JFDA188_proof ■ 5 January 2015 ■ 1/9 journal of food and drug analysis xxx (2015) 1e9 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 2 journal of food and drug analysis xxx (2015) 1e9 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 À80C 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 À20 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 Â g for 10 mi- 111 47 powders were stored at À20C 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 Â 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.