification (LOQ) LOD and LOQ were calculated based on the standard deviation of the responses and the slope using three independent analytical curves, as defined by Boligon et al. (2013). LOD and LOQ were calculated as 3.3 and 10 r/S, respectively, where r is the standard deviation of the response and S is the slope of the calibration curve.
Free radicals involved in the process of lipid peroxidation are considered to play a major role in numerous chronic pathologies such as cancer and cardiovascular diseases (Halliwell, Gutteridge, & Cross, 1992). DPPH is considered to be a model of a stable lipophilic radical. A chain reaction of lipophilic radicals is initiated by lipid autoxidation. Antioxidants react with DPPH, reducing the number of DPPH free radical to the number of their available hydroxyl groups. Therefore the absorption at 517 nm is proportional
Fig. 1. Antioxidant activity of aqueous extract of sugarcane (a) DPPH radical scavenging activity of different genotypes of sugarcane leaves. (b) DPPH radical scavenging activity of different genotypes of sugarcane juice. Values are mean ± SD (n = 3).
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to the amount of residual DPPH. It is visually noticeable as a discoloration from purple to yellow. Among different genotypes NSG-555, S-2003-US-633, CPF-237 and CPF-246 significantly displayed higher DPPH radical scavenging activities in leaves (Fig 1a) whereas NSG-45, CPF-198, CSSG-668, S-2003-US-694 also showed good antioxidant activities in juice (Fig 1a and b). It is interesting that the aqueous extracts of these genotypes displayed antioxidant activity higher than 50% even at a low concentration of 25 lg/ml. The extract concentration that cause 50% scavenging of DPPH (IC50 value) are shown in Table 1. The IC50 is inversely proportional to the scavenging activity of the extract. The IC50 values of different genotypes of sugarcane varied from 20.82 to 27.47 lg/ml which indicates the high radical scavenging activity of sugarcane. The extracts from the leaves of different genotypes, CPF-246 and NSG-55 demosntrated the highest antioxidant activity (IC50, 20.82 lg/ml) whereas, Lho 84-153 relatively showed less antioxidant activity (IC50, 27.47 lg/ml). NSG-45, NSG-555, CPF198, S-2003-US-633 displayed similar IC50 (Table 1). In comparison to the leaves, juices displayed lesser antioxidant activity (IC50 values in the range of 63.95–200 lg/ml). NSG-55 displayed the highest antioxidant activity (IC50, 63.95 lg/ml) (Table 1). The IC50 values of S-2003-US-718 and Rb-72 could not be calculated as it was higher than 200 lg/ml (Table 1). The results obtained in this study have clearly indicated the high antioxidant activity of sugarcance cultivars and suggest their use in diseases arising from free radicals. The antioxidant activity of sugarcane juice is also supported by the literature (Fabiana et al., 2008; Joaquim et al. 2006; Kadam et al., 2008). The antioxidant activity of aqueous extract sugarcane leaves is supported by the studies of Pei-Ying Yu (2009) where three varieties of sugarcane displayed significant total antioxidant activity, reducing activity and liposome peroxidation inhibition studies. 3.2. Total antioxidant activity In the phosphomolybdenum assay, which is a quantitative method to evaluate water-soluble and fat-soluble antioxidant capacity (total antioxidant capacity), the extracts demonstrated electron-donating capacity showing its ability to act as chain terminators, transforming relative free radical species into more stable non-reactive products (Dorman, Kosar, Kahlos, Holm, & Hiltunen, 2003). Allhorn, Klapyta, and Akerstrom (2005) reported that the reducing property can be a novel antioxidation defense mechanism, possibly through the ability of the antioxidant compound to reduce transition metals. Therefore, the higher reducing ability of the sugarcane extracts may have contributed to the higher antioxidant activity. The total antioxidant activity of the extract (equivalent to ascorbic acid) ranged between 62.3 and 80.26 lg/ ml. CPF-246 was found to be the potential candidate of antioxidant
Table 1 Extract concentration that causes 50% scavenging (IC50) for DPPH radical assay. Genotypes
IC50 (lg/ml) for leaves
IC50 (lg/ml) for juice
NSG-45 CPF-198 Lho 83-153 S-2003-US-633 CSSG-668 S-2003-US-718 S-2003-US-160 CO-1148 CPF-246 S-2003-US-694 Rb-72 CPF-237 NSG-555
25.34 25.53 27.47 21.04 23.63 22.12 26.89 24.43 20.82 22.62 25.78 23.33 20.82
100 88.82 89.31 198 142.34 >200 148 89.28 92.95 81.96 >200 182.84 63.95
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activity (80.26 lg/ml) whereas, Co-1148 showed the least antioxidant activity (62.3 lg/ml). S-2003-US-718, Rb-72, S-2002-US-160, and CPF-198 demonstrated the similar antioxidant activities (Fig 2a). In comparison to the leaves juices showed higher reducing activity as their antioxidant activity varied between 70.43 and 99.9 lg/ml. Co-1148 and CPF-246 displayed the highest reducing activities (Fig 2b). The high reducing activity of juice may be due to the high content of ascorbic acid which is less in leaves. 3.3. Protection against DNA damage In order to study the protective effects of sugarcane aqueous extract on hydroxyl radical-mediated DNA strand breaks, thirteen genotypes of sugarcane were used in the site specific DNA damage assay. Incubation of plant DNA with FeSO4 and H2O2 for 40 min in water bath resulted in producing hydroxyl ions, whereby indicating that both sin gle-strand and double-strand DNA breaks can be induced by FeSO4/H2O2 at the indicated concentrations and incubation time (Fig 3). The gel pattern of DNA exposed to FeSO4 + H2O2, in the presence and the absence of extracts of sugarcane is presented in Fig. 3. The control showed the absence of specific band in treated DNA (shredding) which indicates DNA damage. Different genotypes of sugarcane extracts at a final concentration of 20 lg/ml significantly reduced the DNA damage (Fig. 3). Hydroxyl radicals generated by the Fenton reaction are known to cause oxidatively induced breaks in DNA strands to yield its fragmented forms. The free radical scavenging activity of sugarcane was studied on genomic DNA. The treatment of supercoiled DNA with Fenton’s reagent directed the alteration of DNA to open circular form. The addition of extracts to the reaction mixture substantially decreased the DNA strand scission and retained the supercoiled form, thus effectively protects DNA even at a low concentration of 20 lg/ml. The genotypes showed their ability to protect against DNA damage induced by hydroxyl radicals. These results are in line with our previous studies (Abbas et al., 2013). Based on the previous data, it is possible that the powerful antioxidant activity of sugarcane aqueous extracts is given by the presence of substances with hydroxyls. In this context, flavonoids possess an ideal structure for the scavenging of free radicals since they present a number of hydroxyls acting as hydrogen donators acting as an important antioxidant agent (Cao, Sofic, & Prior, 1997). In fact, polyphenols are an important group of pharmacologically active compounds. They are considered to be the most active antioxidant derivatives in plants (Edenharder & Grunhage, 2003). However, it has been shown that the phenolic content does not necessary follow the antioxidant activity. Antioxidant activity is generally the result of the combined activity of a wide range of compounds, including phenolics, peptides, organic acids and other components (Gallardo, Jimenez, & Garcia-Conesa, 2006). This is first report to date on the protective effect of sugarcane leaves on DNA damage. Kadam et al. (2008) reported that sugarcane juice has protective role against radiation induced DNA damage. Sugarcane extracts and juices are able to exhibit the protection on DNA damage caused by hydroxyl radical, which might be due to their chelating activity on iron or hydroxyl radical scavenging or both (Sabir et al., 2012). 3.4. Content of phenolics, flavonoids and HPLC characterization of phenolic compounds The HPLC phenolic profile of S. officinarum in infusion of leaves was also acquired. HPLC analysis is shown in Fig. 4. The samples of S. officinarum (CPF-246) displayed the highest antioxidant activity, therefore, its phenolic profile was determined. The results revelaed that the infusion contains several compounds in addition to gallic acid (retention time-tR 10.97 min, peak 1), catechin (tR = 16.03 min,
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Fig. 2. Total antioxidant activity of sugarcane measured by phosphomolybdenum reduction assay. (a) Antioxidant activity of different genotypes of sugarcane leaves. (b) Antioxidant activity of different genotypes of sugarcane juice. Values are mean ± SD (n = 3).
Fig. 3. Effect of different varieties of sugarcane on DNA damage induced with Fe + H2O2 (Control). (a) Protective effect of different genotypes of sugarcane leaves (b). Protective effect of different genotypes of sugarcane juice. The lanes (2-14), FeSO4 and H2O2 in the presence of different varieties of sugarcane. From left to right Control, NSG45, CPF-198, Lho 83-153, CPF-246, CSSG-668, S-2003-US-718, Rb-72, S-2002-US-160, CO-1148, NSG-555, S-2003-US-694, S-2003-US-633 and CPF-237.
peak 2), chlorogenic acid (tR = 19.56 min, peak 3), caffeic acid (tR = 22.17 min, peak 4), ferulic acid (tR = 24.13 min, peak 5), epicatechin (tR = 28.04 min, peak 6), ellagic acid (tR = 32.17 min, peak
7), cumaic acid (tR = 36.09 min, peak 8), rutin (tR = 38.11 min, peak 9), quercitrin (tR = 41.05 min, peak 10), isoquercitrin (tR = 43.39 min, peak 11) and quercetin (tR = 47.83 min, peak 12). The content
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has effectively protected the DNA damage. Among different cultivars CPF-246, S-2003-US-633, CPF-237 showed the highest antioxidant and protection against DNA damage. The investigation results that these cultivars can be used not only as accessible source of natural antioxidants but also as an ingredient of the functional food and control of degenerative diseases. However, further detailed studies are required to explain their mechanism of action.
References Fig. 4. HPLC phenolic profile of Saccharum officinarum (CPF-246) infusion. Gallic acid (peak 1), catechin (peak 2), chlorogenic acid (peak 3), caffeic acid (peak 4), ferulic acid (peak 5), epicatechin (peak 6), ellagic acid (peak 7), cumaric acid (peak 8), rutin (peak 9), quercitrin (peak 10), isoquercitrin (peak 11) and quercetin (peak 12).
Table 2 Phenolics and flavonoids composition of Saccharum officinarum (CPF-246) leaves. Compounds
LOQ
LOD
mg/g
Infusion %
lg/mL
lg/mL
Phenolics Ferulic acid Chlorogenic acid Caffeic acid Cumaric acid Gallic acid Ellagic acid
14.63 ± 0.03d 3.27 ± 0.02b 9.16 ± 0.01c 11.65 ± 0.03f 4.38 ± 0.02a 9.03 ± 0.02c
1.46 0.32 0.91 1.16 0.43 0.90
0.017 0.029 0.034 0.011 0.018 0.008
0.01 0.095 0.112 0.036 0.059 0.027
Flavonoids Quercitrin Rutin Isoquercitrin Quercetin Catechin Epicatechin
10.96 ± 0.02f 3.34 ± 0.01b 3.98 ± 0.01b 5.81 ± 0.01e 4.51 ± 0.01a 5.78 ± 0.01e
1.09 0.33 0.39 0.58 0.45 0.57
0.035 0.038 0.016 0.023 0.005 0.013
0.115 0.124 0.052 0.075 0.016 0.042
Results are expressed as means ± SD (n = 3). Averages followed by different letters differ by Turkey test p < 0.05.
of phenolics and flavonoids are shown in Table 2. Ferulic acid (14.63 ± 0.03 mg/g), cumaric acid (11.65 ± 0.03 mg/g), quercetrin (10.96 ± 0.02 mg/g), caffeic acid (9.16 ± 0.01 mg/g) and ellagic acid (9.03 ± 0.02 mg/g) were detected as major compounds. Whereas, catechin (4.51 ± 0.01 mg/g), epicatechin (5.78 ± 0.01 mg/g), gallic acid (4.38 ± 0.02 mg/g), quercetin (5.81 ± 0.01), isoquercetrin (3.98 ± 0.01 mg/g), gallic acid (4.38 ± 0.02 mg/g) and chlorogenic acid (3.27 ± 0.02 mg/g) relatively showed minor contribution. This is the first comprehensive phytochemical study of sugarcane leaves. Earlier studies have shown the presence of phenolics, luteolin 8-C-rhamnosylglucoside, diosmetin-8-glucoside, vitexin, orientin, tricin-7-O-neohesperidoside and tricin derivatives in the juice of sugarcane (Fabiana et al., 2008). Lee, Chen, Yu, Wang, and Duh (2012) reported the presence of flavonoids, benzoic acid, chlorogenic acid, caffeic acid and vitexin in leaves of different genotypes of sugarcane. The observed antioxidant activity and protecting ability of sugarcane against DNA damage is due to the phenolic acids (ferulic acid, gallic acid, chlorogenic acid, caffeic acid and cumaric acid) and flavonoids (quercetrin, catechin, epicatechin, rutin and quercetin). The exogenous antioxidants from sugarcane extracts may act directly or indirectly with the internal antioxidant system for synergetic effects to protect several diseases linked to free radicals such as heart diseases, neurodisorders and other stress related disorders. 4. Conclusions In conclusion the results of present study revealed that the different cultivars of sugar cane are rich source of antioxidants and
Abbas, S. R., Ahmad, S. D., Sabir, S. M., Shah, A. H., Shahid, A., Gohar, M., et al. (2013). Antioxidant activity, repair and tolerance of oxidative DNA damage in different cultivars of sugarcane (Saccharum officinarum) leaves. Australian Journal of Crop Science, 7, 40–45. Allhorn, M., Klapyta, A., & Akerstrom, B. (2005). Redox properties of the lipocalin alpha-1microglobulin: Reduction of cytochrome c, hemoglobin, and free iron. Free Radical Biology and Medicine, 38, 557–567. Aslam, S. M., & Khan, M. A. (2001). All about sugarcane crop. Pakistan: Nuclear institute of Agriculture Tandojam. Boligon, A. A., Kubiça, T. K., Mario, D. B., Brum, T. F., Piana, M., Weiblen, R., et al. (2013). Antimicrobial and antiviral activity-guided fractionation from Scutia buxifolia Reissek extracts. Acta Physiologiae Plantarum. http://dx.doi.org/ 10.1007/s11738-013-1259-0. Boligon, A. A., Sagrillo, M. R., Machado, L. F., Filho, O. S., Machado, M. M., da Cruz, I. B. M., et al. (2012). Protective effects of extracts and flavonoids isolated from Scutia buxifolia Reissek against chromosome damage in human lymphocytes exposed to hydrogen peroxide. Molecules, 17, 5757–5769. Cao, G., Sofic, E., & Prior, R. L. (1997). Antioxidant and pro-oxidant behavior of flavonoids: Structure–activity relationships. Free radicals Biology and Medicine, 22, 749–760. Craig, W. (1997). Phytochemicals. Guardians of our health. Journal of the American Dietetic Association, 97, S199–S204. Dorman, H. J. D., Kosar, M., Kahlos, K., Holm, Y., & Hiltunen, R. (2003). Antioxidant properties and composition of aqueous extracts from Mentha species, hybrids, varieties, and cultivars. Journal of Agriculture and Food Chemistry, 51, 4563–4569. Edenharder, R., & Grunhage, D. (2003). Free radical scavenging abilities of flavonoids as mechanism of protection against mutagenicity induced by tert-butyl hydroperoxide or cumene hydroperoxide in Salmonella typhimurium TA102. Mutation Research, 540, 1–18. Fabiana, C. V., Renata, C., Tatiana, O. D., & Janete, H. Y. (2008). HPLC microfractionation of flavones and antioxidant (radical scavenging) activity of Saccharum officinarum L.. Journal of Brazilian Chemical Society. http://dx.doi.org/ 10.1590/S0103-50532008000500014. Gallardo, C., Jimenez, L., & Garcia-Conesa, M. T. (2006). Hydroxycinnamic acid composition and in vitro antioxidant activity of selected grain fractions. Food Chemistry, 99, 455–463. Halliwell, B., Gutteridge, J. M. C., & Cross, C. E. (1992). Free radicals, antioxidants and 481 human disease: Where are we now? Journal of Laboratory and Clinical Medicine, 119, 598–620. Hatano, T., Kagawa, H., Yasuhara, T., & Okuda, T. (1998). Two new flavonoids and other constituents in licorice root; their relative astringency and radical scavenging effects. Chemical and Pharmaceutical Bulletin, 36, 2090–2097. Hertog, M. G., Feskens, E. J., Hollman, P. C., Katan, M. B., & Kromhout, D. (1993). Dietary antioxidant flavonoids and risk of coronary heart disease: The Zutphen elderly study. Lancet, 342, 1007–1011. Joaquim, M. D. A., Maurício, D. V. N., Adyary, F. L., Franco, M. L., & Maria, I. G. (2006). Antioxidant activity of phenolic compounds from sugar cane (Saccharum officanarum L.) juice. Plant Foods for Human Nutrition, 61, 187–192. Kadam, U. S., Ghosh, S. B., Strayo, D., Suprassana, P., Devasagayam, T. P. A., & Bapat, V. A. (2008). Antioxidant activity in sugar cane juice and its protective role against radiation induced DNA damage. Food Chemistry, 106, 1154–1160. Kromhout, D. M. G., Aravanis, C., Blackburn, H., Buzina, R., Fidanza, F., & Giampaoli, S. (1995). Flavonoid intake and long-term risk of coronary heart disease and cancer in the seven countries study. Archives of Internernal Medicine, 155, 381–386. Lee, C. P., Chen, Z. T., Yu, P. Y., Wang, Y. C., & Duh, P. D. (2012). Identification of bioactive compounds and comparison of apoptosis induction of three varieties of sugarcane leaves. Journal of Functional Foods, 4, 391–397. Li, M., & Midmore, D. J. (1999). Estimating the genetic relationships of Chinese water chestnut (Eleocharis dulcis (Burm. f.) Hensch.) cultivated in Australia, using random amplified polymorphic DNAs (RAPDs). The Journal of Horticultural Science and Biotechnology, 74, 224–231. Mira, A., Syeda, S., Farhana, I., Dilara, F., Rasheda, A., Dilruba, N., et al. (2011). A survey of medicinal plants used by the traditional medicinal practitioners of Khulna City, Bangladesh. American Eurasian Journal of Sustainable Agriculture, 5, 177–195. Pei-ying yu. (2009). Antioxidant and anticancer activities of Sugarcane (Saccharum officinarum L.) leaf of different varieties. MSc. (thesis). Graduate Institute of Biotechnology, China, p. 183. Ponamari, G., Sathishkumar, R., & Lakshmi, P. T. V. (2011). The effects of drying treatments on the contents of antioxidants in Cardiospermum halicacabum Linn. International Journal of Pharma and Biosciences, 2, B304–B313.
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Porto, C. D., Calligaris, S., Celloti, E., & Nicoli, M. C. (2000). Antiradical properties of commercial cognacs assessed by the DPPH test. Journal of Agriculture and Food Chemistry, 48, 4241–4245. Prieto, P., Pineda, M., & Aiguel, M. (1999). Spectrophotometer quantization of antioxidant capacity through the formation of Phosphomolybdenum complex: Specific application to the determination of vitamin E. Analytical Biochemistry, 269, 337–341. Sabir, S. M., Ahmad, S. D., Hamid, A., Khan, M. Q., Athyade, M. L., Santos, D. B., et al. (2012). Antioxidant and hepatoprotective activity of ethanolic extracts of Solidago microglossa containing polyphenolic compounds. Food Chemistry, 131, 741–747.
Seow, M. H., Amru, N. B., & Chandran, S. (2012). Antioxidant activity, phenolic and flavonoid contents in the leaves of different varieties of sweet potato (Ipomoea batatas). Australian Journal of Crop Science, 6, 375–380. Stoner, G. D., & Mukhtar, H. (1995). Polyphenols as cancer chemopre ventive agents. Journal of Cellular Biochemistry, 22, 169–180. Yeung, S. Y., Lan, W. H., Huang, C. S., Lin, C. P., Chan, C. P., & Chang, M. C. (2002). Scavenging property of three cresol isomers against H2O2, hypochlorite, superoxide and hydroxyl radicals. Food and Chemical Toxicology, 40, 1403–1413.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Phenolic profile, antioxidant potential and DNA damage protecting activity of sugarcane (Saccharum officinarum) Syed Rizwan Abbas a, Syed Mubashar Sabir b,⇑, Syed Dilnawaz Ahmad c, Aline Augusti Boligon d, Margareth Linde Athayde d a
Department of Plant Breeding and Molecular Genetics, Faculty of Agriculture, University of Poonch Rawalakot, Azad Kashmir, Pakistan Department of Chemistry, University of Poonch Rawalakot, Azad Kashmir, Pakistan University of Azad Jammu and Kashmir, Muzaffarabad, Azad Kashmir, Pakistan d Phytochemical Research Laboratory, Department of Industrial Pharmacy, Federal University of Santa Maria, Build 26, Room 1115, Santa Maria CEP 97105-900, Brazil b c
a r t i c l e
i n f o
Article history: Received 8 July 2013 Received in revised form 16 September 2013 Accepted 19 September 2013 Available online 29 September 2013 Keywords: Sugarcane DNA damage Antioxidant activity HPLC analysis Hydroxyl radical
a b s t r a c t The present study investigated the antioxidant and phenolic composition of sugarcane. The leaves and juices of thirteen varieties of sugarcane were studied for their antioxidant activity and protective effect on DNA damage. 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH) assay was used to determine the radical scavenging activities in leaves and juices. Different varieties of sugarcane showed good antioxidant properties, IC50 values ranged from 20.82 to 27.47 lg/ml for leaves and from 63.95 to higher than 200 lg/ml for juice. The leaves and juice possess strong ability to protect against DNA damage induced by hydroxyl radical generated in Fenton reaction. The major phenolic acids, some flavonoid aglycone and glycosides were identified in leaves by high performance liquid chromatography. Ferulic acid (14.63 ± 0.03 mg/g), cumaric acid (11.65 ± 0.03 mg/g), quercetrin (10.96 ± 0.02 mg/g), caffeic acid (9.16 ± 0.01 mg/g) and ellagic acid (9.03 ± 0.02 mg/g) were prédominant in infusion of sugarcane. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction In the last several decades, there has been a huge interest in identifying free radical scavengers or antioxidants that can inhibit or retard the oxidative DNA damage. These antioxidants supplied by diets, include vitamin C, vitamin E, carotenoids (b-carotene, acarotene, b-cryptoxanthin, lutein, zeaxanthin and lycopene) to several polyphenolic compounds including flavo noids (catechins, flavonols, flavones and isoflavonoids) can impede carcinogenesis by scavenging free radicals or interfering with the binding of carcinogens to DNA (Craig, 1997; Stoner & Mukhtar, 1995). Accordingly, antioxidants, abundant in foods, have received the great attention and have been studied extensively, since they can reduce risks for cardiovascular disease or several types of cancers (Hertog, Feskens, Hollman, Katan, & Kromhout, 1993; Kromhout et al., 1995). Sugarcane is an important cash crop of Pakistan. It is mainly grown for sugar production. It is an important source of income and employment for the farming community of the country. It also forms essential item for industries like sugar, chip board, paper, barrages, confectionery, uses in chemicals, plastics, paints, synthetics, fiber, insecticides and detergents (Aslam & Khan, 2001). Sugar⇑ Corresponding author. Tel.: +92 3455171876; fax: +92 05824960007. E-mail address: [email protected] (S.M. Sabir). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.09.113
cane, popularly known as noble cane, due to its high sucrose content and low fiber content is one of the important industrial crops of the world. The sugarcane juice contains flavonoids such as apigenin, luteolin and tricin derivatives and among phenolics, hydroxycinnamic, caffeic and sinapic acid, representing a total content of around 160 mg/L (Joaquim, Maurício, Adyary, Franco, & Maria, 2006) whereas sugarcane leaves contains luteolin-8-C(rhamnosylglucoside) as major compound with radical scavenging activity (Fabiana, Renata, Tatiana, & Janete, 2008). It is principal raw material for the sugar industry as 70% of the world’s sugar comes from sugarcane. When the sugar is produced, the sugarcane leaf is removed prior to a series of manufacturing procedures. The leaves are by products of agro-industries and are wasted or used as feedstuff and fertilizer. Sugarcane leaves are commonly discarded or burned. However, the disposal has become a matter of serious concern. Hence, more rational uses of sugarcane leaves are necessary. Besides sugar production, large number of population in the tropics and subtropics relishes its juice and consume raw cane. In the Pakistan chewing raw sugarcane is recommended for sound and healthy body. Both the roots and stems of sugarcane are used in medicine to treat skin and urinary tract infections, as well as for bronchitis, heart conditions, loss of milk production, cough, anemia, constipation as well as general debility. It is also used for jaundice and lowering blood pressure (Mira et al., 2011). The DPPH radical assay has been widely used to test the ability of compounds
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as free-radical scavengers or hydrogen donors and to evaluate the antioxidative activity of plant extracts and foods (Porto, Calligaris, Celloti, & Nicoli, 2000; Seow, Amru, & Chandran, 2012). In view of the potential role of sugarcane as a dietary source of flavonoids as well as its possible use as functional food, this study was aimed to evaluate the antioxidant activity of thirteen varieties of sugarcane, their phenolic and flavonoid contents and to examine their protective effect on hydroxyl radical mediated DNA damage. To our knowledge this is first report on the antioxidant and DNA protecting ability of these sugarcane cultivars.
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aqueous extract of sugarcane. The mixture was shaken vigorously and left to stand for 30 min in the dark, and the absorbance was measured at 517 nm. The capacity to scavenge the DPPH radical was calculated using the following equation: (%) = [(Ao A1)/ Ao)] 100, Where, Ao is the absorbance of the control reaction and A1 is the absorbance of the sample itself. The IC50 values (Extract concentration that cause 50% scavenging) were determined from the graph of scavenging effect percentage against the extract concentration. All determinations were carried out in triplicate. 2.5. Total antioxidant activity assay
2. Material and methods 2.1. Chemicals and reagents 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH Sigma, 90.0%), 1,1,1-tris (hydroxymethyl) ethane (Tris) and Low melting agarose were purchased from Sigma and Aldrich Chemicals (St, Louis, MO, USA). Ethanol (95%), iron sulphate, phosphate buffer, hydrogen peroxide, sodium acetate and sodium carbonate were purchased from Bio-Chemical (Lahore). Methanol, acetic acid, gallic acid, chlorogenic acid, caffeic acid, ferulic acid, cumaric acid and ellagic acid were purchased from Merck (Darmstadt, Germany). Quercetin, quercitrin, isoquercitrin, rutin, catechin and epicatechin were acquired from Sigma Chemical Co. (St. Louis, MO, USA). All the chemicals and reagents were of analytical grade. 2.2. Sugarcane samples Studies were conducted on thirteen genotypes of sugarcane which were collected from different growing areas of Pakistan. Genotypes selected for experiments were NSG-45, CPF-198, Lho 83-153, CPF-246, CSSG-668, S-2003-US-718, Rb-72, S-2002-US160, CO-1148, NSG-555, S-2003-US-694, S-2003-US-633 and CPF237. These genotypes were chosen for their frequent cultivation in the area and use by local communities. All genotypes were grown under glass house conditions in Faculty of agriculture, University of Poonch Rawalakot Pakistan. Mature sugarcane leaves (4 months old) yellowish-green in color were harvested from aerial parts of plants. Three samples of leaves were chosen from each plant.
The assay was based on the reduction of molybdenum, Mo(VI)– Mo(V) by the extract and subsequent formation of a green phosphate/Mo(V) complex at acidic pH (Prieto, Pineda, & Aiguel, 1999). The extract (0.1 mg/ml) was mixed with 3 ml of the reagent solution (0.6 M H2SO4, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were incubated at 95 °C for 90 min. The mixture was cooled to room temperature and the absorbance of the solution was measured at 695 nm. 2.6. DNA extraction DNA was extracted from leaves of sugarcane with new modified method (Li & Midmore, 1999). DNA of all sample were stored at 20 °C. The presence of DNA was confirmed by gel electrophoresis (1.5%). 2.7. Site-specific hydroxyl radical-mediated DNA strand breaks The site specific hydroxyl radical-mediated DNA strand breaks were measured by the procedure described by Yeung et al. (2002) with some minor modifications. Briefly, 0.5 lg DNA was incubated with 1 ll of 1 mM FeSO4, 1 ll of 10% H2O2, 3 ll of 100 lg/ml of aqueous extracts of sugarcane, and the final volume was made up to 15 ll with 50 mM phosphate buffer (pH 7.0). The mixture was incubated in water bath at 37 °C for 30 min. After the incu bation, the sample was immediately loaded in a 1.5% agarose gel along with 3 ll ethidim bromide, containing 40 mM Tris, 20 mM sodium acetate and 2 mM EDTA, and electrophoresed in a horizontal slab apparatus in Tris/boric/EDTA gel buffer. The gel was then photographed under UV light.
2.3. Preparation of sugarcane extracts
2.8. HPLC–DAD- analysis of phenolic compounds
The leaves were air dried in order to remove the moisture. The leaves of sugarcane (5 g) were ground and soaked in boiling water (250 ml) for 15 min, allowed to cool and filtered using whatman filter paper. The highly significant total phenolic content and antioxidant activity has been recorded in plant sample extracted with distilled boiling water (Ponamari, Sathishkumar, & Lakshmi, 2011). The obtained residue was further extracted twice and finally the whole extract was concentrated. The extract weight and percentage yield were found to be 0.5–1.5%, respectively. The serial dilution of the extract was made to obtain the desired concentration of plant for experiment. Sugarcane juice was extracted from the genotypes and filtered to obtain the juices.
High performance liquid chromatography (HPLC–DAD) was performed with a Shimadzu Prominence Auto Sampler (SIL-20A) HPLC system (Shimadzu, Kyoto, Japan), equipped with Shimadzu LC-20AT reciprocating pumps connected to a DGU 20A5 degasser with a CBM 20A integrator, SPD-M20A diode array detector and LC solution 1.22 SP1 software. Reverse phase chromatographic analyses were carried out under gradient conditions using C18 column (4.6 mm 250 mm) packed with 5 lm diameter particles; the mobile phase was water containing 2% acetic acid (A) and methanol (B), and the composition gradient was: 5% (B) for 2 min; 25% (B) until 10 min; 40%, 50%, 60%, 70% and 80% (B) every 10 min; following the method described by Boligon et al. (2012) with slight modifications. Saccharum officinarum (sugarcane) infusion leaves and mobile phase were filtered through 0.45 lm membrane filter (Millipore) and then degassed by ultrasonic bath prior to use, the extracts of sugarcane were analyzed at a concentration of 30 mg/ml. The flow rate was 0.7 ml/min and the injection volume was 50 ll. The sample and mobile phase were filtered through 0.45 lm membrane filter (Millipore) and then degassed by ultrasonic bath prior to use. Stock solutions of standards references were prepared in the HPLC mobile phase at a concentration range
2.4. Antioxidant activity by DPPH radical scavenging The antioxidant activities of the sugarcane extracts were measured using the stable DPPH radical according to the method of Hatano, Kagawa, Yasuhara, and Okuda (1998). Briefly 0.25 mM solution of DPPH radical (0.5 ml) was added to the sample solution in ethanol (1 ml) at different concentrations (25–300 lg/ml) of
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of 0.025–0.300 mg/ml catechin, epicatechin, quercetin, quercitrin, isoquercitrin and rutin, and 0.045–0.500 mg/ml for gallic, chlorogenic, caffeic, ferulic, cumaric and ellagic acids. Quantification was carried out by integration of the peaks using the external standard method, at 257 nm for gallic acid, 280 nm for catechin and epicatechin, 325 nm for ellagic acid, chlorogenic acid, caffeic acid, ferulic acid and cumaric acid, and 365 for quercetin, quercitrin, isoquercitrin and rutin. The chromatography peaks were confirmed by comparing its retention time with those of reference standards and by DAD spectra (200–600 nm). All chromatography operations were carried out at ambient temperature and in triplicate.
2.10. Statistical analysis The results were expressed as means ± standard deviation. The data was analyzed by one way AOVA and different group means were compared by Duncan’s multiple range (DMR) test where necessary. P < 0.05 was considered significant in all cases. The software Package Statistica was used for analysis of data.
3. Results and discussion 3.1. DPPH radical scavenging activity
2.9. Limit of detection (LOD) and limit of quantification (LOQ) LOD and LOQ were calculated based on the standard deviation of the responses and the slope using three independent analytical curves, as defined by Boligon et al. (2013). LOD and LOQ were calculated as 3.3 and 10 r/S, respectively, where r is the standard deviation of the response and S is the slope of the calibration curve.
Free radicals involved in the process of lipid peroxidation are considered to play a major role in numerous chronic pathologies such as cancer and cardiovascular diseases (Halliwell, Gutteridge, & Cross, 1992). DPPH is considered to be a model of a stable lipophilic radical. A chain reaction of lipophilic radicals is initiated by lipid autoxidation. Antioxidants react with DPPH, reducing the number of DPPH free radical to the number of their available hydroxyl groups. Therefore the absorption at 517 nm is proportional
Fig. 1. Antioxidant activity of aqueous extract of sugarcane (a) DPPH radical scavenging activity of different genotypes of sugarcane leaves. (b) DPPH radical scavenging activity of different genotypes of sugarcane juice. Values are mean ± SD (n = 3).
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to the amount of residual DPPH. It is visually noticeable as a discoloration from purple to yellow. Among different genotypes NSG-555, S-2003-US-633, CPF-237 and CPF-246 significantly displayed higher DPPH radical scavenging activities in leaves (Fig 1a) whereas NSG-45, CPF-198, CSSG-668, S-2003-US-694 also showed good antioxidant activities in juice (Fig 1a and b). It is interesting that the aqueous extracts of these genotypes displayed antioxidant activity higher than 50% even at a low concentration of 25 lg/ml. The extract concentration that cause 50% scavenging of DPPH (IC50 value) are shown in Table 1. The IC50 is inversely proportional to the scavenging activity of the extract. The IC50 values of different genotypes of sugarcane varied from 20.82 to 27.47 lg/ml which indicates the high radical scavenging activity of sugarcane. The extracts from the leaves of different genotypes, CPF-246 and NSG-55 demosntrated the highest antioxidant activity (IC50, 20.82 lg/ml) whereas, Lho 84-153 relatively showed less antioxidant activity (IC50, 27.47 lg/ml). NSG-45, NSG-555, CPF198, S-2003-US-633 displayed similar IC50 (Table 1). In comparison to the leaves, juices displayed lesser antioxidant activity (IC50 values in the range of 63.95–200 lg/ml). NSG-55 displayed the highest antioxidant activity (IC50, 63.95 lg/ml) (Table 1). The IC50 values of S-2003-US-718 and Rb-72 could not be calculated as it was higher than 200 lg/ml (Table 1). The results obtained in this study have clearly indicated the high antioxidant activity of sugarcance cultivars and suggest their use in diseases arising from free radicals. The antioxidant activity of sugarcane juice is also supported by the literature (Fabiana et al., 2008; Joaquim et al. 2006; Kadam et al., 2008). The antioxidant activity of aqueous extract sugarcane leaves is supported by the studies of Pei-Ying Yu (2009) where three varieties of sugarcane displayed significant total antioxidant activity, reducing activity and liposome peroxidation inhibition studies. 3.2. Total antioxidant activity In the phosphomolybdenum assay, which is a quantitative method to evaluate water-soluble and fat-soluble antioxidant capacity (total antioxidant capacity), the extracts demonstrated electron-donating capacity showing its ability to act as chain terminators, transforming relative free radical species into more stable non-reactive products (Dorman, Kosar, Kahlos, Holm, & Hiltunen, 2003). Allhorn, Klapyta, and Akerstrom (2005) reported that the reducing property can be a novel antioxidation defense mechanism, possibly through the ability of the antioxidant compound to reduce transition metals. Therefore, the higher reducing ability of the sugarcane extracts may have contributed to the higher antioxidant activity. The total antioxidant activity of the extract (equivalent to ascorbic acid) ranged between 62.3 and 80.26 lg/ ml. CPF-246 was found to be the potential candidate of antioxidant
Table 1 Extract concentration that causes 50% scavenging (IC50) for DPPH radical assay. Genotypes
IC50 (lg/ml) for leaves
IC50 (lg/ml) for juice
NSG-45 CPF-198 Lho 83-153 S-2003-US-633 CSSG-668 S-2003-US-718 S-2003-US-160 CO-1148 CPF-246 S-2003-US-694 Rb-72 CPF-237 NSG-555
25.34 25.53 27.47 21.04 23.63 22.12 26.89 24.43 20.82 22.62 25.78 23.33 20.82
100 88.82 89.31 198 142.34 >200 148 89.28 92.95 81.96 >200 182.84 63.95
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activity (80.26 lg/ml) whereas, Co-1148 showed the least antioxidant activity (62.3 lg/ml). S-2003-US-718, Rb-72, S-2002-US-160, and CPF-198 demonstrated the similar antioxidant activities (Fig 2a). In comparison to the leaves juices showed higher reducing activity as their antioxidant activity varied between 70.43 and 99.9 lg/ml. Co-1148 and CPF-246 displayed the highest reducing activities (Fig 2b). The high reducing activity of juice may be due to the high content of ascorbic acid which is less in leaves. 3.3. Protection against DNA damage In order to study the protective effects of sugarcane aqueous extract on hydroxyl radical-mediated DNA strand breaks, thirteen genotypes of sugarcane were used in the site specific DNA damage assay. Incubation of plant DNA with FeSO4 and H2O2 for 40 min in water bath resulted in producing hydroxyl ions, whereby indicating that both sin gle-strand and double-strand DNA breaks can be induced by FeSO4/H2O2 at the indicated concentrations and incubation time (Fig 3). The gel pattern of DNA exposed to FeSO4 + H2O2, in the presence and the absence of extracts of sugarcane is presented in Fig. 3. The control showed the absence of specific band in treated DNA (shredding) which indicates DNA damage. Different genotypes of sugarcane extracts at a final concentration of 20 lg/ml significantly reduced the DNA damage (Fig. 3). Hydroxyl radicals generated by the Fenton reaction are known to cause oxidatively induced breaks in DNA strands to yield its fragmented forms. The free radical scavenging activity of sugarcane was studied on genomic DNA. The treatment of supercoiled DNA with Fenton’s reagent directed the alteration of DNA to open circular form. The addition of extracts to the reaction mixture substantially decreased the DNA strand scission and retained the supercoiled form, thus effectively protects DNA even at a low concentration of 20 lg/ml. The genotypes showed their ability to protect against DNA damage induced by hydroxyl radicals. These results are in line with our previous studies (Abbas et al., 2013). Based on the previous data, it is possible that the powerful antioxidant activity of sugarcane aqueous extracts is given by the presence of substances with hydroxyls. In this context, flavonoids possess an ideal structure for the scavenging of free radicals since they present a number of hydroxyls acting as hydrogen donators acting as an important antioxidant agent (Cao, Sofic, & Prior, 1997). In fact, polyphenols are an important group of pharmacologically active compounds. They are considered to be the most active antioxidant derivatives in plants (Edenharder & Grunhage, 2003). However, it has been shown that the phenolic content does not necessary follow the antioxidant activity. Antioxidant activity is generally the result of the combined activity of a wide range of compounds, including phenolics, peptides, organic acids and other components (Gallardo, Jimenez, & Garcia-Conesa, 2006). This is first report to date on the protective effect of sugarcane leaves on DNA damage. Kadam et al. (2008) reported that sugarcane juice has protective role against radiation induced DNA damage. Sugarcane extracts and juices are able to exhibit the protection on DNA damage caused by hydroxyl radical, which might be due to their chelating activity on iron or hydroxyl radical scavenging or both (Sabir et al., 2012). 3.4. Content of phenolics, flavonoids and HPLC characterization of phenolic compounds The HPLC phenolic profile of S. officinarum in infusion of leaves was also acquired. HPLC analysis is shown in Fig. 4. The samples of S. officinarum (CPF-246) displayed the highest antioxidant activity, therefore, its phenolic profile was determined. The results revelaed that the infusion contains several compounds in addition to gallic acid (retention time-tR 10.97 min, peak 1), catechin (tR = 16.03 min,
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Fig. 2. Total antioxidant activity of sugarcane measured by phosphomolybdenum reduction assay. (a) Antioxidant activity of different genotypes of sugarcane leaves. (b) Antioxidant activity of different genotypes of sugarcane juice. Values are mean ± SD (n = 3).
Fig. 3. Effect of different varieties of sugarcane on DNA damage induced with Fe + H2O2 (Control). (a) Protective effect of different genotypes of sugarcane leaves (b). Protective effect of different genotypes of sugarcane juice. The lanes (2-14), FeSO4 and H2O2 in the presence of different varieties of sugarcane. From left to right Control, NSG45, CPF-198, Lho 83-153, CPF-246, CSSG-668, S-2003-US-718, Rb-72, S-2002-US-160, CO-1148, NSG-555, S-2003-US-694, S-2003-US-633 and CPF-237.
peak 2), chlorogenic acid (tR = 19.56 min, peak 3), caffeic acid (tR = 22.17 min, peak 4), ferulic acid (tR = 24.13 min, peak 5), epicatechin (tR = 28.04 min, peak 6), ellagic acid (tR = 32.17 min, peak
7), cumaic acid (tR = 36.09 min, peak 8), rutin (tR = 38.11 min, peak 9), quercitrin (tR = 41.05 min, peak 10), isoquercitrin (tR = 43.39 min, peak 11) and quercetin (tR = 47.83 min, peak 12). The content
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has effectively protected the DNA damage. Among different cultivars CPF-246, S-2003-US-633, CPF-237 showed the highest antioxidant and protection against DNA damage. The investigation results that these cultivars can be used not only as accessible source of natural antioxidants but also as an ingredient of the functional food and control of degenerative diseases. However, further detailed studies are required to explain their mechanism of action.
References Fig. 4. HPLC phenolic profile of Saccharum officinarum (CPF-246) infusion. Gallic acid (peak 1), catechin (peak 2), chlorogenic acid (peak 3), caffeic acid (peak 4), ferulic acid (peak 5), epicatechin (peak 6), ellagic acid (peak 7), cumaric acid (peak 8), rutin (peak 9), quercitrin (peak 10), isoquercitrin (peak 11) and quercetin (peak 12).
Table 2 Phenolics and flavonoids composition of Saccharum officinarum (CPF-246) leaves. Compounds
LOQ
LOD
mg/g
Infusion %
lg/mL
lg/mL
Phenolics Ferulic acid Chlorogenic acid Caffeic acid Cumaric acid Gallic acid Ellagic acid
14.63 ± 0.03d 3.27 ± 0.02b 9.16 ± 0.01c 11.65 ± 0.03f 4.38 ± 0.02a 9.03 ± 0.02c
1.46 0.32 0.91 1.16 0.43 0.90
0.017 0.029 0.034 0.011 0.018 0.008
0.01 0.095 0.112 0.036 0.059 0.027
Flavonoids Quercitrin Rutin Isoquercitrin Quercetin Catechin Epicatechin
10.96 ± 0.02f 3.34 ± 0.01b 3.98 ± 0.01b 5.81 ± 0.01e 4.51 ± 0.01a 5.78 ± 0.01e
1.09 0.33 0.39 0.58 0.45 0.57
0.035 0.038 0.016 0.023 0.005 0.013
0.115 0.124 0.052 0.075 0.016 0.042
Results are expressed as means ± SD (n = 3). Averages followed by different letters differ by Turkey test p < 0.05.
of phenolics and flavonoids are shown in Table 2. Ferulic acid (14.63 ± 0.03 mg/g), cumaric acid (11.65 ± 0.03 mg/g), quercetrin (10.96 ± 0.02 mg/g), caffeic acid (9.16 ± 0.01 mg/g) and ellagic acid (9.03 ± 0.02 mg/g) were detected as major compounds. Whereas, catechin (4.51 ± 0.01 mg/g), epicatechin (5.78 ± 0.01 mg/g), gallic acid (4.38 ± 0.02 mg/g), quercetin (5.81 ± 0.01), isoquercetrin (3.98 ± 0.01 mg/g), gallic acid (4.38 ± 0.02 mg/g) and chlorogenic acid (3.27 ± 0.02 mg/g) relatively showed minor contribution. This is the first comprehensive phytochemical study of sugarcane leaves. Earlier studies have shown the presence of phenolics, luteolin 8-C-rhamnosylglucoside, diosmetin-8-glucoside, vitexin, orientin, tricin-7-O-neohesperidoside and tricin derivatives in the juice of sugarcane (Fabiana et al., 2008). Lee, Chen, Yu, Wang, and Duh (2012) reported the presence of flavonoids, benzoic acid, chlorogenic acid, caffeic acid and vitexin in leaves of different genotypes of sugarcane. The observed antioxidant activity and protecting ability of sugarcane against DNA damage is due to the phenolic acids (ferulic acid, gallic acid, chlorogenic acid, caffeic acid and cumaric acid) and flavonoids (quercetrin, catechin, epicatechin, rutin and quercetin). The exogenous antioxidants from sugarcane extracts may act directly or indirectly with the internal antioxidant system for synergetic effects to protect several diseases linked to free radicals such as heart diseases, neurodisorders and other stress related disorders. 4. Conclusions In conclusion the results of present study revealed that the different cultivars of sugar cane are rich source of antioxidants and
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