58 34 1 1.1 中 药 材 和 标 准 品 ; 1,8 - 试 验 菌 种 (Flavobacterium columnare ) ; (Aeromonas sobria) (A. caviae) (Edwardsieda tarda) 培 养 基 (5 g) (5 g) (l0 g), (

34 1 Vol. 34, No.1 2010 1 ACTA HYDROBIOLOGICA SINICA Jan., 2 0 1 0 DOI: 10.3724/SP.J.1035.2010.00057 郑 曙 明 黄 建 军 吴 青 沙 莎 (, 402460) :, ;,, GF254,.... (7..3..1), -, ;,.... (7..3..1),, ; 1,8-,, 10.47,, : > ( 1,8 -)>>>, : ; ; ; ; 中 图 分 类 号 : S917 文 献 标 识 码 : A 文 章 编 号 : 1000-3207(2010)01-0057-08,, [1] ; Aeromonas hydrophila [2] ; 10,,, [3] ; 3,, 0.1102 g/kg [4] ;, [5],,,, 82d [6],,,, ( ) (TLC),,, 收 稿 日 期 : 2008-06-02, 修 订 日 期 : 2009-03-16 基 金 项 目 : (KJ070618) 通 讯 作 者 : (1957 ),, ;, ; Tel: 023-46751115; E-mail: zhsm22@163.com

58 34 1 1.1 中 药 材 和 标 准 品 ; 1,8 - 试 验 菌 种 (Flavobacterium columnare ) ; (Aeromonas sobria) (A. caviae) (Edwardsieda tarda) 培 养 基 (5 g) (5 g) (l0 g), (0.5 g) (0.2 g)(0.2 g) (0.5 g),,,, ph 7.6 7.8 1.5%, 115 30min 1.2 样 品 液 的 制 备 : 2 g, 70% 16 ml, ph 2, 76 90min, 3,,, 100 ml,,, 3,,, 10 ml, :,,,,, 10 ml, ( 1,8- ), 4, : 35 24h,, 0.9 1.5 /ml, 复 方 有 效 成 分 的 薄 层 分 析, R, R : 10 cm 20 cm,,,, 939, 95% GF254 15 g, 0.5,, 0.3 mm,, 110 0.5h, : 2 cm, 1.5 cm, 2 3 mm,, : 9,,.... (7..3..1) 20 ml,,,, 1 2 cm,,, : -, 1%,, Rf 柱 层 析 分 离 复 方 有 效 成 分 : 2.0 g,, :, 30 g,,,, :, 2.0 g,,, :,.... (7..3..1) :,, TLC,

1 : 59 体 外 抑 菌 试 验 : 1.5 /ml,, 6 mm, 30 μl 37 24h,, 3 : 1.5 /ml, 6 mm, 30 μl 37 24h,,,, - 12 15 17 21 24 30 33 47 49 77 80 93 1 TLC Fig. 1 The singles and the compound TLC chart 1 2. ; 3 4. ; 5 6. ; 7 8. 1, 2. Chinese nutgall; 3, 4. Chinese compound nutgall; 5, 6. Rhubarb; 7, 8. Scutellaria 2 2.1 1, 3, 3,,,, 2,,,, R, TLC R 1,8-2.2,.... (7..3..1), 1min, TLC, 93 ( 3) A-G, 1 11 1,8 2 TLC Fig. 2 Chinese compound nutgall TLC chart A. ; B. ; C. ; D. ; E. ; F. ; G. 1,8 - A. Tannins; B. Gallic acid; C. Quercetin; D. Baicalin; E. Aloe emodin; F. Emodin; G. 1,8-2 Hydroxy anthraquinone 2.3 ( 1) :,,,,, ( 4 5) : > ( 1,8 - )>>>

60 34 Germ Aeromonas sobria Aeromonas caviae Edwrdsieda tarda Flavobacterium columnare 3 TLC Fig. 3 Result of the separation samples by column chromatography with TLC detection G. 1,8-1 11. 1,8 - D. 24 30. C. 37 47. B. 49 77. A. 80 93. G. Standard 1,8-2 hydroxy anthraquinone 1 11. Separation sample 1, 8-2 hydroxy anthraquinone D. Standard baicalin 24 30. Separation sample baicalin C. Standard quercetin 37 47. Separation sample quercetin B. Standard gallic acid 49 77. Separation sample gallic acid A. Standard tannins 80 93. Separation sample tannins Tab. 1 表 1 复 方 五 倍 子 抗 菌 剂 各 有 效 成 分 的 抑 菌 作 用 Antibacterial action of the effective components of Chinese compound nutgall Anthraquinone Quercetin Quercetin Antibacterial ring (mm) Gallic acid Anthraquinone Chinese compounds nutgall 18 16 10 28 0 30 16 15 8 25 0 28 20 18 13 30 0 35 17 17 10 26 0 30 2.4 : ( 6), : ( 7), : ( 8), : ( 9),

1 : 61 4 Fig. 4 Antibacterial effect of the components of Chinese compound nutgall to Aeromonas sobria 1. ; 2. ; 3. ; 4. ; 5. ; 6. ; 7. 1. Gallic acid; 2. Anthraquinone; 3. Baicalin; 4. Quercetin; 5. Tannins; 6. Chinese compound nutgall; 7. Chinese nutgall; the same as follows 8 Fig. 8 Antibacterial result of allic acid and quercetin Fig. 9 9 Antibacterial result of anthraquinones and baicalin 5 Fig. 5 Antibacterial effect of the components of Chinese nutgall to Aeromonas sobria 6 Fig. 6 Antibacterial result of gallic acid and anthraquinones 7 Fig. 7 Antibacterial result of gallic acid and baicalin 3 3.1, [8] 1,8 -, (84 MRSA 28 MSSA) [9]., ( ), 10.47,,,,,,, [8],

62 34,,,,, 4.2% 28 8 [9],, MIC 0.3125 0.125 mg/ml [10], [11] 1, 8-, 1.51%,, DNA DNA, [12], [13],, [9],,,,,,,,, 3.2,,, ;,, 5 mm 4 mm,,, 3 mm,, [14],,, [15].... (7..3..1),, ;.. (7..3) (7..3),,, ;.. ( 1..1 6..4 7..3),,, ;.... ( 7..3..1 7..3..0.5),,.... (7..3..1),,,,,,,,,,,,,,,, :

1 : 63 1, 8-,,, ;,, R, R, 3.3,,,,, ;,,,, ;,,,,,,,,.... (7..3..1),,,,,.... (7.. 3..1),, TLC : [1] Li Q, Zhang Y J, Hua R Q, et al. Antibacterial experiment of 23 Chinese medicinal herbs and compound prescriptions on 3 bacteria from the gut of Carassius auratus in vitro [J]. Freshwater Fisheries, 2007, 37(4): 7 11 [,,,. 23 3., 2007, 37(4): 7 11] [2] Wang W B, Fang P, Lin X T, et al. The immunoregulative effects of liquorice extract on crucian [J]. Acta Hydrobiologica Sinica, 2007, 31(5): 654 660 [,,,.., 2007, 31(5): 654 660] [3] Zhang H B, Zhang S X. Primary studies on the drug resistance of Aeromonas Sobria popoff and veron to 10 kinds of Chinese herbs [J]. Journal of Xinyang Normal University, 2003, 16(1): 39 41 [,. 10. ( ), 2003, 16(1): 39 41] [4] Jiang L, Zheng S M. Inhibition and toxicity of gall to 3 species bacteria [J]. Reservoir Fisheries, 2005, 25(6): 89 90 [,. 3., 2005, 25(6): 89 90] [5] Jiang L, Zheng S M. Pharmacodynamic study on Chinese nutgall against bacterial septicemia of Carp (Cyprinus carpio) [J]. Chinese Agricultural Science Bulletin, 2006, 22(5): 460 464 [,.., 2006, 22(5): 460 464] [6] Zhou Y, Zheng S M. Study of thermostability of compound galla oral liquid by classical homeothermia [J]. Journal of Southwest Agricultural University, 2006, 28(6): 961 965 [,.. ( ), 2006, 28(6): 961 965] [7] Li Z X, Wang X H, Zhang M M, et al. In vitro antibacterial activity of ethanol-extract of Galla chinensis against Staphycoccus aureus [J]. Traditional Chinese Drug Research & Clinical Pharmacology, 2005, 16(2) : 103 105 [,,,.., 2005, 16(2) : 103 105] [8] Shi B, Di Y, He Y J, et al. Pharmacology activation of tannins [J]. Chinese Traditional and Herbal Drugs, 1998, 29(7): 487 490 [,,,.., 1998, 29(7): 487 490] [9] Wang X P, Zhen L, Zhou Z T, et al. Effects of the compositions of 28 Chinese herbs on the 8 bacteria in oral cavity [J]. Chinese Journal of Integrated Traditional and Western Medicine, 2005, 6(6): 240 244 [,,,.., 2005, 6(6): 240 244] [10] Liu I X, Durham D G, Richards RM. Baicalin synergy with beta-lactam antibiotics against methicillin-resistant Staphylococcus aureus and other beta-lactam-resistant strains of S. aureus [J]. Pharmacy and Pharmacology, 2000, 52(3):

64 34 361 366 [11] Krakauer T, Li B Q, Young H A. The flavonoid baicalin inhibits superantigen-induced inflammatory cytokines and chemokines [J]. FEBS Letters, 2001, 500(1 2) : 52 55 [12] Li S J, Dong X H, Wu H X, et al. Progress for research of pharmacology effect of rhubarb and active ingredient [J]. Medical Recapitulate, 2005, 11(1): 76 79 [,,,.., 2005, 11(1): 76 79] [13] Woo S W, Nan J X, Lee S H, et al. Aloe emodin suppresses myofibroblas2 tic differentiation of rat hepaticstellate cells in primary culture [J]. Pharmacol Txicol, 2002, 90(4): 193 198 [14] Xie P S, Yan Y Z. Optimization of the TLC of protoberberine alkaloids and fingerprint valuation of the cortidis rhizome [J]. Planar Chromatography Modern TLC, 1992, 5(5): 302 307 [15] Pharmacopoeia committee of The People's Republic of China Health Department. Chinese Pharmacopoea TLC ATLAS of Traditional Chinese Herb Drugs [M]. Guangzhou: Guangdong Science and Technology Publishing House Press. 1993, 12 16 [. [M]. :. 1993, 12 16] STUDIES ON SEPARATION AND ANTIBACTERIAL ACTIVITY OF THE EFFECTIVE COMPONENTS OF CHINESE HERBAL COMPOUNDS GALLNUT FOR FISHERY ANTIMICROBIAL AGENT ZHENG Shu-Ming, HUANG Jian-Jun, WU Qing and SHA Sha (Fish Breeding and Healthy Culture Research Center, Southwest University, Chongqing 402460) Abstract: The effective components of Chinese Herbal Compounds Gallnut for fishery antimicrobial agent are separated and identified by thin-layer chromatography and column chromatography. Through antibacterial test and joint drug test of several aquatic animal pathogens in vitro, the antibacterial strength of active ingredients of the compound and the inter-relationship between the drug components are determined. The mechanism of Chinese Herbal Compounds Gallnut is supplied with the theoretical basis and the quality control is provided for its reference. The lamina is made with silica gel GF254 and the developing agent is spread with benzene: ethanol: formic acid (7:3:1). Moreover, it is colorized with NH 3 -FeCl 3 and the result should be observed under sunlight. The monomer is isolated through dry silicon column loaded and it is eluted with benzene: ethanol: formic acid (7:3:1). The active ingredients of Chinese Herbal Compounds Gallnut are identified for physical and chemical properties and by thin-layer chromatography analysis. The results shows that the effective components of Chinese Herbal Compounds Gallnut are tannins, gallic acid, quercetin, baicalin, aloe emodin, emodin, 1, 8-2 hydroxy anthraquinone. The most content monomer is gallic acid by UV spectrophotometry for 10.47%. The antibacterial action for Aeromonas sobria, A. caviae, Edwrdsieda torda and flavobacterium columnare is tested. Aeromonas sobria selected as the object and the main active ingredients for two matched, antibacterial action in vitro is tested. Antibacterial order of the active ingredients is as follows: gallic acid > anthraquinone > baicalin > quercetin > tannide. There is synergy between gallic acid and anthraquinone, there is cumulative effect between gallic acid and baicalin, gallic acid and quercetin, anthraquinone and baicalin, respectively. Key words: Chinese compound gallnut; Effective component; Thin-layer chromatography; Column chromatography; Antibacterial activity