Antioxidant Activities and Contents of Flavonoids And

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Available online at www.sciencedirect.com 65 66 1 ScienceDirect 67 2 68 3 69 4 70 5 journal homepage: www.jfda-online.com 71 6 72 7 73 8 74 9 Original Article 75 10 76 11 Antioxidant activities and contents of flavonoids 77 12 78 13 and phenolic acids of Talinum triangulare extracts 79 14 80 15 81 16 and their immunomodulatory effects 82 17 83 18 a b c b 84 19 Q19 Dung Y. Liao , Yu C. Chai , Sue H. Wang , Chih W. Chen , 85 * 20 Ming Shiun Tsai a, 86 21 87 a 22 Department of Bioindustry Technology, Da-Yeh University, Dacun, Changhua, Taiwan 88 23 b Department of Health Food, Chung Chou University of Science and Technology, Yuanlin, Changhua, Taiwan 89 24 c Department of Biomedical Sciences, Chung Shan Medical University, Taichung City, Taiwan 90 25 91 26 92 27 article info abstract 93 28 94 29 95 Q1 Article history: In this study, leaves and stems of Talinum triangulare were sequentially extracted with 30 96 Received 19 April 2014 phosphate buffer solution to obtain PTL and PTS (phosphate buffered extracts of T. trian- 31 97 Received in revised form gulare leaves and stems), with 75% ethanol to obtained ETL and ETS (ethanol extracts of T. 32 98 14 July 2014 triangulare leaves and stems), or with 90C boiling water to obtain WTL and WTS (water 33 99 34 Accepted 17 July 2014 extracts of T. triangulare leaves and stems). We investigated the antioxidant activities of 100 ' 35 Available online xxx various T. triangulare extracts, analyzed the extracts stimulations on human mononuclear 101 36 cell (MNC) growth and secretion of cytokines (interleukin-1 beta, interferon-g, and tumor 102 37 Keywords: necrosis factor-a) and nitric oxide, and then assayed their subsequent inhibitions on human 103 38 antioxidant activities leukemic U937 cell growth. Results indicated that extracts of T. triangulare showed significant 104 39 human leukemic U937 cells antioxidant activities. Among these extracts, WTS showed the highest stimulatory effect on 105 40 human MNC growth. The secretion levels of interleukin-1 beta, interferon-g, and tumor 106 41 human mononuclear cells immunomodulatory activity necrosis factor-a in the conditioned medium, wherein human MNC was treated with 500 mg/ 107 42 108 Talinum triangulare extracts mL WTS for 72 hours, were 1275, 859, and 2222 pg/mL, respectively. All conditioned media 43 109 obtained from human MNCs cultured with various T. triangulare extracts showed significant 44 110 T. triangulare 45 inhibition against U937 cell growth of over 40%. These results suggest that 111 46 extracts may be used in health foods for their immunomodulatory potential. 112 47 Copyright © 2015, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan 113 48 LLC. All rights reserved. 114 49 115 50 116 51 117 52 118 53 America, and much of South America. Its common names 119 54 1. Introduction include Waterleaf, Cariru, Surinam purslane, Philippine 120 55 121 56 spinach, Ceylon spinach, Florida spinach, Potherb fame Talinum triangulare (Jacq.) is also called Talinum fruticosum (L.) flower, Lagos bologi, and Sweetheart [1e3]. T. triangulare is 122 57 123 Juss. It is native plant in Mexico, the Caribbean, the Central widely grown in tropical regions as a leaf vegetable. 58 124 59 125 60 * Corresponding author. Department of Bioindustry Technology, Da-Yeh University, Number 168, University Road, Dacun, Changhua 126 61 51591, Taiwan. 127 62 E-mail address: [email protected] (M.S. Tsai). 128 63 http://dx.doi.org/10.1016/j.jfda.2014.07.010 129 64 1021-9498/Copyright © 2015, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan LLC. All rights reserved.

Please cite this article in press as: Liao DY, et al., Antioxidant activities and contents of ﬂavonoids and phenolic acids of Talinum triangulare extracts and their immunomodulatory effects, Journal of Food and Drug Analysis (2015), http://dx.doi.org/10.1016/ j.jfda.2014.07.010 JFDA188_proof ■ 5 January 2015 ■ 2/9

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1 Originating from the tropical Africa as a terrestrial herbaceous WTS (water extracts of T. triangulare leaves and stems, 66 2 plant, it is now widely cultivated as a medicinal and food crop respectively). All samples were stored at 80C until use. 67 3 68 in India, South America, other parts of Asia, and Nigeria [4e6]. 4 69 Several articles have shown that T. triangulare can increase 2.1.2. Reagents 5 70 stamina and function as an immunostimulant [7,8].In Roswell Park Memorial Institute (RPMI) 1640 and fetal bovine 6 71 7 Taiwan, T. triangulare has been used in the treatment and serum (FBS) were purchased from Hyclone (Logan, UT, USA). 72 8 prevention of hepatic ailments and cancer in folk medicine. Sodium bicarbonate, HEPES, L-glutamine, and sodium pyru- 73 9 Some vegetable extracts have been found to exhibit func- vate were purchased from Sigma Chemical Co. (St. Louis, MO, 74 10 tional uses; in certain cases, they function as antioxidants USA). Trypan blue was obtained from e-Bioscience (CA, USA). Q2 75 e 11 [5,9 12], angiotensin I-converting enzyme inhibitors [13,14], Potassium ferricyanide (K3Fe(CN)6), Trolox, a,a-diphenyl-b- 76 12 antibiotics [15], and cancer cells' inhibitors [16e18]. Many picrylhydrazyl (DPPH), 2,2-azinobis-(3-ethylbenzthiazoline-6- 77 13 human diseases are known to be related to free radicals. The sulfonate) (ABTS), EDTA, and a-tocopherol were also pur- 78 14 free radical scavenging medicines are antioxidants in nature. chased from Sigma Chemical Co. 79 15 80 Hence, a relationship exists between human health and minor 16 81 nutrients exhibiting antioxidant activities, such as vitamin C, 2.1.3. Flavonoids and phenolic acid contents in different T. 17 82 b 18 vitamin E, -carotene, flavonoids, and other antioxidants [19]. triangulare extracts 83 19 Furthermore, numerous studies (epidemiological, case- T. triangulare extracts were analyzed for flavonoids and 84 20 control, or prospective and retrospective cohort) related to phenolic acid compositions via high-performance liquid 85 21 dietary antioxidant intake have been linked to reducing can- chromatography (L-2130; Hitachi Co., Japan) using an RP- Q3 86 22 cer risks [17,20]. 18GP250 column (L, 250 mm; ID, 4.6 mm) and UV/Vis de- 87 23 In investigating antioxidant activities, studies on vegeta- tectors. Flavonoids and phenolic acid were detected at 280 nm 88 24 bles' effects in immunomodulation and cancer-cell-growth and separated using a linear gradient elution program with a 89 25 inhibition may also yield a deeper insight into their func- mobile phase containing solvent A (10% methanol with 0.05% 90 26 91 tionality. Vegetable extracts with high antioxidant activities formic acid) and solvent B (70% methanol with 0.05% formic 27 92 can also be used for food preservation. Therefore, in this acid). The gradient program is shown in Table S1. The flow Q4 28 93 study, we investigated the extracts of T. triangulare for their rate used was set at 1.0 mL/min throughout the gradient. 29 94 30 antioxidant activities as well as their inhibitory effects on the Identification and quantification were accomplished by 95 31 growth of human leukemic U937 cells, by using an indirect comparing the retention time of peaks in the methanol- 96 32 model and studying their immunomodulatory activities. containing solutions to that of the standard compounds. 97 33 98 34 99 35 2.2. Antioxidant activities 100 36 2. Materials and methods 101 37 2.2.1. Reducing power 102 38 103 2.1. Materials A method developed by Chiang and Chang [19] for a reducing 39 104 power test was used. In brief, sample solutions, a-tocopherol, 40 105 41 2.1.1. T. triangulare samples and butylated hydroxyanisole methanolic solutions (positive 106 42 Fresh T. triangulare was obtained from Chungliao (Nantou, control group) were spiked with 2.5 mL of phosphate buffer 107 43 Taiwan). T. triangulare samples were separated into leaf and and 2.5 mL of 1% potassium ferricyanide. Mixtures were kept 108 44 stem, cleaned and immediately stored at 20 C overnight, in a 50 C water bath for 20 minutes, cooled by placing it in 20 C 109 45 then lyophilized for 48 hours. Following lyophilization, the water bath for 5 minutes, spiked with 2.5 mL of 10% tri- 110 46 dried samples were crushed to 30 mesh powders. The dried chloroacetic acid, and then centrifuged at 800 g for 10 mi- 111 47 powders were stored at 20C in darkness. nutes. The supernatant (5 mL) was mixed with 5 mL of 112 48 The extracts were prepared using the methods described distilled water and 1 mL of 0.1% ferric chloride. The absor- 113 49 114 by Nagai and Inoue [21] and Guo et al [22] with modifications. bance at 700 nm was then detected with a spectrophotometer 50 115 Three grams of leaf and stem powders was suspended and after reaction for 10 minutes; higher absorbance (A ) repre- 51 700 116 extracted with 30 volumes of 10 mM of sodium phosphate sents stronger reducing power. 52 117 53 buffer (pH 7.0) then shaken at 4 C for 24 hours. Extracts were 118 54 centrifuged at 4000 g for 30 minutes, and the supernatants 2.2.2. DPPH radical scavenging activity 119 55 were pooled, lyophilized, and denoted as PTL (phosphate The scavenging effect on DPPH free radical was measured 120 56 buffered extract of T. triangulare leaf) or PTS (phosphate buff- following the method described by Shimada et al [23] with 121 57 ered extract of T. triangulare stem). The residues were extrac- some modifications. Sample solutions (5 mL) and a-tocopherol 122 58 ted with 30 mL 75% ethanol at 4C for 24 hours. Extracts were methanolic solutions (positive control group) were added to 123 59 centrifuged at 4000 g for 30 minutes, and the supernatants 1 mL of 1 mM DPPH in methanolic solution. The mixture was 124 60 were pooled, lyophilized, and denoted as ETL (ethanol extract shaken and left to stand for 30 minutes at room temperature. 125 61 126 of T. triangulare leaves) and ETS (ethanol extract of T. triangu- The absorbance rates of the resulting solution and the positive 62 127 lare stems). Three grams of leaf and stem powders was control group was measured at 517 nm. The lower absorbance 63 128 64 extracted with distilled water at 90 C for 2 hours. The extracts (A517) represents a higher DPPH scavenging activity, which is 129 e 65 were centrifuged at 4000 g for 30 minutes, and the super- expressed as [1 (test sample absorbance/blank sample 130 natants were pooled, lyophilized, and denoted as WTL and absorbance)] 100%.

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1 2.2.3. Ferrous ion chelating ability were filtered to obtain condition media (CMs). The concen- 66 2 The method described by Decker and Welch [24] was adopted. trations of cytokines including interleukin-1 beta (IL-1b), 67 3 68 Five milliliters of the test solutions, including sample and interferon-gamma (IFN-g), and tumor necrosis factor-alpha 4 69 EDTA solutions, was spiked with 0.1 mL of 2mM FeCl and (TNF-a) were determined using commercial enzyme-linked 5 2 70 immunoassay kits (Bender, USA). 6 0.2 mL of 5mM ferrozine solutions. After reaction for 10 mi- Q5 71 7 nutes, the absorbance at 562 nm of the resulting solutions was 72 8 recorded. The higher ferrous ion chelating ability of the test 2.4. Measurement of NO productions in MNC-CMs 73 9 sample gave a lower absorbance (A562). The percentage of 74 10 ferrous ion chelating ability is expressed as [1 e (test sample NO productions in T. triangulare extracts-treated MNC-CMs 75 11 absorbance/blank sample absorbance)] 100. were determined [28]. After an incubation of 24 and 72 hours, 76 12 100 mL of the MNC-CMs was mixed with an equal volume of 77 13 78 2.2.4. Trolox equivalent antioxidant capacity Griess reagent (1% sulfanilamide/0.1% NED/2.5% H PO )ina 14 3 4 79 The ABTS scavenging activity was determined using the Tro- 96-well flat-bottomed microtiter plate and incubated at room 15 80 lox equivalent antioxidant capacity (TEAC) method according 16 temperature for 15 minutes. The absorbance was read at 81 to Yeh and Yen [25]. The TEAC assay is based on the capacity 17 540 nm by a microplate reader (mQuant; BIO-TEK Instruments, 82 ,þ 18 to quench ABTS radical formation relative to Trolox. Briefly, Inc., USA). The nitrite concentrations were determined by Q6 83 e 19 0.1 mL of extracts representing 1.0 10.0 mg/mL dry weight of comparison with the sodium nitrite standard curve. 84 20 plants was incubated for 45 seconds with 0.9 mL ABTS solu- 85 21 tion, and ABTS absorbance was measured at 734 nm. Data are 2.4.1. T. triangulare extract-treated MNC-CM affecting 86 22 expressed as Trolox equivalents, based on standard curves of human leukemia cell growth 87 23 1.0e3.5 mmol Trolox. The human leukemia U937 cell line was obtained from the 88 24 89 Bioresource Collection and Research Center in Taiwan. Cells 25 90 were cultured in RPMI 1640, supplemented with 10% heat- 26 2.3. Cell assays 91 inactivated FBS, penicillin (100 units/mL), streptomycin 27 92 m 28 2.3.1. Cell survival of human mononuclear cells by T. (100 g/mL), HEPES (10mM), sodium pyruvate (1mM), and so- 93 29 triangulare extracts dium bicarbonate (10mM). Cells were subsequently incubated 94 at 37C in a humidified atmosphere containing 5% CO . Then, 30 Human peripheral blood was obtained (in the laboratory) from 2 95 1 105 cells/mL U937 cells were seeded in a 12-well microtiter 31 healthy adult volunteers between the ages of 22 and 25 years. 96 32 plate and treated with different T. triangulare extracts-treated 97 Human mononuclear cells (MNCs) were recovered by adding 33 MNC-CMs (fresh cell medium/CM ¼ 4:1, v/v) for 5 days. The 98 blood to FicolleHypaque solution (1.077 g/mL; Sigma Chemical 34 99 number of viable cells was counted using a trypan blue dye 35 Co.) and centrifuging at 400 g for 30 minutes. A suspension 100 5 exclusion test [29]. Growth inhibition (%) was calculated with 36 of 5 10 cells/mL in an RPMI 1640 medium was prepared. 101 the following equation: 37 MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium 102 38 bromide) assays for determining the growth of MNCs were 103 39 modified from those of Liao et al [26] and Chen et al [27]. MNCs 104 Growth inhibition (%) ¼ [1(Number of live cells after 40 were incubated with various concentrations of different T. 105 incubation for 5 days/total cells before incubation)] 100 41 triangulare extracts in medium, medium only, and medium 106 42 with same extract volume of phosphate buffer saline (PBS; 107 43 108 control) at 37 C in a humidified atmosphere containing 5% CO2 44 2.5. Statistical analysis 109 for 24 and 72 hours, respectively. Following PBS washing, 25 mL 45 110 of MTT reagent was added in FBS-free medium, and cells were 46 The data were subjected to analysis of variance using Statistic 111 incubated for 4 hours. One hundred microliters of MTT lysis 47 Analytical System (SAS) [30]. Duncan's range tests were used Q7 112 48 buffer (25 mL of N,N-dimethyl formamide and 0.67 g/mL so- for paired comparison of means. Results were considered 113 49 dium dodecyl sulfate) was added, and then cells were incu- significant if the associated p value is < 0.05. 114 50 bated for 16 hours. After centrifugation, supernatants were 115 51 assayed for absorbance at 570 nm. The growth index was 116 52 calculated using the following equation: 117 53 3. Results 118 54 119 55 ¼ 3.1. Flavonoids and phenolic acid contents in different T. 120 Growth index (A570 of sample stimulated cells/A570 of 56 triangulare extracts 121 medium control cells) 57 122 58 Flavonoids are the most common and widely distributed 123 59 2.3.2. Evaluation of cytokine productions in T. triangulare group of plant phenolic compounds. In this study, the nar- 124 60 extracts-treated MNCs' condition media ingin, hesperisin, diosmin, quercetin, and hesperetin contents 125 61 126 The cytokine test for the T. triangulare extracts was determined of different T. triangulare extracts were analyzed, and the re- 62 127 according to Chen et al [27]. In brief, a suspension of 1 106 sults are shown in Table 1. The naringin content of WTS was 63 128 ± m 64 cells/mL in an RPMI 1640 medium was prepared. MNCs were the highest (263.7 0.59 g/100 mg) and that of PTL was the 129 65 stimulated with different T. triangulare extracts at various lowest (ND). The decreasing sequence of naringin contents of Q8 130 concentrations for 24 and 72 hours, respectively. Cell media the leaf extracts was ETL > WTL > PTL; for the stem extracts,

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1 66 e 2 Table 1 Contents of various flavonoids in different extracts of Talinum triangulare. 67 3 Naringin Hesperisin Diosmin Quercetin Hesperetin 68 4 69 (mg/100 mg) (mg/100 mg) (mg/100 mg) (mg/100 mg) (mg/100 mg) 5 70 6 ETL 144.9 ± 0.68c 0.12 ± 0.01b ND 0.53 ± 0.02e ND 71 b a e a 7 ETS 150.5 ± 0.62 ND 0.12 ± 0.01 0.51 ± 0.05 31.6 ± 0.32 72 d a b a 8 WTL 121.5 ± 1.90 0.14 ± 0.01 0.05 ± 0.01 2.72 ± 0.06 ND 73 ± a ± b ± a ± c 9 WTS 263.7 0.59 0.12 0.01 0.12 0.01 0.64 0.01 ND 74 ± b 10 PTL ND ND ND 1.43 0.01 ND 75 ± e ± d 11 PTS 42.6 1.37 ND ND 0.56 0.02 ND 76 12 All values are presented as mean ± SD; within the same column values not sharing a common superscript letter are significantly different from 77 13 one another by Duncan's range test (p < 0.05; n ¼ 3). 78 14 ETL ¼ ethanol extract of T. triangulare leaves; ETS ¼ ethanol extract of T. triangulare stems; ND ¼;SD¼ standard deviation; WTL ¼ water extract of Q13 79 15 T. triangulare leaves; WTS ¼ ethanol extract of T. triangulare stems. 80 16 81 17 82 18 83 the decreasing sequence was WTS > ETS > PTS. The hesper- in PTL (ND). WTS showed the highest ferulic acid content 19 84 isin contents of ETL, WTL, and WTS were 0.12 ± 0.01, (70.84 ± 0.35 mg/100 mg) and PTL the lowest (ND). The 20 85 0.14 ± 0.01, and 0.12 ± 0.01 mg/100 mg, respectively. The dio- decreasing sequence of all catechin, chlorogenic acid, caffeic 21 86 ± 22 smin contents of ETS, WTL, and WTS were 0.12 0.01, acid, and ferulic acid contents of the leaf extracts was 87 ± ± > > 23 0.05 0.01, and 0.12 0.01 mg/100 mg, respectively. The WTL ETL PTL. For the stem extracts, the decreasing 88 24 quercetin content of WTL was the highest (2.72 ± 0.06 mg/ sequence of catechin, caffeic acid, and ferulic acid contents 89 25 100 mg) and that of ETS was the lowest (0.51 ± 0.05 mg/ was WTS > ETS > PTS; but for chlorogenic acid content, it was 90 26 100 mg). The decreasing sequence of quercetin contents of the ETS > WTS > PTS. 91 27 leaf extracts was WTL > PTL > ETL; for the stem extracts, the 92 28 decreasing sequence was the same as that of the leaf extracts. 3.2. Antioxidant activities of different T. triangulare 93 29 94 However, hesperetin was only detectable in ETS extracts 30 95 (31.6 ± 0.32 mg/100 mg). 31 96 It is well known that phenolic compounds belong to the 32 As these T. triangulare extracts contained various and signifi- 97 33 bioactive components of plant products and have good cant amounts of flavonoids and phenolic acids, we examined 98 34 health-promoting activities. In this study, contents of gallic their antioxidant activities. The yields of different extracts 99 35 acid, protocatechuic acid, catechin, vanillic acid, chlorogenic (WTL, WTS, PTL, PTS, ETL, and ETS) were 14.45%, 16.38%, 100 36 acid, caffeic acid, and ferulic acid were detected in different T. 9.51%, 11.87%, 12.26%, and 13.48%, respectively. The WTS and 101 37 triangulare extracts, and the results are shown in Table 2.WTS ETS of T. triangulare extracts were shown to have the greatest 102 38 had the highest gallic acid content (334.80 ± 1.52 mg/100 mg), DPPH and ferrous ion chelating ability relative to other T. tri- 103 39 whereas PTL had the lowest level (0.75 ± 0.02 mg/100 mg). The angulare extracts (Table 3). As for the reducing power of ex- 104 40 105 decreasing sequence of gallic acid contents of the leaf extracts tracts, absorbance at 700 nm ranged between 0.19 and 0.49 at a 41 106 was WTL > ETL > PTL; for the stem extracts, the decreasing sample concentration of 1 mg/mL. The highest activity was in 42 107 sequence was WTS > PTS > ETS. Both protocatechuic acid and 43 ETL (0.49), and the lowest was in PTL (0.24; Table 3). For the 108 vanillic acid were not detected in all T. triangulare extracts. The 44 stem extracts, PTS showed the highest activity (0.38), and WTS 109 ± 45 Q9 highest levels of catechin (17.13 0.01 mg/100 mg), chloro- showed the lowest (0.19) (Table 3). The DPPH radical scav- 110 ± 46 genic acid (55.33 0.53 mg/100 mg), and caffeic acid enging activities ranged between 5.71% and 60.93%. The 111 47 (8.32 ± 0.22 mg/100 mg) were all found in WTL, and the lowest decreasing sequence of DPPH radical scavenging activities of 112 48 113 49 114 50 115 e 51 Table 2 Contents of various phenolic acids in different Talinum triangulare extracts. Q14 116 52 Hydroxybenzoic acids Hydroxycinnamic acids 117 53 118 Gallic acid Protocatechuic acid Catechin Vanillic acid Chlorogenic acid Caffeic acid Ferulic acid 54 119 55 (mg/100 mg) (mg/100 mg) (mg/100 mg) (mg/100 mg) (mg/100 mg) (mg/100 mg) (mg/100 mg) 120 56 ETL 13.84 ± 0.33d ND 13.15 ± 0.01c ND 17.49 ± 0.73d 3.79 ± 0.29c 15.03 ± 0.11d 121 57 ETS 12.63 ± 0.42e ND 12.43 ± 0.29d ND 22.83 ± 0.39b 2.44 ± 0.09d 29.54 ± 0.50b 122 58 WTL 64.48 ± 0.55b ND 17.13 ± 0.01a ND 55.33 ± 0.53a 8.32 ± 0.22a 17.90 ± 0.22c 123 59 WTS 334.80 ± 1.52a ND 14.23 ± 0.11b ND 18.67 ± 0.40c 5.57 ± 0.15b 70.84 ± 0.35a 124 60 PTL 0.75 ± 0.02f ND ND ND ND ND ND 125 Q12 61 PTS 30.42 ± 0.25c ND 11.01 ± 0.02e ND 4.01 ± 0.02e 0.96 ± 0.02e 11.01 ± 0.02e 126 62 127 All values are means ± SD. Values not sharing a common letter (superscript) within the same column are significantly different from one 63 another by Duncan's range test (p < 0.05; n ¼ 3). 128 64 ETL ¼ ethanol extract of T. triangulare leaves; ETS ¼ ethanol extract of T. triangulare stems; ND ¼;SD¼ standard deviation; WTL ¼ water extract of 129 65 T. triangulare leaves; WTS ¼ ethanol extract of T. triangulare stems. 130

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1 66 e 2 Table 3 Antioxidant activities of different extracts of Talinum triangulare. 67 3 Samples DPPH scavenging (%) Reducing power (absorbance at 700 nm) Ferrous ion chelating ability (%) TEAC value 68 4 (1 mg/mL) (mM Trolox) 69 5 70 ETL 43.57 ± 0.05b 0.49a 40.93 ± 0.37b 2.15 ± 0.007a 6 71 ETS 60.93 ± 0.87a 0.27c 69.30 ± 1.70a 2.05 ± 0.000b 7 c c b b 72 WTL 41.20 ± 0.86 0.28 47.26 ± 0.47 2.05 ± 0.000 8 WTS 60.10 ± 0.50a 0.19d 63.94 ± 1.25a 2.17 ± 0.003a 73 9 PTL 5.71 ± 0.04e 0.24c 19.49 ± 0.11c 2.18 ± 0.002a 74 10 PTS 8.34 ± 0.01d 0.38b 25.03 ± 0.64c 2.15 ± 0.004a 75 11 76 Data with different letters (superscript) within the same column are significantly different (p < 0.05). 12 77 DPPH ¼ a,a-diphenyl-b-picrylhydrazyl; ETL ¼ ethanol extract of T. triangulare leaves; ETS ¼ ethanol extract of T. triangulare stems; ND ¼; Q15 13 78 SD ¼ standard deviation; TEAC ¼ Trolox equivalent antioxidant capacity; WTL ¼ water extract of T. triangulare leaves; WTS ¼ ethanol extract of 14 79 T. triangulare stems. 15 80 16 81 17 82 > 18 the leaf and stem extracts was ETS and WTS ETL and cytokines to enhance human immunity. MNCs were treated 83 > ' 19 WTL PTS and PTL (Table 3). The extracts ferrous ion with various concentrations of WTL and WTS for 24 and 72 84 20 chelating ability ranged between 19.49% and 69.30%. The hours, respectively. The growth indexes of MNCs were 85 21 decreasing sequence of the ferrous ion chelating ability of the calculated using treatments with medium only as 1.00. Table 4 86 22 leaf and stem extracts was similar to DPPH radical scavenging illustrates that both WTL and WTS stimulated MNC growth. 87 23 activities (Table 3). Meanwhile, the TEAC value ranged be- The MNC stimulations increased with time and dosage. WTS 88 24 tween 2.05 and 2.18 mmol Trolox. None of the extracts showed showed stronger MNC stimulating effects than WTS. Thus, 89 25 90 any significant differences. the highest growth index (1.79) was achieved by a treatment 26 91 From the results shown in Table 3, we may deduce that the using WTL at 1000 mg/mL for 72 hours. However, the same 27 92 stem extracts of T. triangulare have greater antioxidant activ- volume of PBS treatment showed no effect on MNC growth. 28 93 29 ities than its leaf extracts. Furthermore, extracts obtained 94 from 90C boiling water and 75% ethanol showed stronger 30 3.4. Inhibitions of human leukemic U937 cell growth by 95 31 antioxidant activities than those from PBS. 96 WTL- and WTS-treated MNC-CMs 32 97 33 98 34 3.3. Effects of various concentrations of WTL and WTS From previous results, we showed that both WTL and WTS 99 35 on growth of MNC stimulated MNC growth. We next tested whether WTL- and 100 36 WTS-treated MNC-CMs can inhibit human leukemic U937 cell 101 37 Because T. triangulare extracts with 90 C boiling water (WTL growth. MNCs were treated with various concentrations of 102 38 and WTS) showed almost the highest concentrations of fla- WTL and WTS for 24 and 72 hours, respectively, to obtain 103 39 vonoids and phenolic acids, and the highest antioxidant ac- different CMs. The inhibitory effects on the growth of human 104 40 tivities, we used WTL and WTS as samples to examine the leukemic U937 cells were studied following different CMs' 105 41 immunomodulatory activities of T. triangulare extracts via an treatments. The experimental results shown in Fig. 1 indicate 106 42 107 indirect mode. First, we tested whether WTL or WTS affected that MNC-CMs prepared from both WTL- and WTS-treated 43 108 growth of MNCs. MNCs are cells existing in the peripheral CMs significantly inhibited the growth of U937 cells when 44 109 blood of healthy adults. It would be beneficial to human health compared with the PBS-treated control group. All WTL- and 45 110 46 if MNCs could be stimulated, given that they can secrete WTS-treated MNC-CMs showed over 40% growth inhibition 111 47 112 48 113 49 Table 4 e Growth index of MNC after incubation with indicated concentrations of Talinum triangulare extracts for 24 and 72 114 50 hours. Q16 115 51 Growth index of MNC 116 52 117 m 53 Sample concentration ( g/mL) 118 54 sample 100 200 500 800 1000 119 55 WTL (24 h) 1.24 ± 0.01b 1.23 ± 0.04b 1.33 ± 0.04a 1.35 ± 0.05a 1.36 ± 0.03a 120 56 WTS (24 h) 1.33 ± 0.01b 1.31 ± 0.03b 1.35 ± 0.02ab 1.41 ± 0.02a 1.43 ± 0.04a 121 57 Medium (24 h): 1.00 ± 0.00 122 58 PBS (24 h): 1.02 ± 0.01 123 59 WTL (72 h) 1.32 ± 0.04b 1.32 ± 0.06b 1.35 ± 0.02ab 1.40 ± 0.02a 1.41 ± 0.07a 124 60 WTS (72 h) 1.67 ± 0.01b 1.65 ± 0.01b 1.74 ± 0.02a 1.70 ± 0.00a 1.79 ± 0.02a 125 61 Medium (72 h): 1.00 ± 0.00 126 62 PBS (72 h): 0.99 ± 0.03 127 63 Data with different letters (superscript) within the same row are significantly different (p < 0.05). 128 64 MNC ¼ mononuclear cell; PBS ¼ phosphate buffer saline; WTL ¼ water extract of T. triangulare leaves; WTS ¼ ethanol extract of T. triangulare 129 65 stems. 130

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1 66 2 67 3 68 4 69 5 70 6 71 7 72 8 73 9 74 10 75 11 76 12 77 13 78 14 79 15 80 16 81 17 82 18 83 19 Fig. 2 e Concentrations of IL-1b in various MNC-CMs 84 e 20 Fig. 1 Growth inhibitions of U937 cells treated with MNC- treated with different Talinum triangulare extracts for 24 85 21 CMs prepared from different Talinum triangulare extracts and 72 hours. Values are expressed as mean ± SD (n ¼ 3). 86 ± 22 for 24 and 72 hours. Values are expressed as mean SD Each letter indicates a statistically significant difference 87 ¼ < 23 (n 3). *p 0.01, statistically significant difference within within each group, p < 0.01. IL-1b ¼ interleukin-1 beta; 88 24 ¼ 89 each group. MNC-CM mononuclear cell-condition MNC-CM ¼ mononuclear cell-condition medium; 25 ¼ 90 medium; SD standard deviation. SD ¼ standard deviation. 26 91 27 92 28 93 29 94 results, it can be concluded that T. triangulare extracts of WTS 30 ratio of U937 cells. The results of U937 growth inhibitions 95 and WTL are capable of stimulating MNCs to secrete cyto- 31 showed time-relateddbut not dosage-relateddeffect because 96 kines. Therefore, WTL- and WTS-treated MNC-CMs can 32 growth inhibitions increased with treatment time but not with 97 33 extract dosage. The MNC-CMs with 500 mg/mL WTS and 800 mg/ significantly inhibit leukemic U937 cell growth. 98 34 mL WTL treatments for 72 hours showed the highest U937 cell 99 35 growth inhibitions of 50.33% and 48.35%, respectively. 3.6. Effect on nitric oxide productions of WTL- and WTS- 100 36 treated MNCs 101 37 3.5. Cytokine concentrations in WTL- and WTS-treated 102 38 103 MNC-CMs As nitric oxide (NO) is an important cellular messenger for 39 many physiological responses including inflammation, we 104 40 105 41 To explain why WTL- and WTS-treated MNC-CMs can signif- 106 42 icantly inhibit leukemic U937 cell growth, we investigated the 107 43 concentrations of three cytokines (IL-1b, IFN-g, and TNF-a)in 108 44 WTL- and WTS-treated MNC-CMs, because these three cyto- 109 45 kines secreted by activated MNCs have been shown to have 110 46 antiproliferative and differentiating effects on human 111 47 leukemic U937 cells [31]. MNCs were treated with various 112 48 concentrations of WTL and WTS for 24 and 72 hours, respec- 113 49 114 tively. The concentrations of cytokines in the supernatants of 50 115 cultured media were then determined. All IL-1b (Fig. 2), IFN-g 51 116 (Fig. 3), and TNF-a (Fig. 4) concentrations in WTL- and WTS- 52 117 53 treated MNC media were significantly increased compared 118 54 with those in PBS-treated control group. The concentrations of 119 55 cytokines in these media also showed time-relateddbut not 120 56 dosage-relateddeffects (as shown in Fig. 1), because concen- 121 57 trations of cytokines increased with treatment time but not 122 58 with extract dosage. The IL-1b concentrations in WTL- and 123 59 WTS-treated MNC-CMs (at 500 mg/mL, for 72 hours) were Fig. 3 e Concentrations of IFN-g in various MNC-CMs 124 60 741.47 and 1275.01 pg/mL, respectively (Fig. 2); the IFN-g treated with different Talinum triangulare extracts for 24 125 61 126 concentrations were 577.89 and 859.48 pg/mL, respectively and 72 hours. Values are expressed as mean ± SD (n ¼ 3). 62 127 (Fig. 3); the TNF-a concentrations were 1862.85 and 2222.29, Each letter indicates a statistically significant difference 63 128 < g ¼ 64 respectively (Fig. 4). At the same concentration and treatment within each group, p 0.01. IFN- interferon-gamma; 129 ¼ 65 time, all cytokine concentrations of MNCs treated with WTS MNC-CM mononuclear cell-condition medium; 130 were higher than those of WTL treatments. From these SD ¼ standard deviation.

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1 MNCs treated for 72 hours showed the highest NO concen- 66 2 trations (1.87e1.92 mM). Because NO could induce the death 67 3 68 of leukemic U937 cells [27], we showed that WTS and WTL 4 69 can stimulate the growth of MNCs (Table 4) and enhance 5 70 cytokine secretions (Figs. 2e4) and NO productions (Table 5) 6 71 7 of MNCs. Therefore, WTS- and WTL-treated MNC-CMs can 72 8 significantly inhibit the growth of leukemic U937 cells 73 9 (Fig. 1). 74 10 75 11 76 12 77 13 4. Discussion 78 14 79 15 Oxidative stress is responsible for more than 100 human dis- 80 16 eases [5] including cancers worldwide. Flavonoids are poly- 81 17 phenols widely distributed in fruits and vegetables, and have 82 18 been shown to be good antioxidants. Phenolic compounds are 83 19 Fig. 4 e Concentrations of TNF-a in various MNC-CMs 84 bioactive components of plants and show health-promoting 20 treated with different Talinum triangulare extracts for 24 85 activities. In this study, we reported that extracts from T. tri- 21 and 72 hours. Values are expressed as mean ± SD (n ¼ 3). 86 angulare showed significant contents of flavonoids (Table 1) 22 Each letter indicates a statistically significant difference 87 and phenolic acids (Table 2), and that T. triangulare extracts 23 within each group, p < 0.01. MNC-CM ¼ mononuclear 88 24 also showed significant antioxidant activities (Table 3). 89 cell-condition medium; SD ¼ standard deviation; 25 Because WTL and WTS could significantly stimulate MNC 90 TNF-a ¼ tumor necrosis factor-alpha. 26 growth (Table 4), secretions of cytokines (Figs. 2e4), and pro- 91 27 ductions of NO (Table 5), both WTL- and WTS-treated MNC- 92 28 CMs showed significant inhibitory effects on human leukemic 93 29 U937 cell growth (Fig. 1). Thus, T. triangulare extracts showed 94 30 were interested in whether WTL and WTS treatment significant immunomodulatory activities that can inhibit 95 31 96 induced NO productions in MNCs. As shown in Table 5,all leukemic U937 cancer cells. Based on these results, we suggest 32 97 NO concentrations in WTL- and WTS-treated MNC media that T. triangulare extracts can be used as healthy foods 33 98 were significantly increased compared with those in the 34 because of their antioxidant activities and immunomodula- 99 PBS-treated control group. The NO concentrations in WTL- 35 tory potential. 100 36 treated groups also showed time-related, but not dosage- Stem extracts of T. triangulare (such as WTS) have higher 101 37 related, effect because NO concentration increased with concentrations of flavonoids and phenolic acids than leaf 102 38 treatment time but not with extract dosage. However, the extracts of T. triangulare (such as WTL) (Tables 1 and 2), and 103 39 NO concentrations in WTS-treated groups showed reversed also show stronger antioxidant activities (Table 3). These re- 104 40 time-related (except for 1000 mg/mL), but not dosage-related, sults are reliable. Developing a new extraction method to 105 41 effect because NO concentrations decreased with treatment obtain more flavonoids and phenolic acids from T. triangulare 106 42 107 time (except for the dosage of 1000 mg/mL) but not with should increase the extract's antioxidant activities. Further- 43 108 extract dosage. MNCs (200 and 500 mg/mL) treated with WTS more, WTS also showed stronger stimulations of MNCs than 44 109 for 24 hours showed the highest NO concentrations WTL at any same dosage and time interval (Table 4), induction 45 110 (2.79 mM). For WTL, all (200, 500, and 800 mg/mL) WTL-treated e 46 of more cytokine secretions (Figs. 2 4), and higher production 111 47 112 48 113 49 Table 5 e Concentrations of nitric oxide in media of MNC incubated with indicated concentrations of Talnum triangulare 114 50 extracts for 24 and 72 hours. 115 51 Concentrations of nitric oxide (mM) 116 52 117 m 53 Sample concentrations ( g/mL) 118 54 Sample 100 200 500 800 1000 119 55 WTL (24 h) 0.98 ± 0.05ab 0.85 ± 0.08b 1.05 ± 0.13a 0.85 ± 0.02b 0.85 ± 0.11b 120 56 WTS (24 h) 2.59 ± 0.05bc 2.79 ± 0.04a 2.79 ± 0.04a 2.46 ± 0.07c 0.78 ± 0.04d 121 57 Medium (24 h): 0.33 ± 0.03 122 58 PBS (24 h): 0.42 ± 0.01 123 59 WTL (72 h) 1.75 ± 0.03b 1.88 ± 0.10a 1.87 ± 0.08a 1.92 ± 0.08a 1.79 ± 0.04b 124 60 WTS (72 h) 1.67 ± 0.01c 1.65 ± 0.01c 1.74 ± 0.02ab 1.70 ± 0.00b 1.79 ± 0.02a 125 61 Medium (24 h): 0.41 ± 0.01 126 62 PBS (72 h): 0.44 ± 0.04 127 63 Data with different letters within the same row are significantly different (p < 0.05). 128 64 MNC ¼ mononuclear cell; PBS ¼ phosphate buffer saline; WTL ¼ water extract of T. triangulare leaves; WTS ¼ ethanol extract of T. triangulare 129 65 stems. 130

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1 of NO at 24 hours (Table 5); in addition, WTS-treated MNC- 66 2 CMs showed stronger inhibitory effects than WTL-treated Conflicts of interest 67 3 68 MNC-CMs on the cell growth of U937 cells (Fig. 1). These re- 4 69 sults are also reliable. They show that antioxidant activities The authors declared no potential conflicts of interest with 5 70 are significantly related to growth inhibition of U937 cells, respect to the research, authorship, and/or publication of this 6 article. 71 7 because the stronger antioxidant activities of T. triangulare 72 8 extracts showed better U937 cell growth inhibitions. The 73 9 secreted cytokines and NO are significantly related to growth Q18 74 10 inhibition of U937 cell, because U937 cell growth inhibition Uncited reference 75 11 increases with increasing concentrations of cytokines and NO. 76 12 Although the NO concentrations in WTS (72 hours) groups [32]. 77 13 were lower than those in WTS (24 hours) groups, except for 78 14 the sample concentration of 1000 mg/mL (Table 5), the con- 79 15 80 centrations of the three examined cytokines secreted by 16 Acknowledgments 81 MNCs in WTS (72 hours) groups were all higher than those in 17 82 e 18 the WTS (24 hours) groups (Figs. 2 4). In combination with the This work was supported in part by the National Science 83 19 effects of NO and three cytokines, the inhibitory effects on Council Taiwan, R.O.C. (grant number: NSC 99-2314-B-212- Q10 84

20 U937 cells in the WTS (72 hours) groups were higher than 001-MY3). Q11 85 21 those in the WTS (24 hours) groups at sample concentrations 86 22 of 100e500 mg/mL, although no difference was found at Table S1. HPLC solvent gradient elution program. 87 23 800e1000 mg/mL (Fig. 1). 88 24 Because the extracts of T. triangulare with stronger anti- 89 25 oxidant activities show stronger immunomodulatory activ- 90 26 91 ities, it is implied that the contents of ﬂavonoids and phenolic Time (min) Eluent solvent (%) 27 92 acids in T. triangulare are responsible not only for its antioxi- 28 AB93 dant activities but also for its immunomodulatory activities. 29 0 100 0 94 30 In addition, the stem of T. triangulare shows more promise 5802095 31 compared with its leaf as a potential healthy food because it 15 70 30 96 32 exhibits higher antioxidant and immunomodulatory activ- 20 70 30 97 33 ities. Further studies will examine whether T. triangulare ex- 30 65 35 98 34 tracts show antioxidant activities and determine the 35 65 35 99 35 mechanism that induces cytokine secretion and NO produc- 45 50 50 100 55 50 50 36 tion in MNCs. 101 65 30 70 37 The MNC-CMs treated with 500 mg/mL WTS for 72 hours 102 38 70 30 70 103 showed the highest U937 cell growth inhibition (at 50.33%). 39 75 0 100 104 Furthermore, the growth inhibition of U937 cells treated with 80 0 100 40 105 85 0 100 41 MNC-CMs prepared with the cold-water extract of Flammulina 106 42 velutipes at 800 mg/mL was 60% [31]; with the water extract of A ¼ 10% methanol and 0.05% formic acid; B ¼ 70% methanol and 107 43 rice at 500 mg/mL, growth inhibition was calculated as 35% 0.05% formic acid. 108 44 [26]; with bovine skimmed colostrums and their hydrolysates HPLC ¼ high-performance liquid chromatography. 109 45 collected on the 2nd day postpartum at 500 mg/mL, it was 50% 110 46 [27]; and with semipuriﬁed polysaccharides of Antrodia 111 47 camphorate at 100 mg/mL, it was 55% [29]. Collectively, these 112 48 references 113 results indicate a similar potential of WTS as immunomodu- Q18 49 114 latory extracts. 50 115 The results of this study show that T. triangulare extracts 51 116 exhibit immunomodulatory effects through the stimulation [1] Fasuyi AO. Nutritional potentials of some tropical vegetable 52 117 leaf meals: chemical characterization and functional 53 of MNCs for secretions of cytokines (IL-1b, IFN-g, and TNF-a) 118 properties. Afr J Biotechnol 2005;5:049e53. 54 and induction of NO into the MNC-CMs. The cytokines (IL-1b, 119 [2] Fasuyi AO. Bio-nutritional evaluations of three tropical leaf IFN-g, and TNF-a) are able to induce the differentiation and 55 vegetables (Telfairia occidentalis, Amaranthus cruentus and 120 56 death of U937 cells [31]. NO is recognized as one of the most Talinum triangulare) as sole dietary protein sources in rat 121 57 versatile players in the immune system. It is involved in the assay. Food Chem 2007;103:757e65. 122 58 pathogenesis and control of infectious diseases, tumors, [3] Uwah EI, Ndahi NP, Ogugbuaja VO. Study of the levels of 123 59 autoimmune processes, and chronic degenerative diseases some agricultural pollutants in soils and water leaf (Talinum 124 60 [33]. Yang and Park [28] reported that antioxidant enzyme triangulare) obtained in maiduguri. Nigeria J Appl Sci Environ 125 61 e 126 inhibitors enhance NO-induced cell death in U937 cells. It 2009;4:71 8. 62 [4] Kumar A, Prasa MNV, Sytar O. Lead toxicity, defense and 127 may be stated from these results that T. triangulare extracts 63 associated indicative biomarkers in Talinum triangulare 128 64 not only inhibit the growth of U937 cells and exhibit their grown hydroponically. Chemosphere 2012;89:1056e65. 129 65 immunomodulatory activities, but also stimulate the growth [5] Liang D, Zhou Q, Gong W, Wang Y, Nie Z, He H, Li J, Wu J, 130 of MNCs. Wu C, Zhang J. Studies on the antioxidant and

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