Antioxidant and Nitrite-Scavenging Capacities of Phenolic Compounds from Sugarcane (Saccharum officinarum L.) Tops

Transcription

1 Moleules 2014, 19, ; doi: /moleules OPEN ACCESS moleules ISSN Artile Antioxidant and Nitrite-Savenging Capaities of Phenoli Compounds from Sugarane (Saharum offiinarum L.) Tops Jian Sun 1, *, Xue-Mei He 2,3, Mou-Ming Zhao 1, *, Li Li 2,3, Chang-Bao Li 2,3 and Yi Dong College of Light Industry and Food Sienes, South China University of Tehnology, Guangzhou , China; waydongyi @163.om Agro-food Siene and Tehnology Researh Institute, Guangxi Aademy of Agriultural Sienes, Nanning , China; s: xuemeihe1981@126.om (X.-M.H.); lili@gxaas.net (L.L.); hangaoli@gxaas.net (C.-B.L.) Guangxi Crop Geneti Improvement Laoratory, Nanning , China * Authors to whom orrespondene should e addressed; s: jiansun@gxaas.net (J.S.); femmzhao@sut.edu.n (M.-M.Z.); Tel.: (M.-M.Z.); Fax: (M.-M.Z.). Reeived: 4 July 2014; in revised form: 15 August 2014 / Aepted: 15 August 2014 / Pulished: 26 August 2014 Astrat: Sugarane tops were extrated with 50% ethanol and frationated y petroleum ether, ethyl aetate (EtOA), and n-utyl alohol suessively. Eight phenoli ompounds in EtOA extrats were purified through silia gel and Sephadex LH-20 olumn hromatographies, and then identified y nulear magneti resonane and eletrospray ionization mass spetra. The results showed that eight phenoli ompounds from EtOA extrats were identified as affei aid, is-p-hydroxyinnami aid, queretin, apigenin, alanin A, australone A, morain M, and 5'-geranyl-5,7,2',4'-tetrahydroxyflavone. The antioxidant and nitrite-savenging apaities of different solvent extrats orrelated positively with their total phenoli (TP) ontents. Amongst various extrats, EtOA extrats possessed the highest TP ontent and presented the strongest oxygen radial asorane apaity (ORAC), 1,1'-diphenyl-2-pirylhydrazyl (DPPH) radial-savenging apaity, 2,2'-azois-3-ethylenthiaazoline-6-sulfoni aid (ABTS) radial-savenging apaity, ferri reduing antioxidant power (FRAP) and nitrite-savenging apaity. Thus, sugarane tops ould e promoted as a soure of natural antioxidant. Keywords: sugarane tops; phenoli ompounds; identifiation; antioxidant apaity; nitrite-savenging apaity

2 Moleules 2014, Introdution Free radials and other reative oxygen speies are produed y oxidative metaolism ontinuously in vivo, resulting in ell ageing, ell death, and tissue damage [1]. With inreasing evidenes showing the involvement of oxidative stress indued y free radials in the development of various diseases suh as skin ageing, atheroslerosis, diaetes, aner and irrhosis [2,3], it has een argued that exess antioxidants may impair signaling of reative oxygen and prodution of lipid oxidation produts [4]. Antioxidants, e.g., phenoli ompounds, are enefiial for postponing ageing, preventing disorder, reduing disease risk and maintaining health y inhiition of lipid peroxidation [4,5]. The ondition of oxidative stress ours when the pro-oxidant/anti-oxidant alane hange in favor of the pro-oxidant, as a result of an overprodution of free radials, suh as the peroxil, alkoxyl, and hydroxyl radials. The inrease of free radials at ellular level leads to DNA damage, protein oxidization and lipid peroxidation, and susequently to ell death via apoptosis or via nerosis. Convining evidene has demonstrated that oxidative stress is involving in the physio-pathologial asis of many proesses, inluding neurodegenerative diseases, ardiovasular diseases, aner, inflammation, and aging. Free radials an result in geneti alterations of ertain ells, thus inreasing the risk of these diseases [6]. Phenoli ompounds exist naturally in vegetales, fruits and grains. These ompounds possess the aility to redue oxidative damage eause they an at as diret antioxidant y donating a hydrogen atom to free radials and y helating metal ions, suh as iron or opper, as well as they an at as indiret antioxidants y upregulating antioxidant enzymes [6,7]. These antioxidant properties of phenoli ompounds are diretly related to their hemial struture, and partiularly to the phenol group [6]. Phenoli ompounds are of interest in pharmaeutial and food industries. Their pharmaologial ations are asried to the free radial savenging and metal ion helating ativities, and their effets on pathways of ell-signaling and on gene expression [8]. The antioxidant apaities of phenoli ompounds are often assessed y the Trolox equivalent antioxidant apaity (TEAC), the ferri reduing antioxidant power (FRAP), the hypohlorite savenging apaity, the deoxyriose method and the opper-phenanthroline-dependent DNA oxidation assays. The multidimensional effiaies of phenoli ompounds differ depending on the mehanism of antioxidant ation in diverse experimental systems. Syntheti phenoli ompounds, e.g., utylated hydroxyanisole (BHA), have een restrited in food industry for arinogeni possiility. Hene, natural antioxidants have een extensively investigated in reent years [9]. Sugarane (Saharum offiinarum L.) is one of the important rops in sutropial and tropial areas. Some varieties are onsumed as fruits. Sugarane is a prinipal sugar soure for the food industry. Approximately 70% of the sugar produed gloally omes from sugarane. Sugarane tops, yielding aout 15% of the total sugarane yield [10], are good soures for natural antioxidants. They are often thrown away, left to rot, urnt, used as forage, or produed into everages. It was reported reently that sugarane tops possessed higher flavonoid ontents than sugarane stems [11]. Although sugarane tops ontain plentiful phenoli ompounds, they have still not een developed and utilized adequately up until now. We reported herein the isolation and strutural identifiation of eight phenoli ompounds (Figure 1) in sugarane tops. Oxygen radial asorane apaity, DPPH radial-savenging apaity, ABTS radial-savenging apaity, ferri reduing antioxidant power and nitrite-savenging apaity of different extrats from sugarane tops were also determined. The results otained will e useful for indiating potential ioativities and

3 Moleules 2014, health enefits of sugarane tops whih an e utilized as a new soure of natural antioxidant in the food industry. These data are also important to illuminate that sugarane top phenolis possess an antioxidant protetion role, whih an postpone ageing and senesing. Figure 1. Phenoli ompounds isolated from EtoA extrat of sugarane tops. R O HO O R R 1 R 2 R 3 1 Caffei aid 3 Queretin H 2 Cis-p-hydroxyinnami aid H 4 Apigenin H H H 5 Alanin A isoprenyl H HO HO HO A O C R 2 R 1 R 3 B O O HO O 6 Australone A HO O O 7 Morain M 8 5'-Geranyl-5,7,2',4'-tetrahydroxy-flavone Figure 2. Total phenoli ontent in different extrats of sugarane tops. TE, PE, EE, BE and WE were the areviations of total extrat, petroleum ether extrat, EtOA extrat, n-bu extrat, and water extrat, respetively. Different letters horizontally (a d) indiated signifiant differenes (p < 0.05) of total phenoli ontent among different extrats aording to Dunan s multiple range test a TP ontent (mg GAE/g) d d 20 0 TE PE EE BE WE 2. Results and Disussion 2.1. Total Phenoli Content TP ontents of various extrats from sugarane tops are shown in Figure 2. Statistial analysis indiated that TP ontents were signifiantly (p < 0.05) affeted y the differene of extrating solvent. TP ontents ranged from ± 0.83 to ± 3.40 mg GAE/g extrats, and the order was as

4 Moleules 2014, follows: EtOA extrat > n-bu extrat > total extrat > petroleum ether extrat > water extrat. The highest TP ontent of EtOA extrat indiated that phenoli omponents in sugarane tops mainly dissolved in EtOA, whih was the optimum solvent for separating and enrihing phenoli ompounds from sugarane top extrats Identifiation of Major Phenoli Compounds in Sugarane Tops Eight phenoli ompounds (Figure 1) were isolated from EtOA extrat with the highest TP ontent. Their hemial strutures were identified as follows: Compound 1: pale yellow powder, mp C, ESI-MS m/z: 179 [M H]. 1 H-NMR (DMSO-d 6, 400 MHz) δ: 9.6 (1H, s, H-9), 7.44 (1H, d, J = 16.0 Hz, H-7), 7.08 (1H, d, J = 3.0 Hz, H-2), 6.99 (1H, dd, J = 3.0, 9.2 Hz, H-6), 6.65 (1H, d, J = 9.0 Hz, H-5), 6.17 (1H, d, J = 16.0 Hz, H-8); 13 C-NMR (DMSO-d 6, 100 MHz) δ: (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), (C-9). Compound 1 was identified as affei aid y omparing NMR and MS data with literature [12]. Compound 2: white needles (H 2 O), mp C, ESI-MS m/z: 164 [M+H] +, 147, 138, 121, 119, 93, 65. IR (KBr) ν max : 3400, 1675, 1606, 1511, 1445, 1240, 1211, 988 m 1. 1 H-NMR (DMSO-d 6, 400 MHz) δ: 7.66 (2H, d, J = 7.4 Hz, H-2, 6), 6.70 (2H, d, J = 7.4 Hz, H-3, 5), 6.62 (1H, d, J = 12.0 Hz, H-7), 5.68 (1H, d, J = 12.0 Hz, H-8); 13 C-NMR (DMSO-d 6, 100 MHz) δ: (C-1), (C-2, 6), (C-3, 5), (C-4), (C-7), (C-8), (C-9). Compound 2 was onfirmed as is-p-hydroxyinnami aid y NMR and MS data with literature [13]. Compound 3: yellow needles (Me), mp C, UV (Me) λ max (log ε): 258, 379. IR (KBr) ν max : 3420, 3370, 3289, 1672, 1615, 1520, 1432, 1360 m 1. ESI-MS m/z: 301 [M H]. 1 H-NMR (DMSO-d 6, 400 MHz) δ: (1H, s, -5), (1H, s, -7), 9.58 (1H, s, -4'), 9.35 (1H, s, -3'), 9.30 (1H, s, -3), 7.68 (1H, d, J = 1.5 Hz, H-2'), 7.55 (1H, dd, J = 9, 1.5 Hz, H-6'), 6.89(1H, d, J = 9 Hz, H-5'), 6.41(1H, d, J = 2 Hz, H-6), 6.19(1H, d, J = 2 Hz, H-6); 13 C-NMR (DMSO-d 6, 100 MHz) δ: 146 (C-2), 136 (C-3), 176(C-4), (C-4a), 157 (C-5), 98.1 (C-6), (C-7), 93.2 (C-8), (C-8a), (C-1'), (C-2'), (C-3'), (C-4'), (C-5'), (C-6'). Compound 3 was identified as queretin y the omparison of NMR and MS data with literature [14]. Compound 4: yellow needles (Me), mp C, ESI-MS m/z: 269 [M H]. IR (KBr) ν max : 3288, 1655, 1606, 1510 m 1. 1 H-NMR (DMSO-d 6, 400 MHz) δ: 7.99 (2H, dd, J = 8.4, 2.4 Hz, H-2', 6'), 6.94 (2H, dd, J = 8.4, 2.4 Hz, H-3', 5'), 6.88 (1H, s, H-3), 6.56 (1H, d, J = 2.4 Hz, H-8), 6.21 (1H, d, J = 2.4 Hz, H-6); 13 C-NMR (DMSO-d 6, 100 MHz) δ: (C-2), (C-3), (C-4), (C-4a), (C-5), 99.8 (C-6), (C-7), 94.3 (C-8), (C-8a), (C-1'), (C-2', 6'), (C-3', 5'), (C-4'). Compound 4 was identified as apigenin y omparing NMR and MS data with literature [15]. Compound 5: yellow powder, ESI-MS m/z: 353 [M H]. 1 H-NMR (DMSO-d 6, 400 MHz) δ: (1H, r s, 5-), 7.18 (1H, d, J = 8.4 Hz, H-6'), 6.56 (1H, d, J = 2.4 Hz, H-3'), 6.51 (1H, dd, J = 8.4, 2.4 Hz, H-5'), 6.31(1H, d, J = 3.2 Hz, H-8), 6.23 (1H, d, J = 3.2 Hz, H-6), 5.11 (1H, t, J = 5.6 Hz,

5 Moleules 2014, H-10), 3.1 (2H, d, J = 6.8 Hz, H-9), 1.56 (3H, s, H-13), 1.42 (3H, s, H-12); 13 C-NMR (DMSO-d 6, 100 MHz) δ: (C-2), (C-3), (C-4), (C-4a), (C-5), 99.4 (C-6), (C-7), 94.3 (C-8), (C-8a), 24.6 (C-9), (C-10), (C-11), 25.8 (C-12), 17.6 (C-13), (C-1'), (C-2'), (C-3'), (C-4'), (C-5'), (C-6'). Compound 5 was identified as alanin A y omparing NMR and MS data with literature [16]. Compound 6: yellow power, ESI-MS m/z: 419 [M H]. 1 H-NMR (Aetone-d 6, 400 MHz) δ: 7.85 (1H, d, J = 8.8 Hz, H-6'), 6.62 (IH, d, J = 1.7 Hz, H-3'), 6.55(1H, dd, J = 8.8, 1.7 Hz, H-5'), 6.71 (1H, d, J = 10 Hz, H-9), 5.71 (1H, d, J = 10 Hz, H-10), 6.46 (1H, s, H-8), 5.13 (1H, t, J = 7.2 Hz, H-15), (1H, m, H-14), (1H, m, H-13), 1.64 (3H, s, H-12), 1.57 (3H, s, H-17), 1.45 (6H, s, H-18); 13 C-NMR (Aetone-d 6, 100 MHz) δ: (C-2), (C-3), (C-4), (C-4a), (C-5), (C-6), (C-7), 95.3 (C-8), (C-8a), (C-9), (C-10), 81.1 (C-11), 27.0 (C-12), 42.2 (C-13), 23.7 (C-14), (C-15), (C-16), 18.0 (C-17), 25.7 (C-18), (C-1'), (C-2'), (C-3'), (C-4'), (C-5'), (C-6'). Compound 6 was identified as australone A y omparing NMR and MS data with literature [17]. Compound 7: rown powder, ESI-MS m/z: 241 [M H]. 1 H-NMR (DMSO-d 6, 400 MHz) δ: 7.38 (1H, d, J = 8.4 Hz, H-4), 7.06 (1H, s, H-3), 6.92 (1H, d, J = 2.0 Hz, H-7), 6.74 (1H, dd, J = 8.4, 2.0 Hz, H-5), 6.67 (2H, d, J = 3.2 Hz, H-2',6'), 6.22 (1H, t, J = 3.2 Hz, H-4'); 13 C-NMR (DMSO-d 6, 100 MHz) δ: (C-2), (C-3), (C-3a), (C-4), (C-5), (C-6), 97.4 (C-7), (C-7a), (C-1'), (C-2'), (C-3'), (C-4'), (C-5'), (C-6'). Compound 7 was identified as morain M y omparing NMR and MS data with literature [18]. Compound 8: yellow powder, UV (Me) λ max (log ε): (0.85), (0.51), (0.29), (0.59). IR (KBr) ν max : 3427, 2929, 1621, 1492, 1278, 1151, 1122, 1060, 968, 822 m 1. ESI-MS m/z: [M H]. 1 H-NMR (Aetone-d 6, 400 MHz) δ: (1H, r s, 5-), 7.70 (1H, s, H-6'), 7.10 (1H, s, H-3), 6.66 (1H, s, H-3'), 6.46 (IH, d, J = 2 Hz, H-8), 6.23 (1H, d, J = 2 Hz, H-6), 5.39 (1H, t, J = 7.3 Hz, H-2''), 5.13 (1H, t, J = 6.0 Hz, H-7''), 3.32 (2H, d, J = 7.6 Hz, H-1''), 2.14 (2H, m, H-6''), 2.06 (2H, m, H-5''), 1.76 (3H, s, H-4''), 1.60 (3H, s, H-10''), 1.57 (3H, s, H-9''); 13 C-NMR (Aetone-d 6, 100Hz) δ: (C-2), (C-3), (C-4), (C-4a), (C-5), 99.5 (C-6), (C-7), 94.6 (C-8), (C-8a), (C-1'), (C-2'), (C-3'), (C-4'), (C-5'), (C-6'), 28.1 (C-1''), (C-2''), (C-3''), 16.2 (C-4''), 40.5 (C-5''), 27.5 (C-6''), (C-7''), (C-8''), 25.8 (C-9''), 17.8 (C-10''). Compound 8 was identified as 5'-geranyl-5,7,2',4'- tetrahydroxy-flavone y omparing NMR and MS data with literature [16] Antioxidant Capaity ORAC Assay The antioxidant ativity of phenoli ompounds and their metaolites in vitro depends upon the arrangement of funtional groups aout nulear struture. Suffiient evidene support that the role of speifi strutural omponents are requisites for free radial savenging, metal helation and oxidant ativity [19]. Both onfiguration and total numer of hydroxyl groups sustantially influene several

6 Moleules 2014, mehanisms of antioxidant ativity. ORAC assay is a reent ut widely aepted analysis method for total antioxidant apaility eause it is more sensitive and effetive than other methods. ORAC values are also used as standard measures for omparing antioxidant ativity of food materials [20,21]. Total antioxidant potential of five sugarane top extrats was measured y ORAC assay (Figure 3A). Statistial analysis indiated that the ategory of extrating solvent signifiantly affeted ORAC values whih varied from 4.76 ± 1.09 μm TE/mg to ± 3.8 μm TE/mg. EtOA extrat possessed the highest (p < 0.05) ORAC value, followed y water extrat, petroleum ether extrat, n-bu extrat, and total extrat. EtOA extrat exhiiting the highest TP ontent and ORAC value (Figures 2 and 3A) suggested that ORAC value was positively related to TP ontent. This ratioination was onfirmed through Tale 1, in whih the orrelation oeffiient of TP ontent and ORAC was Figure 3. Antioxidant ailities of different extrats of sugarane tops. ORAC assay (A), DPPH radial-savenging apaities assay (B), ABTS radial-savenging apaities assay (C) and FRAP assay (D). TE, PE, EE, BE, and WE were the areviations of total extrat, petroleum ether extrat, EtOA extrat, n-bu extrat and water extrat, respetively. Different letters horizontally (a e) indiated signifiant differenes (p < 0.05) of antioxidant ailities among different extrats aording to Dunan s multiple range test. 140 a A 500 B a ORAC value (um TE/mg) DPPH (um TE/mg) d 0 TE PE EE BE WE 0 TE PE EE BE WE 1200 a C 1800 a D ABTS value (um TE/mg) d FRAP value(um TE/ mg) d e TE PE EE BE WE TE PE EE BE WE Tale 1. Correlation analysis of total phenoli ontent and antioxiant ailities. TP Content ORAC FRAP Correlation oeffiient Signifiane (ilateral) DPPH Savenging Capaities ABTS Savenging Capaities Nitrite-Savenging Capaities

7 Moleules 2014, DPPH and ABTS Radial Savenging Capaities Oxidative damage aused y free radials may e related to aging and diseases suh as atheroslerosis, diaetes, aner, and irrhosis. Phenoli ompounds an effiiently ath hydrogen atom and provide hydrogen atom to free radials through phenoli oxhydryl, and then phenoli ompounds transform into stale phenoxy radial to inhiit oxidant development [22,23]. Total free radial-savenging apaities of five extrats from sugarane tops were measured using ommerially availale stale free radials DPPH (Figure 3B) and ABTS (Figure 3C). All five extrats presented DPPH radial-savenging apaity to some extent. Amongst them, EtOA extrat showed the strongest savenging aility, followed y n-bu extrat, petroleum ether extrat, water extrat, and total extrat. DPPH radial savenging values varied from 56.2 ± 7.7 μm TE/mg to ± 12.4 μm TE/mg. The order of ABTS radial-savenging apaity was EtOA extrats > n-bu extrats > petroleum ether extrats > total extrats > water extrats. Both DPPH and ABTS radial-savenging apaities were signifiantly (p < 0.05) orrelated with TP ontent (Tale 1), indiating that the main hemial sustanes for savenging free radial in sugarane tops were phenoli omponents. The similar result was found in sugarane juie y Kadam et al. [24] who reported that EtOA extrat from sugarane juie exhiited the high TP ontent and the strong free radial savenging apaity. Phenoli omponents from sugarane tops, ontaining more hydroxyl groups, exhiited very high aility to savenge DPPH and ABTS radials. The hemistry of phenoli omponents from sugarane tops is preditive of their free radial savenging ativity eause the redution potentials of phenoli radials are lower than those of DPPH and ABTS radials, implying that phenoli ompounds may not only deativate these oxyl speies, ut also inhiit deleterious onsequenes of the reations [25] FRAP Assay In FRAP assay, antioxidant power refers as reduing aility whih is measured y potassium ferriyanide redution method. EtOA extrat had the highest FRAP value, followed y n-bu extrat, water extrat, total extrat, and petroleum ether extrat (Figure 3D). FRAP value varied from ± 17.6 μm TE/mg to ± 32.3 μm TE/mg. FRAP value was signifiantly (p < 0.05) orrelated with TP ontent (Tale 1). It was reported that reduing aility was generally assoiated with the presene of redutones. The antioxidant ation of redutones was ased on the reaking of the free-radial hain y donating a hydrogen atom. The phenoli omponents of sugarane tops extrat may at as redutones y donating eletrons, reating with free radials to onvert them to more stale produts and terminating the free radial hain reation [26] Nitrite-Savenging Capaity Nitrite-savenging apaities of different extrat from sugarane tops are shown in Figure 4. Statistial analysis showed that the ategory of extrating solvent signifiantly influened nitrite-savenging apaity. EtOA extrat presented the highest (p < 0.05) nitrite-savenging ratio among five extrat, followed y n-bu extrat, total extrat, petroleum ether extrat, and water extrat. The nitrite-savenging ratio of EtOA extrat approahed to that of vitamin C, indiating that EtOA extrat of sugarane tops was proaly a good nitrite-savenging additive for the food industry.

8 Moleules 2014, From Tale 1, nitrite-savenging apaity was signifiantly related to TP ontent (p < 0.05), suggesting that phenoli omponents played an important role in savenging nitrite. This finding was similar to the previous report y Liu et al. [27], who found that the flavonoid-enrihed extrat of Maydis stigma had signifiant savenging aility on nitrite. Phenoli ompounds ould inhiit the formation of N-nitroso-dimethylamine due to their hydroxyl groups. At low ph, the nitrite was onverted to nitrous aid and susequently to N 2 O 3, whih ould e rapidly redued to NO y phenoli ompounds that existed in sugarane tops. Consequently, the formation of N-nitrosamine was inhiited, and the nitrite was also savenged. Figure 4. Nitrite-savenging apaity of different extrats of sugarane tops. TE, PE, EE, BE, and WE were the areviations of total extrat, petroleum ether extrat, EtOA extrat, n-bu extrat, and water extrat, respetively. Different letters horizontally (a d) indiated signifiant differenes (p < 0.05) of nitrite-savenging apaity among different extrats aording to Dunan s multiple range test. 100 Ratio of nitrite-savenging(%) d d d a 0 TE PE EE BE WE V 3. Experimental Setion 3.1. Plant Materials Sugarane variety of ROC22 (one of mainly ultivated variety in China) was hosen as plant material. Sugarane materials were otained from a ane experimental ase in Guangxi Sugarane Researh Institute, Guangxi Aademy of Agriultural Sienes on 20 Novemer, The healthy sugarane tops were ut, sun-dried and rushed into powder. The dried powdered plant material was stored at room temperature (25 C) in a desiator until further analysis Chemials and Reagents Asori aid, ABTS diammonium salt, DPPH, fluoresein (FL), Folin Cioalteu reagent, trolox (6-hydroxy-2,5,7,8-tetramethylhroman-2-aroxyli aid), and 2,2'-azois (2-amodinopropane) dihydrohloride (AAPH) were purhased from Sigma Chemial Co. (St. Louis, MO, USA). Other reagents were of analytial grade.

9 Moleules 2014, Preparation of Sugarane Tops Extrats The dry powder of sugarane tops (4.1 kg) was extrated with 50% ethanol (Et) with the solid/liquid rate of 1/10 (m:v) for 90 min at 70 C. Et extrat was evaporated to dryness y a RE52AA rotary evaporator (Yarong Equipment Co., Shanghai, China) under redued pressure at 50 C. The residue was dissolved in water and frationated y petroleum ether. Petroleum ether-solule fration was olleted and the residue was extrated y ethyl aetate (EtOA). EtOA-solule fration was olleted and the residue was further extrated n-utyl alohol (n-bu). Petroleum ether-solule fration, EtOA-solule fration, n-bu-solule fration, and water-solule fration were otained. All the frations were onentrated separately under redued pressure and freeze-dried to get petroleum ether extrat, EtOA extrat, n-bu extrat, and water extrat. The rude 50% Et extrat was expressed as total extrat. Five different extrats (1 mg) were preisely weighed, dissolved in 1 ml of 50% Et, and then diluted for further analysis Determination of Total Phenoli Content Total phenoli (TP) ontent in sugarane tops extrats was measured y the method of Dorman et al. [28]. TP ontent was alulated aording to the standard urve for galli aid solutions and expressed as milligrams of galli aid equivalents per gram different sugarane tops extrats (mg GAE/g) Isolation and Identifiation of Major Phenoli Compounds EtOA extrat (38 g, this extrat possessed the highest TP ontent) from sugarane tops was purified using silia gel olumn hromatography (900 g, mesh), eluting with gradient mixtures of hloroform-methanol (100:0 100:100, v/v) to otain 10 frations (A J). Fration C (8 g) was separated y silia gel olumn (100 g, mesh) using petroleum ether-aetone (100:0 70:30, v/v) as eluents to afford 8 frations (C1 C8). Fration C2 and C5 was further sujeted to Sephadex LH-20 olumn hromatography eluting with methanol to afford ompound 1 (4 mg) and ompound 2 (6 mg). Fration D (3.4 g) was purified using Sephadex LH-20 olumn eluting with hloroform-methanol (1:1, v/v) to give ompound 3 (5 mg), ompound 4 (7 mg) and ompound 5 (5 mg). Fration E (7.2 g) was sujeted to silia gel olumn hromatography (100 g, mesh) eluting with hloroform-methanol (9:1, v/v) to give 7 frations (E1 E7). Fration E2 (1.2 g) was further purified using Sephadex LH-20 olumn eluting with methanol to give ompound 6 (8 mg), ompound 7 (5 mg), and ompound 8 (4 mg). The hemial strutures of isolated phenoli ompounds were identified y nulear magneti resonane (NMR) spetrosopy, eletrospray ionization mass spetra (ESI-MS), and infrared (IR) spetrosopy. NMR spetra were measured on a Bruker AV-400 spetrometer (Bruker Biospin, Rheinstetten, Germany) ( 1 H-NMR, 400 MHz; 13 C-NMR, 100 MHz). Chemial shift values (δ) were reorded in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal standard. ESI-MS spetra were reorded on a Finnigan LCQ Advantage Max ion trap mass spetrometer (Finnigan MAT, San Jose, CA, USA). IR spetra were determined on a Jaso FT/IR-480 plus Fourier

10 Moleules 2014, transform (Jaso, Tokyo, Japan). Melting points (mp) were reorded on a XT-4 miro melting point apparatus (Beijing Teh Instrument Co. Ltd, Beijing, China) without orretion Antioxidant and Nitrite-Savenging Capaity Assays Oxygen Radial Asorane Capaity ORAC was determined y the modified methods of Cao and Prior [29], Ana et al. [30], and Lin et al. [31]. The assay was arried out on a Varioskan Flash spetral san multimode plate reader (Thermo Fisher Sientifi, Thermo Eletron Co., Waltham, MA, USA), using 96-well polystyrene white miroplates with an exitation wavelength of 485 nm and an emission wavelength of 530 nm. The fluoresein sodium salt (FL) stok solution (39.9 μm) was kept at 4 C in the dark, and was diluted to μm fresh FL working solution. A total of g of AAPH was aurately weighed and made into a mm solution, whih was kept in an ie ath. All aove hemials were dissolved in 75 mm sodium phosphate uffer (ph 7.4). A total of 25 μl of sample (different solvent extrats) and 25 μl of 75 mm sodium phosphate uffer (lank) were added in a well of 96-well mirotitre, and then 75 μl of FL working solution was added. The mixture was preinuated for 10 min at 37 C. Finally, 100 μl of AAPH solution was added rapidly. The miroplate was plaed in the reader and automatially shaken prior to eah reading. Fluoresene was measured every minute for 70 min. Trolox solution ( μm) was used as positive ontrol and measured in every assay. ORAC value was quantified using the regression equation etween Trolox onentration and net area under urve (AUC). The ORAC values, expressed as μm trolox equivalents (μm TE/mg) were alulated y applying the following formula: AUC = 0.5(f 0 + f n ) + (f 1 + f f i + + f n 1 ) where f 0 was initial FL reading at time 0 and f i was FL reading at time i. Net AUC was alulated as AUC sample AUC lank DPPH Radial-Savenging Capaity DPPH radial-savenging apaity was determined aording to the modified method of Luo et al. [32] and expressed as Trolox equivalent. Two milliliters of sample were added into 2 ml of 0.2 mm DPPH solution (in 50% ethanol). The asorane at 517 nm of the mixture was measured after 30 min of inuation at 25 C. The asorane of lank and ontrol were otained y replaing DPPH solution or sample with ethanol, respetively. DPPH radial savenging ratio (%) was alulated as [1 (A sample A lank )/A ontrol ] 100. Where, A sample was asorane of sample, A lank was asorane of lank and A ontrol was asorane of ontrol. DPPH radial savenging value was determined using a standard urve of Trolox ( mm) and the results were expressed as miromolar Trolox equivalent per milligram extrats (μm TE/mg).

11 Moleules 2014, ABTS Radial-Savenging Capaity ABTS radial-savenging apaity was determined aording to the modified method of Roerta et al. [33]. This method was ased on the apaity of antioxidant to inhiit ABTS radial ation (ABTS + ) ompared with Trolox. ABTS + stok solution was produed y dissolving ABTS in 2.45 mm potassium persulfate solution and keeping the mixture in the dark for h at room temperature. The stok solution was diluted using 10 mm sodium phosphate uffer (ph 7.4) to otain the working solution with an asorane of 0.7 at 734 nm. A total of 40 μl of sample was mixed with 4 ml of ABTS + working solution for 30 s efore measurement at 734 nm. ABTS + savenging ratio (%) was alulated as (1 A sample /0.7) 100. ABTS + savenging value was determined using a standard urve of Trolox (0 1 mm) and the results were expressed as miromolar Trolox equivalent per milligram extrats (μm TE/mg) Ferri-Reduing Antioxidant Power FRAP was onduted aording to the modified method of Jayaprakasha et al. [34]. One milliliter of sample, 2.5 ml of 0.2 M sodium phosphate uffer (ph 6.6) and 2.5 ml of 1% potassium ferriyanide were mixed in a 10 ml test tue. After inuation for 20 min at 50 C, 2.5 ml of 10% trihloroaeti aid was added into the mixture. Two milliliters of upper layer were taken out and mixed with 2 ml of distilled water and 0.1% ferri hloride. The asorane was measured at 700 nm after 10 min. A high asorane of reation mixture indiated a strong reduing power. The reduing power value of sample was expressed as miromolar Trolox equivalent per milligram extrats (μm TE/mg) Nitrite-Savenging Capaity Assay Nitrite-savenging apaity was evaluated y the method of hydrohlori aid naphthalene ethylenediamine oloration. One milliliter of sample or 1 ml of 50% ethanol (lank) was mixed with 1 ml of 5 mg/l nitrite solution and 1 ml of itri aid uffer (ph 3). After reating for 30 min at 37 C, 1 ml of 4 g/l amino enzene sulfoni aid sodium (in 20% hydrohlori) was added in the mixture, and then 0.5 ml of 2 g/l hydrohlori aid naphthalene ethylenediamine (in water) was also added after 3 min. The mixture was reated for 15 min and measured at 538 nm. Asori aid was used as positive ontrol. Nitrite-savenging ratio (%) = (1 A sample /A lank ) Statistial Analysis All experiments were performed in tripliate (n = 3) and the results were expressed as mean ± standard deviation. Statistial analysis was arried out y SPSS 18 statistial software (SPSS In., Chiago, IL, USA). A differene was onsidered statistially signifiant when p < Conlusions Eight phenoli ompounds were isolated from EtOA extrat of sugarane tops. The effets of different extrats (i.e., total extrat, petroleum ether extrat, EtOA extrat, n-bu extrat and water

12 Moleules 2014, extrat) on iologial ativities (antioxidant and nitrite savenging apaities) were investigated in vitro. Amongst them, EtAO extrat from sugarane tops possessed the strongest antioxidant and nitrite savenging apaities. Further investigation will e performed to study the orrelation etween antioxidant ativity and hemial struture of sugarane top phenolis. Antioxidant and nitrite savenging apaities of individual phenoli ompounds from sugarane tops will e determined in following researh. The funtional produts will e also explored from sugarane tops. Aknowledgments The authors appreiate the finanial supports from National Projet for Introdution of Foreign Tehnology and Personnel Management (grant no. S ), Tehnology Foundation for Seleted Overseas Chinese Sholar, Ministry of Personnel of China (grant no. [2012]250), Guangxi Key Laoratory Constrution Projets (grant no ), Guangxi Sientifi Researh and Tehnologial Development Projets (grant no. Gui Ke He ; Gui Ke Gong ), and Fundamental Researh Funds from Gangxi Aademy of Agriultural Sienes (grant no. 2013YT02). Author Contriutions Jian Sun and Mou-Ming Zhao onduted the experimental design. Jian Sun and Xue-Mei He arried out the experiment and prepared the manusript. Jian Sun and Li Li revised and approved the final version of the manusript. Chang-Bao Li and Yi Dong ontriuted helpful disussion and sientifi advie during the preparation of manusript. Conflits of Interest The authors delare no onflit of interest. Referenes 1. Takashima, M.; Horie, M.; Shihiri, M.; Hagihara, Y.; Yoshida, Y.; Niki, E. Assessment of antioxidant apaity for savenging free radials in vitro: A rational asis and pratial appliation. Free Radi. Biol. Med. 2012, 52, Suryo Rahmanto, A.; Pattison, D.I.; Davies, M.J. Photo-oxidant-indued inativation of the selenium-ontaining protetive enzymes thioredoxin redutase and glutathione peroxidase. Free Radi. Biol. Med. 2012, 53, Choi, D.B.; Cho, K.-A.; Na, M.-S.; Choi, H.-S.; Kim, Y.-O.; Lim, D.-H.; Cho, S.J.; Cho, H. Effet of amoo oil on antioxidative ativity and nitrite savenging ativity. J. Ind. Eng. Chem. 2008, 14, Niki, E. Antioxidant apaity: Whih apaity and how to assess it? J. Berry Res. 2011, 4, Niki, E. Do antioxidants impair signaling y reative oxygen speies and lipid oxidation produts? FEBS Lett. 2012, 586, Sun, J.; Nagendra, P.K.; Amin, I.; Yang, B.; You, X.; Li, L. Polyphenols: Chemistry, Dietary Soures and Health Benefits; Nova Siene Pulishers: New York, NY, USA, 2013; pp

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