XIE Tianyan 1, 2, HE Kaize 1**, ZHAO Hai 1, PU Qiang 1 & FANG Yang 1

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1 DOI: /SP.J Chin J Appl Environ Biol ( 2 ) : * 1, 2 1** Lemnaceae Oxford Cup MIC zh0018 zh0224zh0003zh0225 HPLC. 95% zh0225mic6.25 mg/ml MIC0.78 mg/ml. 9.8% 8.1% zh % 17.2%. HPLC. zh CLC Q Antimicrobial activities and flavonoid contents of the from four strains of duckweed* XIE Tianyan 1, 2, HE Kaize 1**, ZHAO Hai 1, PU Qiang 1 & FANG Yang 1 1 Chengdu Institute of Biology, Chinese Academy of Science, Chengdu, 641, China 2 University of Chinese Academy of Science, Beijing, 049, China Abstract Duckweed (Lemnaceae) is a plant which grows in the sewage surface teeming with rich microbes, and it is proved that duckweed has a protective mechanism against microbes. In order to study the antibacterial mechanism of duckweeds and provide the basis for screening anti-bacterial substances and studying the infection mechanism of plant and animal pathogens or human pathogenic microorganisms, we first extracted using solvents with different polarities four strains of duckweed belonging to Lemna minor L. zh0018, Landoltia punctata zh0224, Spirodela polyrrhiza (L.) Schleid zh0003 and zh0225. The antimicrobial activities of their were compared by Oxford Cup method and the minimum inhibitory concentration (MIC) measurement. Meanwhile, the contents and the composition of total flavonoids were determined by spectrophotometry combined with high performance liquid chromatography (HPLC) analysis. The results showed that the methanol and 95% ethanol of the duckweed had different degrees of antimicrobial activities against, Staphylococcus aureus, Pseudomonas aeruginosa, and. The antimicrobial activity of the methanol from S. polyrrhiza was the strongest (MIC: 6.25 mg/ml), followed by L. minor L. ; the ethyl acetate phase had the strongest activity (MIC: 0.78 mg/ml) followed by the n-butanol phase. The methanol of L. minor L. had the highest level of total flavonoids content, 9.8% of the total, followed by the 8.1% content of the methanol of L. punctata. In addition, the ethyl acetate phase of S. polyrrhiza zh0225 methanol had the highest content of total flavonoids (22.46%) followed by the 17.2% content of the n-butanol. In HPLC measurement, the composition of flavonoids showed tremendous difference among the duckweed, with the two strains of S. polyrrhiza having similar composition with Received: Accepted: *2011BAD22B03 KSCX2-EW-J-22 Y2C5021 Supported by the Sci-tech Pillar Project of China (2011BAD22B03), the Major Projects of Knowledge Innovation Program of Chinese Academy of Sciences (KSCX2-EW-J-22), and the Western Light Talent Cultivation Program of Chinese Academy of Sciences (Y2C5021) ** Corresponding author ( hekz@cib.ac.cn)

2 different contents. The results suggested an enormous potential of S. polyrrhiza zh0225 as an excellent duckweed species for active substance extraction and pharmaceutical production. Keywords duckweed; ; antimicrobial activities; total flavonoids Lemnaceae [1]. [2] [3] [4]. [5]. [6-7] [8]. [9]. [10-11] [12-13]. [14].. 95% Lemna minor L. Landoltia punctata Spirodela polyrrhiza (L.) Schleid zh0018 zh0024 zh0003 zh % 1: h.. mg/ml. 1:10 3: cereus Nesterenkonia Pseudomonas aeruginosa Klebsiella peneumoniae Pantoea agglomerans Acinetobacter haemolytius Acinetobacter baumanii Candida albicans Penicillium italicum Wehmer Penicillium digitatum Phytophthora capsici Leon /ml [15] MIC [15] 3. [15] 20 mm mm mm++ 10 mm+8 mm- MIC [16] [17]. HPLC 0.22 μm %13.31% 13.7% - mg/ml 95%. 26.6% %45% 4.23% 15.41% zh0225 zh MIC 1A 1B mg/ml zh0224zh0003 zh0225 MIC mg/ml. 567 zh000395% Chin J Appl Environ Biol

3 1 zh0018 Table 1 Extraction rates and inhibition zone diameters of from Lemna minor zh0018 (ρ/m g ml - 1) Methanol 95% 95% Ethanol Water Methanol Ethyl acetate N-butanol Yield (r/%) Pseudomonas aeruginosa cereus Candida albicans 2 zh0224 Table 2 Extraction rates and inhibition zone diameters of from Landoltia punctata zh0224 Methanol Ethyl acetates N - butanol extractes Yield (r/%) (+) 1.4(+) 1.2(+) 1.1(+) Pseudomonas aeruginosa 0.85(+) 3 zh0003 Table 3 Extraction rates and inhibition zone diameters of from Spirodela polyrrhiza (L.) Schleid zh0003 Methanol 95% 95% Ethanol Ethyl acetates Methanol Ethyl acetates N-butanol Yield (r/%) (++++) 2.0(+++) Pseudomonas aeruginosa 4 zh0225 Table 4 Extraction rates and inhibition zone diameters of from Spirodela polyrrhiza (L.) Schleid zh0225 Methanol 95%95% Ethanol Ethyl acetates Ethyl acetates N-butanol Ethyl acetates Yield (r/%) (+++) 1.8(+++) 2.1(++++) 2.1(++++) 1.9(+++) 1.8(+++) 2.1(++++) 1.9(+++) Staphylococcus aureus Pseudomonas aeruginosa 1.3( ++) 1.9(+++) 1.3(+++) 1.8(+++) 1.8(++) 1.1(+) 1.2(+) 1.9(+++) Chin J Appl Environ Biol

1 48 h. AB. Fig. 1 Antimicrobial activity against of methanol of different concentrations from Spirodela polyrrhiza (L.) Schleid in 48 hours. A: inhibition zone; B: growth curve.

25 3.13 1.56 0.78 0.39 6 zh0225mic Table 6 Antimicrobial activity at different concentrations and MIC of methanol from Spirodela polyrrhiza (L.) Schleid zh0225 Methanol Ethyl acetates N-butanol 2.

4 1 48 h. AB. Fig. 1 Antimicrobial activity against of methanol of different concentrations from Spirodela polyrrhiza (L.) Schleid in 48 hours. A: inhibition zone; B: growth curve. 5 zh0003mic Table 5 Antimicrobial activity at different concentrations and MIC of from Spirodela polyrrhiza (L.) Schleid zh0003 Methanol 95% Methanol 95% Ethanol Ethyl acetates N-butanol zh0225mic Table 6 Antimicrobial activity at different concentrations and MIC of methanol from Spirodela polyrrhiza (L.) Schleid zh0225 Methanol Ethyl acetates N-butanol 2.1(++++) 2.3(++++) (+++) 2.1(++++) 1.0(++) (++++) (++++) (++++) zh %MIC Table 7 Antimicrobial activity at different concentrations and MIC of 95% ethanol from Spirodela polyrrhiza (L.) Schleid zh %95% Ethanol Ethyl acetates N-butanol 1.8(+++) Chin J Appl Environ Biol

5 mg/ml zh mg/ml zh % 95%95% mg/ml mg/ ml+++ mg/ml y = x R 2 = % 9.845%9.39%5.97% 95%. zh0003 zh %8.1%5.94%6.78%. 1.0%22.46%17.2%1.6%. 2 1zh00182zh % 3zh00184zh0224 5zh00036zh02257 zh02258zh02259 zh022510zh0225. Fig. 2 Total flavonoids content of duckweed. 1: methanol of Lemna minor L.zh0018; 2: 95% ethanol of Lemna minor L; 3: water of Lemna minor L.; 4: methanol of Landoltia punctata; 5: methanol of Spirodela polyrrhiza zh0003; 6: methanol of Spirodela polyrrhiza zh0225; 7: petroleum of Spirodela polyrrhiza; 8: Ethyl acetates of Spirohela polyrrhiza; 9: of Spirodela polyrrhiza; 10: raffinate of Spirodela polyrrhiza HPLC 95%. 3 zh0225 zh00034a. zh022595% 8 Table 8 Antimicrobial activity of duckweed against several human, animal and plant pathogen strains Duckweed zh0018 Lemna minor L. zh0018 methanol zh0224 Landoltia punctata zh0224 methanol zh0225 Spirodela polyrrhiza zh0225 methanol zh0003 Spirodela polyrrhiza zh0003 methanol zh % Spirodela polyrrhiza zh % ethanol zh % Spirodela polyrrhiza zh % ethanol Spirodela polyrrhiza Ethyl acetates Spirodela polyrrhiza N-butanol Nesterenkonia Acinetobacter haemolytius Acinetobacter baumanii Pantoea agglomerans Klebsiella. peneumoniae Candida albicans penicillium italicum Wehmer Phytophthora Penicillium capsici Leon digitatum 0.85(+) 1.8(+++) 1.4(+++) 1.4(+++) 0.85(+) 1.1(+) 2.0(++++) 0.85(+) Chin J Appl Environ Biol

6 3 zh0003azh0225bhplc. [18] kromasil C nm10 μl1 ml min min 30%-40%20-30 min 40%-53%30-40 min 53%-90%40-50 min 90%. Fig. 3 The HPLC atlas of from Spirodela polyrrhiza zh0003 (A) and zh0225 (B). Flavonoids analysis conditions [18] : kromasilc18 column; UV detector monitored at 254 nm; injection volume: 10 μl; flow rate: 1.0 ml min -1. The mobile phase consisted of methanol and water, with gradient elution program as 30-40% methanol for 0-20 min, 40-53% for min, 53-90% for min and 90% for min Azh0003zh0225Bzh0225Czh0003. Fig. 4 Content of four kinds of flavonoids from Spirodela polyrrhiza. A: zh0003 andzh0225 ; B: methanol and ethanol of z h0003; C: methanol ext racts and ethanol of zh Bzh00034C. zh0018 zh0224 zh0003zh0225 zh0225 zh C-8-C- HPLC C- 8-C- HPLC. kromasilc18254 nm10 μl 0.6 ml min min 30%-40%30-45 min 40%- 53%45-60 min 53%-90%60-65 min 90%. Fig. 5 The HPLC atlas of standard substance of Luteolin 8-C-glucoside, Apigenin 8-C-glucoside, and Spirodela polyrrhiza. Kro masilc18 column: UV detector monitored at 254 nm; injection volume: 10 μl; flow rate: 0.6 ml min -1. The mobile phase consisted of methanol and water, with the following gradient elution program: 30-40% methanol for 0-30 min, 40-53% for min, 53-90% for min and 90% for min. 3.1 [19]. [20]. Chin J Appl Environ Biol

7 95%.. zh003zh0225 zh0225zh0003. zh0225 MIC 0.78 mg/ml. 3.2 [10-11] [20] [12]. y = x R 2 = %.. HPLC 8-C- 8-C-HPLC. [References] 1,,,,,.., 2010, 45 (4): Harvey RM, Fox JL. Nutrient removal using Lemna minor [J]. Water Pollut Control Fed, 1973, 45 (9): Zirschky J, Reed SC. The use of duckweed for waste water treatment [J]. Water Pollut Control Fed, 1988, 60: , [J]., 8, 12 (10): [Qian JH. Scavenging and application of aquatic plant to water pollutant [J]. J Hebei Agric Sci, 8, 12 (10): 73-74] 5,,,,,,. [J]., 2010, 38 (16): [Luo TC, Hou ET, Lu F, Gong J, Ni SF, Cui YT, Sun FY. Overview of pharmacological research on Spirodela polyrhiza (L.) Schleid [J]. J Anhui Agricult Sci, 2010, 38 (16): ] 6 Gao JP, Garrison AW, Hoehamer C, Mazur C, Wolfe NL. Uptake and phytotransformation of o, p -DDT and p, p -DDT by axenically cultivated aquatic plants [J]. J Agric Food Chem, 0, 48 (12): Gao J, Garrison AW, Hoehamer C, Mazur C, Wolfe NL. Uptake and phytotransformation of organophosphorus pesticides by axenically cultivated aquatic plants [J]. J Agric Food Chem, 0, 48 (12): Eid M, Kandil M, Moursy EB, Sayed G. Bioassays of duck weed vegetation [J]. Intern J Trop Insect Sci, 1992, 13 (5): [D]. :, 4 [Wang Y. Research on extraction and isolation of polysaccharide from Herba Spirodelae seu Lemnae and its primary pharmacodynamics [D]. Chongqing: Chongqing University, 4] 10,,,,. [J]., 1999, 30 (2): [Ling Y, He BZ, Bao YY. Studies on the chemical constituents of common ducksmeat (Spirodela polyrrhiza ) [J]. Chin Trad Herb Drugs, 1999, 30 (2): 88-89] 11,,,,,.., 2010, 27 (11): ,.., 9, 20 (4): [Peng L, Li ZM. The protective effect of Spirodela polyrrhiza (L.) Schleid extract on ECV- 304 injury induced by hydrogen peroxide [J]. Lishizhen Med Mat Med Res, 9, 20 (4): ] 13. [D]. :, 6 [Peng L. Study on extraction and purifieation technology of total flavonoids from Spirodela polyrrhiza (L.) Schleid and the bioactivity [D]. Chongqing: Chongqing University, 4] 14.. [J], 4 (2): [Zhu T. The mechanism of duckweed in the prevention and cure of gill rot, enteritis and skin rot of grass carp [J]. J Trad Chin Veter Med, 4 (2): 19-20] 15. [J].. 5, 23 (4): [Huang XD. The comparative studies on antibacterial activities of by different extracting solvents from the Syzygium buxifolium leaves [J]. J Wuhan Bot Res, 5, 23 (4): ] 16,,. - [J]., 1996 (4): [Yuan XM, Jiang MW, Hu ZZ. Colorimetric determination of Flavones in Hawthorn and its products [J]. Food Ferment Ind (4): 27-32] 17,. [J]. (), 2012, 10 (1): Qiao X, He WN, Xiang C, Han J, Wu LJ, Guo DA, Ye M. Qualitative and quantitative analyses of flavonoids in Spirodela polyrrhiza by highperformance liquid chromatography coupled with mass spectrometry [J]. Phytochem Anal, 2011, 22: Zhang Y, Hu Yb, Yang BY, Ma F, Lu P, Li LM,Wan CS, Rayner S, Chen SY. Duckweed (Lemna minor) as a model plant system for the study of human microbial pathogenesis [J]. PloS ONE, 2010, 5 (10): e [J]., 2011, 32 (11): [Huang XD. Total flavonoid contents, antibacterial activities and antioxidant activities of four from Dimocarpus longan seeds [J]. Food Sci, 2011, 32 (11): 43-47] Chin J Appl Environ Biol

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