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3 - 3 - BIA DNA 24% MTT 16 N350 Streptomyces purpurascens var. marinus N350 DMSO λ233nm 252nm 480nm 494nm 05% 540nm 579nm OD 01NNaOH E556nm/E596nm=111 HPLC N350 LD 50 30mg/kg P388 KB IC ng/mL 250ng/mL 10mg/kg S % ILS 829% N350 1L 30g 40g CaCO 3 5g NaNO 3 1g K 2 HPO 4 1g MgSO 4 7H 2 0 1g 10% 500mL 60mL 28 5d 1186mg/L BIA

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5 - 5 - STUDY ON THE ANTITUMOR AGENT FROM A MARINE ACTINOMYCETES, N350 ABSTRACT In this work, an improved BIA spot test was utilized as an antitumor prescreen model. 16 strains, out of all 655 tested marine microorganisms isolated from the intertidal zone of the Xiamen coast, Taiwan Strait, were found having positive induction activity. Moreover, strong activity of an actinomycetes, N350 was observed using BIA liquid test in parallel with MTT assay. The N350 strain was subsequently taxonomically proposed as Streptomyces purpurascens var. marinus, basing on its morphological features, culture characteristics, physiological and biochemical charac- teristics. One of characteristics of this antitumor agent is similar to ph indicator, and it is soluble in methanol, ethanol, benzene, dimethyl sulfoxide and acidic water but insoluble in water and petroleum benzine. The induction activity of the separated spots was detected by BIA in situ after developed in the TLC (solvent system: chloroform-methanol (3:2)). Then the crude antitumor substance was preliminarily purified and the Rf 0.73 spot, which has identical chemical color development results with those of anthracyclines, such as adriamycin, was found to be bioactive. The other physic-chemical properties of this antitumor agent, including UV/VIS spectrum( 233nm, 252nm, 480nm, 494nm ), the absorptance at 540nm and 579nm turning to be higher when solved in 0.5% Mg(OAC) 2 -MeOH while the color of the solution becoming purple, the ratio of E556nm/E596nm being 1.11 in 0.1N NaOH-MeOH and its aglycone having the same retention time of HPLC as daunomycinone, demonstrated that it belongs to the same group of hydroxyl anthra- quinone chromophores as daunomycin. This antitumor substance exhibits inhibitory activity against Gram-positive bacteria. It also presents strong cytotoxicity to P388 and KB cell in vitro, IC 50 being 156ng/mL and 250ng/mL respectively, while showing LD 50 30mg/kg in the acute toxic experiment to mice. In addition, its inhibitory activities against S180 solid tumor and Ehrlich Ascites carcinoma in mice, inhibition rate and ILS being 34.2% and 82.9% respectively, showed a promising applicable value. The optimal fermentation conditions were established. With 10% inoculum and 500mL Erlenmeyer flask containing 60mL fermentation medium, which was made from artificial sea water and in each liter consists of 30g soybean powder, 40g soluble starch, 5g CaCO 3, 1g NaNO 3, 1g K 2 HPO 4, 1g MgSO 4 7H 2 O, the fermentation was carried out at 28 for 5 days on a rotary shaker and the production of the active substance was 118.6mg/L. Keywords: Marine actinomycetes; Antitumor agent; BIA

6 [1] / /10 [2] [4] Dougherty Gardner [6] Welch Farber VP-16 FT [6] DNA C [7] 1 DNA DNA JM-216 [7] [6] Pt DNA [5] [3]

7 - 7-2 Taxotere [8] Vinorelbine [9,10] Rhizoxin [11] (Rhizopus sinensis) 16 IC 50 ng/ml 10 Combrestain 3 Iododoxorubicin MX-2 Anthrapyrazoles [13] Mitoxantrone Losoxantrone CI-941 [14] 4 DNA DNA MST-16 [15] CPT-11 5 DNA DNA DNA A-T DNA Carzalesin U A DNA U [17] L1210 [18] 6 Sulofenur LY LY LY Suramin PDGF TGF EGF FGF Bombesin Somatostatin [16] [20] 8 PTK Genistein Erbstatin Lavendustin 4- PTK 9 C PKC PKC PKC Staurosporine Calphostin Staurosporine [21] PKC A PKA 8- Ilmofosine Vorozole 1 [22] A RA [23,24] [12] [19]

8 TTNPB arotinoid RA 1000 [25] MGI D 3 camp - - D HMBA N- NMF [11] NCI [27,28,29] 40~45% [30] [26]

9 [31] 100 Omura [32] 71% [33] Bergmann [34] [35] 3700 Harada H Cladophoropsis vaucheriaeformis L1210 NIH-3T3 C. Vaucheriaeformis L1210 [36,37] Callystatin A KB IC ng/mL [38] Ecteinascidin-743(ET-743, NSC ) [39] Aerophysinin 1 [40] Didemnin B [41] Dercitin [42] Bryostatin 1 [43] Punaglandin [44] Namenamicin [45] Echthinaschidine [33] 1945 C [46] [47] Claude ZoBell [31] Rosenfeld ZoBell 1958 Grein Meyers [48] Burkholder Pentabro- mpseudiline [31]

10 Okami 1975 Sagami Chainia Purpurogena SS-228Y [49,50,51] C-237 Macrolactin A [52] B16-F10 IC 50 35µg/mL IC 50 =50µg/mL HIV(IC 50 =10µg/mL) Altemicidin Streptomyces Sioyaensis SA-1758 L1210 IMC IC µg/mL 082µg/mL [53,54] Acklins Aspergillamides A B HCT-116 IC 50 =16ng/mL [55] CAÑEDO LM [56] Bacillus sp. PhM-PHD-090 Isocoumarin PM P388 A-549 HT-29 MEL-28 IC µM Palaemon [31] Macrodactylus Monaghan [57] [58] Dercitamide Dercitus sp. Cystodytes sp. Diketopiperazine Tedania ignis [59] Okadaic acid P388 L1210 IC µg/ml 012mg/kg [60] lima Halichondria okadai [61] Prorocentrum Pconcavum Dinophysis acuminata [62] Okadaic acid Sagami Flavobacterium ugliginosum MP-55 Marinactan S180 10~50mg/kg 10 79~90% Amphidinium sp. 3 Amphidinolide A B C L1210 IC µg/mL [33] [63,64] Moore Scheuer Zooanthid Palythoatoxica [65] 1/10 ROMERO F. Thiocoraline L-13-ACM2-092 P388 A-549 MEL-28 [66,67] DNA RNA

11 - 11-1% [68] 1% 5% [31] [69] µg/mL 80%~100% P388 2 BIA PIA BIP DNA [70] 1953 Lwoff λ DNA DNA λ DNA

12 SP10 BIA [71] Lein 1962 BIA -LacZ DNA [72] Moreau [73] BIA Steinburg [74] DNA PIA DNA 3 mechanism-oriented 90% DNA DNA DNA DNA DNA DNA HTCA [75] 72

13 [73] [76] 2 SDI Test MTT Methy-thiazolyltetrazolium Formazan H H-TdR 3 H-TdR cpm 4 Colorimetric Assay-MTT [77,78] SDI Test Mosmann MTT 1988 Paull XTT MTT MTT XTT Bsattke MTT MTS [76] 5 ATP MMC 5-Fu BLM ADM cddp 3 ATP ATP 4 ATP ATP FCM [79] NAG AgNOR [80,81,82] 1 100% 2 6 [69] NCI [22] 60

14 %

15 BIA (Escherichia coli.)br513 (Staphylococcus aureus) (Bacillus subtilis) (Saccharomyces cerevisiae) (Escherichia coli) 2 P388D1 KB 2 MTT RR BNG 6-bromo-2-naphthyl- -D-galactopyranoside Sigma ONPG o-nitrophenyl- -D-galactopyranoside X-Gal 5-Bromo-4-Chloro- 3-Indolyl- -D-Galactopyranoside ( ) C Kyowa Hakko Kogyo Co.Ltd DMSO DNA 3 1 Sigma Farmitalia Carlo Erba Milano-Italy) 1 1 mol/l Tris 850mL 1211g Tris 1000mL 2 50 Medium E 100g MgSO 4 7H 2 O 1000g Citric acid H 2 O 5000g K 2 HPO g NaH 2 PO ml min 3 Medium A 105g K 2 HPO 4 45g KH 2 PO 4 1g (NH 4 ) 2 SO 4 05g 1000 ml min

16 ZCM 61g Na 2 HPO 4 7H 2 O 55g NaH 2 PO 4 H 2 O 075g KCl 0246g MgSO 4 7H 2 O 27mL - 25mg 1000mL ph PBS Na 2 HPO 4 144g KH 2 PO 4 024g KCl 02g NaCl 8g ph mL min 6 D-Hank s NaCl 80g KCl 040g KH 2 PO 4 006g Na 2 HPO 4 H 2 O 060g NaHCO mL min 7 002%Versene EDTA 01g D-Hank s 500mL 37%NaHCO 3 ph min 8 D-Hank s % SDS-001N HCl SDS 100g 1N HCl 10mL 1000mL 10-1mL 10mL 11 05g 100mL 12-1mL 50mL 05mL 13 NaCl 298g KCl 073g MgCl 2 6H 2 O 107g MgSO 4 7H 2 O 54g CaCl 2 2H 2 O 11g 1000mL 14 5mg/mL DNA [83] 10mg DNA 2mL 4 20min 22 18h -70 (2) 1 RMPI 1640 Gibco BRL 2 LB NaCl 10g 5g 10g 1000mL ph min 20g LBE NaCl 10g 5g 10g 1M Tris 5mL 1000mL ph min 4mL 50 Medium E 10mL 20% 20g 20g NaCl 05g KNO 3 1g K 2 HPO 4 3H 2 O 05g MgSO 4 7H 2 O 05g FeSO 4 7H 2 O 001g 15~20g 1000mL ph 74~ min PDA 200g 30min 20 g ph 68~ mL 20g 20g 15g 5g 25g CaCO 3 1g 1000mL ph 75~80 25g 10g 5g CaCO 3 3g (NH 4 ) 2 SO 4 25g 1000mL ph 30g 40g CaCO 3 5g NaNO 3 1g K 2 HPO 4 1g MgSO 4 7H 2 O 1g 1000mL ph 15g 10g 20g 40g CaCO 3 5g 1000mL ph 20g 15g 5g 25g CaCO 3 1g

17 Degree papers are in the Xiamen University Electronic Theses and Dissertations Database. Full texts are available in the following ways: 1. If your library is a CALIS member libraries, please log on and submit requests online, or consult the interlibrary loan department in your library. 2. For users of non-calis member libraries, please mail to etd@xmu.edu.cn for delivery details.

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