中華民國雜草會刊 35:1-14, DOI: /WSSROC (1)-001 酶 * 3* (Limnophila rugosa (Roth) Merr.) 酶 a b α,α-diphenyl-β-picrylhy

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1 中華民國雜草會刊 35:1-14, DOI: /WSSROC (1)-001 酶 * 3* (Limnophila rugosa (Roth) Merr.) 酶 a b α,α-diphenyl-β-picrylhydrazyl (DPPH) 酶 0.1X 0.5X 1X 酶 28.1% 46.4% 72.3% 酶 0.5X 酶 8.38 mg ml -1 酶 86% IC mg ml -1 IC µg ml -1 酶 : 酶

2 2 中華民國雜草學會會刊第三十五卷第一期中華民國 103 年 6 月 Effects of nitrogen application rates on antioxidative activity of Limnophila rugosa (Roth) Merr. and its inhibitory activity on xanthine oxidase Ming-Huang Hsu 1, Zhi-Wei Yang 2, Shu-Fong Huang 3, Chi-Ming Yang 4, Tzong-Fu Kuo 5*,and Wen-Dar Huang 3* 1 Refining and Manufacturing Research Institute, CPC Corporation, Taiwan, Chiayi 600, Taiwan 2 Taoyuan District Agricultural Research and Extension Station, Council of Agriculture, Taoyuan 239, Taiwan 3 Department of Agronomy, National Taiwan University, Daan, Taipei 106, Taiwan 4 Biodiversity Research Center, Academia Sinica, Nankang, Taipei 115, Taiwan 5 School of Veterinary Medicine, National Taiwan University, Daan, Taipei 106, Taiwan Abstract Limnophila rugosa (Roth) Merr. is scattered mainly in the lower altitude mountain of Taiwan near ponds, paddy fields, lakes, wetlands, and humid environment. As a native herb with many potential developments, L. rugosa is traditionally used as a medicinal plant. This study focused the effects of various nitrogen application rates (0.1X, 0.5X and 1X N of Kimura solution) on antioxidative activities of L. rugosa. and its inhibitory activity on xanthine oxidase (XO). The increase of chlorophyll index caused by raising chlorophyll a and b and carotenoid contents with increasing nitrogen rate was found. The antioxidative activities including α,α-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging activity, Fe 2+ ion chelating activity and reducing power were significantly increased by increasing nitrogen rate. An improvement of XO activity inhibition was found with increasing nitrogen rate. The ethanol extracts of L. rugosa treated with 0.1X, 0.5X and 1X nitrogen rate inhibited XO by 28.1%, 46.4% and 72.3%, respectively. The inhibition effect of these extracts increased with the increase of extract concentration. The 8.38 mg ml -1 extract of L. rugosa from 0.5X nitrogen treatment inhibited xanthine oxidase by 86% with an IC 50 of 5.87 mg ml -1 compared with an IC 50

3 Weed Sci. Bull. 35(1), June of 3.80 µg ml -1 for allopurinol. This is also the first report to demonstrate that L. rugosa has an ability to inhibit XO activity. The results of this study will provide useful information for medical care and agriculture technology. Keywords: Limnophila rugosa, nitrogen, antioxidative activity, xanthine oxidase. (free radicals) (reactive oxygen species, ROSs) (Schöneich, 1999 Valko et al., 2007) (Chen and Yen, 1998) E (vitamin E) C (vitamin C) (β-carotene) (Waltzl, 1996) 酶 (xanthine oxidase, XO) (Tung and Chang, 2010; Tung et al., 2010) 酶 (Granger et al., 1981; Rasmussen et al., 2000) (hyperuricemia) (Strazzullo and Puig, 2007) 酶酶 (Kong et al., 2000; Hsieh and Tung, 2010; Tung and Chang, 2010; Tung et al., 2010) Limnophila rugosa (Roth) Merr. (Magnoliopsida) (Scrophulariales) (Scrophulariaceae) (Limnophila) (Wu and Chen, 2004)

4 4 中華民國雜草學會會刊第三十五卷第一期中華民國 103 年 6 月 (Guan, 2004) (Gorai et al., 2013) (Gorai et al., 2013; Huang et al., 2011; Linh and Thach, 2011) 酶酶酶 50 cm X 7 30/25 ( / ) 70% / 12/12 hr 1X 1X 0.5X 0.1X (CRD) 酶 LI-COR LI-6400XT % 400 µmol mol -1 CO 2 0-2,000 µmol m -2 s -2 PFD (photon flux density) (net photosynthetic rate, P N ) (stomatal conductance, g s ) (transpiration rate, E) CO 2 (intercellular CO 2 concentration, Ci) (water use efficiency, WUE, P N /E) (carboxylation efficiency, CE, P N /Ci) Konica Minolta SPAD-520

5 Weed Sci. Bull. 35(1), June Yang et al. (1998) 0.01 g 80% 2,264 g nm nm nm Chl a (chlorophyll a) Chl b (chlorophyll b) Car (carotenoid) Porra et al. (1989) Chl a Chl b Holm (1954) Car α,α-diphenyl-β-picrylhydrazyl (DPPH) (Fe 2+ ion) (reducing power) 80% 4 3,000 rpm 5 DPPH Shimada et al. (1992) 1 ml (15 mg ml -1 ) 0.25 ml 10 mm DPPH nm DPPH (%)=[1 (A 517,sample /A 517,blank )] 100 (Fe 2+ ion) Dinis et al. (1994) 1 ml (0.8 mg ml -1 ) 3.7 ml 80% 0.1 ml 2 mm FeCl 2 4H 2 O ml 5 mm ferrozine nm (%)=[1 (A 517,sample /A 517,blank )] 100 Oyaizu (1986) 1mL (3 mg ml -1 ) 1 ml 0.2 M sodium phosphate butter (ph 6.6) 1 ml 1% K 3 Fe(CN) ml 10% (TCA) 3,000 rpm 10 2 ml 2 ml 0.4 ml 0.1% FeCl 3 6H 2 O nm = A 700,sample /A 700,blank 酶 Kong et al. (2000) 95% 酶 50 µl 35 µl 50 mm phosphate buffer (ph 7.5) 30 µl 0.1 U ml -1 xanthine oxidase µl 150 µm xanthine µl 1N HCl 295 nm 酶 (%) (allopurinol) ± (mean ± SD) SAS 9.1 (SAS Institute, 1999) Fisher's LSD (least significant difference) 5%

6 6 中華民國雜草學會會刊第三十五卷第一期中華民國 103 年 6 月 µmol CO 2 m -2 s µmol m -2 s -1 PFD µmol m -2 s µmol CO 2 m -2 s µmol CO 2 m -2 s -1 PFD 200-2,000 µmol m -2 s µmol CO 2 m -2 s µmol CO 2 m -2 s -1 ( 1A) 200 µmol m -2 s -1 ( 1B 1C) CO 2 PFD 500 µmol m -2 s -1 CO µmol mol -1 ( 1D) µmol m -2 s -1 PFD 800 µmol m -2 s -1 PFD 2,000 µmol m -2 s -1 PFD ( 2) C3 C4 1,800 µmol m -2 s -1 C3 (Taiz and Zeiger, 2010; Zuo et al., 2010) ( 1) Chl a 6,187 µg g -1 7,591 µg g -1 Chl b 1,638 µg g -1 2,245 µg g -1 Car 1,469 µg g -1 1,712 µg g DPPH ( 2) 1X DPPH 71.5% 0.5X 0.1X X 0.5X 1X 42.2% 72.3% 84.7% 1.0X 0.1X 0.5X X 0.5X 1.0X X 0.1X 0.5X

7 Weed Sci. Bull. 35(1), June A C 5.0 PN (µmol CO2 m -2 s -1 ) E (mmol H2O m -2 s -1 ) gs (mol H2O m -2 s -1 ) B D Ci (µmol mol -1 ) PPFD (µmol m -2 s -1 ) (A) P N (B) g s (C) E (D) CO 2 Ci Fig 1. Light-response curves of (A) leaf CO 2 assimilation rate (P N ), (B) stomatal conductance (g s ), (C) transpiration (E), and (D) intracellular CO 2 concentration (Ci) in L. rugosa. Values are mean ± SD of four replicates.

8 8 中華民國雜草學會會刊第三十五卷第一期中華民國 103 年 6 月 1. a b Table 1. Changes of Chl a, Chl b and Car contents and Chl index of L. rugosa treated with nitrogen at different rates. Nitrogen rate Chl a (µg g -1 ) Chl b (µg g -1 ) Car (µg g -1 ) Chl index 0.1X 6187±223 c 1638±94 c 1469±39 c 28.1±2.8 c 0.5X 7147±326 b 2046±138 b 1636±57 b 34.1±1.5 b 1X 7591±287 a 2245±115 a 1712±50 a 38.8±1.1 a * Means followed by the same letter within columns are not significant based on LSD test (n=4, p = 0.05). WUE (µmol CO2/mmol H2O) A B CE (mol m -2 s -1 ) PPFD (µmol m -2 s -1 ) 2. (A) (B) Fig 2. Light-response curves of (A) water use efficiency (WUE) and (B) instantaneous carboxylation efficiency (CE) in L. rugosa. Values are mean ± SD of four replicates.

9 Weed Sci. Bull. 35(1), June 酶 0.1X 0.5X 1X 酶 28.1% 46.4% 72.3% 1.0X 0.1X 0.5X ( ) 酶 28~38 酶 20~80% 酶 ( 3) 酶 0.5X 酶 8.38 mg ml -1 酶 86% 5.87 mg ml -1 ( 4) IC µg ml -1 DPPH 酶 ( 1 2) (Hopkins, 1999) (Satureja hortensis) (Teucrium polium) (Cosmos caudatus) (Triticum aestivum) (Alizadeh et al., 2010; Azaizeh et al., 2005; Hassan et al., 2012; Sun et al., 2011) (Eryngium cretium) (Amaranthus cruentus var. Aztek) (Brassica chinensis) (Azaizeh et al., 2005; Skwarylo-Bednarz and Krzepilko, 2009; Zhu et al., 2009) 2. DPPH 酶 Table 2. Changes of DPPH radicle scavenging activity, Fe 2+ ion chelating activity, reducing power and XO inhibitory activity of L. rugosa treated with different nitrogen rate. IC 50 Nitrogen rate DPPH scavenging activity (%) Fe 2+ ion chelating activity (%) Reducing power (A 700 ) XO inhibition (%) 0.1X 27.4±14.0 b 42.2±9.6 b 1.13±0.24 b 2.81±6.6 c 0.5X 40.1±12.7 b 72.9±9.2 a 1.54±0.49 b 46.4±3.7 b 1X 71.5±7.1 a 84.7±8.1 a 2.32±0.14 a 72.3±6.1 a * Means followed by the same letter within columns are not significant based on LSD test (n=4, p = 0.05).

10 10 中華民國雜草學會會刊第三十五卷第一期中華民國 103 年 6 月 100 XO inhibition (%) R² = Chl index 3. 酶 Fig. 3. Relationship between Chl index and XO inhibitory activity of L. rugosa treated with nitrogen at different rates. 100 XO inhibition (%) Concentration (mg ml -1 ) 4. 酶 0.5X Fig 4. The concentration-dependent XO inhibitory acitivity in response to extract of L. rugosa harvested from 0.5X nitrogen rate treatment.

11 Weed Sci. Bull. 35(1), June 酶 (purine) 苷苷酶 (adenine) (guanine) (hypoxanthine) (xanthine) 酶 (Granger et al., 1981; Rasmussen et al., 2000) (hyperuricemia) (Kong et al., 2000; Strazzullo and Puig, 2007) 酶 (allopurinol) (Gilman et al., 1985; Fields et al., 1996) (Hande et al., 1984; Urban et al., 1995) 酶 (Coccinia grandis) (Datura metel) (Strychnos nux-vomica) Pistacia integerrima (Vitex negundo) (Ahmed et al., 2008; Umamaheswari et al., 2007) (Cinnamomum cassia) (Chrysanthemum indicum) (Lycopus europaeus) (Polygonum cuspidatum) (Kong et al., 2000) (Morinda citrifolia) (Acacia confusa) 酶 (Hsieh and Tung, 2010; Palu et al., 2009; Tung and Chang, 2010; Tung et al., 2010) (flavonoids) 萜 (terpenoids) 苷 (glycosides) (alkaloids) (Acharya et al., 2013; Gorai et al, 2013) (Huang et al., 2011) (Gorai et al., 2013) 酶酶 ( 4) 酶 ( 2) 酶酶

12 12 中華民國雜草學會會刊第三十五卷第一期中華民國 103 年 6 月 Acharya R, R Padiya, ED Patel, CR Harisha, VJ Shukla (2013) Phytochemical study of an ethnomedical plant Limnophila rugosa Roth. (Merr) (Scrophulariaceae) whole plant. Ann. Ayurvedic Med. 2: Ahmed NS, M Farman, MH Najmi, KB Mian, A Hasan (2008) Pharmacological basis for use of Pistacia integerrima leaves in hyperuricemia and gout. J. Ethnopharmacol. 117: Alizadeh A, M Khoshkhui, K Javidnia, O Firuzi, E Tafazoli, A Khalighi (2010) Effects of fertilizer on yield, essential oil composition, total phenolic content and antioxidant activity in Satureja hortensisi L. (Lamiaceae) cultivated in Iran. J. Med. Plants Res. 4: Azaizeh H, P Ljubuncic, I Portnaya, O Said, U Cogan, A Bomzon (2005) Fertilization-induced changes in growth parameters and antioxidant activity of medicinal plants used in traditional Arab medicine. Oxf. J. 2: Chen HY, GC Yen (1998) Free radicals, antioxidant defenses and human health. Nutr. Sci. J. 23(1): Gorai D, SK Jash, RK Singh, A Sarkar, S Majhi (2013) Chemical and pharmacological aspects of Limnophila rugosa: An update. Int. J. Nat. Prod. Res. 3: Granger DN, G Rutili, JM McCord (1981) Superoxide radicals in feline intestinal ischemia. Gastroenterology 81: Guan ZB (2004) Introduction and cultivation of Limnophila rugosa. Chinese Wild Plant Resour. 23: Hande KR, RM Noone, WJ Stone (1984) Severe allopurinol toxicity: description and guidelines for prevention in patients with renal insufficiency. Am. J. Med. 76: Hassan SA, S Mijun, UK Yusoff, PB Ding, PEM Wahab (2012) Nitrate, ascorbic acid, mineral and antioxidant activities of Cosmos caudatus in Response to organic and mineral-based fertilizer rates. Molecules 17:

13 Weed Sci. Bull. 35(1), June Hopkins WG (1999) Introduction to plant physiology. John Wiley & Sons, Inc. New York, USA. Hsieh CY, ST Chang (2010) Antioxidant activities and xanthine oxidase inhibitory effects of phenolic phytochemicals from Acacia confuse twigs and branches. J. Agric. Food Chem. 58: Huang XD, XK Huang, YH Li (2011) Chemical composition and antioxidant activities in vitro of the essential oils from Limnophila rugosa leaves in Fujian. J. Quanzhou Norm. Univ. 29: Kong LD, Y Cai, WW Huang, CHK Cheng, RX Tan (2000) Inhibition of xanthine oxidase by some Chinese medicinal plants used to treat gout. J. Ethnopharmacol. 72: Linh NT, LN Thach (2011) Study of the essential oil of Limnophila Rugosa (Roth.) Merr. in the south of Vietnam. J. Essent. Oil Bear. Pl. 14: Palu A, SX Deng, B West, J Jensen (2009) Xanthine oxidase inhibition effects of noni (Morinda citrifolia) fruit juice. Phytother. Res. 23: Rasmussen JT, MS Rasmussen, TE Petersen (2000) Cysteines involved in the interconversion between dehydrogenase and oxidase forms of bovine xanthine oxidoreductase. J. Dairy Sci. 83: Schöneich C (1999) Reactive oxygen species and biological aging - a mechanistic approach. Exp. Gerontol. 34: Skwarylo-Bednarz B, A. Krzepilko (2009) Effect of various NPK fertilizer doses on total antioxidant capacity of soil and amaranth leves (Amaranthus cruentus L.) Int. Agrophysics 23: Strazzullo P, JG Puig (2007) Uric acid and oxidative stress: relative impact on cardiovascular risk. Nutr. Metab. Cardiovasc. Dis. 17: Sun XM, T Zhang, NL Chen, K Zhang (2011) Study on antioxygenic system in leaves of three spring wheat cultivars with different drought-resistant capabilities under different soil moisture content and nitrogen levels. Arid Zone Res. 28: Taiz L, E Zeiger (2010) Plant Physiology. 5th ed. Sinauer Associates, Inc., Sunderland, Massachusetts, USA. 782 pp. Tung YT, ST Chang (2010) Inhibition of xanthine oxidase by Acacia confuse extracts and their phytochemicals. J Agric. Food Chem. 58:

14 14 中華民國雜草學會會刊第三十五卷第一期中華民國 103 年 6 月 Tung YT, CA Hsu, CS Chen, SC Yang, CC Huang, ST Chang (2010) Phytochemicals from Acacia confuse heartweed extracts reduce serum uric acid levels in oxonate-induced mice: their potential use as xanthine oxidase inhibitors. J. Agric. Food Chem. 58: Umamaheswari M, K AsokKumar, A Somasundaram, T Sivashanmugam, V Subhadradevi, TK Ravi (2007) Xanthine oxidase inhibitory activity of some Indian medicinal plants. J. Ethnopharmacol. 109: Urban T, E Maquarre, C Housset, C Chouaid, E Devin, B Lebeau (1995) Allopurinol hypersensitivity. A possible cause of hepatitis and mucocutaneous eruptions in a patient undergoing antitubercular treatment. Rev. Mal. Respir. 12: Valko M, D Leibfeitz, J Moncol, MTD Cronin, M Mazur, J Telser (2010) Free radicals and antioxidants in normal physiological functions and human disease. Intl. J. Biochem. Cell Biol. 39: Waltzl, B (1996) Health-promoting effects of phytochemicals. In Proceeding of IUFOST 96 regional symposium. Wu ZY, SC Chen (2004) Flora of China. Science Press, Beijing, China. Zhu WF, XY Lin, CW Jin, YS Zhang, P Fang (2009) Effects of nitrogen application rates on antioxidant contents and antioxidative activities in Chinese cabbage (Brassica chinensis L.). J. Zhejiang Univ. (Agric. & Life Sci.) 35: Zuo YM, HJ Chen, ZF Yang, QQ Deng, J Tang (2010) The diurnal variation of light parameters and the main impact factors of Cassava in South China. Chinese Agric. Sci. Bull. 26:

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