鏈霉菌H41-55發(fā)酵菌絲體的化學成分

李先盛， 鄭新恒， 楊宇， 林壁潤， 周光雄

(1.暨南大學 藥學院中藥及天然藥物研究所∥廣東省普通高校中藥和天然藥物藥效物質(zhì)基礎(chǔ)重點實驗室， 廣東 廣州 510632；2. 廣東省農(nóng)業(yè)科學院 植物保護研究所∥廣東省植物保護新技術(shù)重點實驗室， 廣東 廣州 510110)

放線菌是一類遺產(chǎn)物質(zhì)中具有高鳥嘌呤和胞嘧啶(G+C)含量的革蘭氏陽性細菌，是海洋微生物中一個重要的類群，廣泛分布在海洋各種環(huán)境中，因產(chǎn)生豐富的活性次生代謝產(chǎn)物而著名.多年來，因近海岸和紅樹林沉積物以及淺海動植物等環(huán)境使放線菌采樣容易，故有較悠久的研究歷史和相對較多的研究報道，并有一定研究深度[1].放線菌能產(chǎn)生多種多樣的化合物，如肽類、大環(huán)內(nèi)酯類、生物堿類、萜類、醌類、有機酸類等，其中有很多化合物具有抗菌、抗腫瘤、抗病毒等活性[2].為從海洋來源放線菌中發(fā)現(xiàn)活性新化合物，本研究就鏈霉菌屬H41-55菌株發(fā)酵菌絲體進行了系統(tǒng)的化學成分研究.首先用高體積分數(shù)的乙醇提取，再用乙酸乙酯萃取獲得乙酸乙酯部位，對該部位進行化學成分的分離和結(jié)構(gòu)鑒定，最終分離并鑒定了15個單體化合物(圖1).該研究成果進一步豐富了鏈霉菌屬放線菌次級代謝產(chǎn)物的多樣性，為后續(xù)活性成分和藥物先導物的發(fā)現(xiàn)奠定了一定的工作基礎(chǔ).

圖1 化合物1～15的化學結(jié)構(gòu)

1 材料與儀器

1.1 鏈霉菌菌株

鏈霉菌H41-55通過培養(yǎng)特征、形態(tài)特征、生理生化特性以及分子生物學分析，鑒定為抗生鏈霉菌 (Streptomycesantibioticus)[3].該菌種分離自廣東省江門市閘坡近海紅樹林的底泥，并保存于廣東省農(nóng)業(yè)科學院植物保護研究所.

1.2 細胞株

人乳腺癌細胞MCF-7、人神經(jīng)膠質(zhì)瘤細胞SF-268和人肺癌細胞NCI-H460均由暨南大學醫(yī)學院提供.

1.3 培養(yǎng)基

菌株培養(yǎng)基配方如下: 可溶性淀粉質(zhì)量20 g、KNO3質(zhì)量1 g、K2HPO4質(zhì)量0.5 g、MgSO4質(zhì)量0.5 g、NaCl質(zhì)量0.5 g、FeSO4質(zhì)量0.01 g、瓊脂質(zhì)量20 g、H2O體積1 000 L、海水晶3 g、pH 7.2～7.4 (用HCl、NaOH調(diào)整).

1.4 種子培養(yǎng)基

高氏一號培養(yǎng)基配方如下：玉米淀粉質(zhì)量3 g、酵母粉質(zhì)量3 g、海水晶質(zhì)量0.25 g、CaCO3質(zhì)量0.15 g、KNO3質(zhì)量0.1 g、MgSO4·7H2O質(zhì)量0.06 g、K2HPO4·3H2O質(zhì)量0.09 g、FeSO4·7H2O質(zhì)量0.002 g、H2O體積100 mL、pH 7.2～7.4 (用HCl、NaOH溶液調(diào)整).

1.5 儀器與試劑

LKYC-2型搖床(杭州郎琨科技有限公司)；Biostat C 30 L發(fā)酵罐(德國貝朗公司)；Agilent 1200型高效液相色譜儀(美國安捷倫公司)；CO2恒溫培養(yǎng)箱(美國Shel-Lab公司)；臺式離心機(德國Sigma公司)；液相分析色譜柱Ultimate XB-C18(5 μm，4.6 mm× 250 mm，美國Welch公司)；Finnigan LCQ Advantage MAX質(zhì)譜儀(美國菲尼根質(zhì)譜公司)；AV- 600和Bruker AV-300型核磁共振儀(瑞士布魯克公司)；液相半制備色譜柱Ultimate XB-C18(5 μm，10 mm× 250 mm，美國Welch公司)；Synergy 酶標儀(美國博騰儀器有限公司).硅膠(200～300目，青島海洋化工廠)；Sephadex LH-20(Pharmacia公司)；色譜級甲醇(山東禹王公司)；核磁用氘代試劑(Merck公司)；液相用水(廣東怡寶公司純凈水)，其他試劑均為分析純；DMSO(德國Sigma公司)；青-鏈霉素(美國Hyclone公司)；胰酶(美國Amresco公司)； 胎牛血清(美國Gibco公司)；四甲基偶氮唑鹽(MTT，德國Sigma公司)； RPMI-1640培養(yǎng)基(美國Gibco公司).

2 實驗方法

2.1 菌株的活化

將低溫保存的菌種H41-55接種到高氏1號固體培養(yǎng)基上，在30 ℃條件下恒溫培養(yǎng)1周.

2.2 種子液培養(yǎng)

于盛有體積為200 mL液體培養(yǎng)基的1 L三角瓶中接種已活化的菌株，28 ℃、165 r/min 搖床培養(yǎng)3 d.

2.3 放大培養(yǎng)

將體積為約1 L的種子培養(yǎng)液轉(zhuǎn)移至裝有20 L發(fā)酵培養(yǎng)基的發(fā)酵罐進行擴大培養(yǎng)，在28 ℃、300 r/min、通氣量15 L/min條件下培養(yǎng)4 d.

2.4 提取分離

菌株發(fā)酵液體積60 L，5 000 r/min 離心10 min后去掉上清液，菌絲體用體積分數(shù)為95%乙醇滲漉提取至上清液無色，減壓蒸干上清液獲得總提取物質(zhì)量521 g，將該提取物加水懸浮，用乙酸乙酯萃取至有機層無色，減壓蒸干乙酸乙酯得質(zhì)量為80 g乙酸乙酯部位.將該部位濕法上樣到硅膠色譜柱上，用石油醚/丙酮系統(tǒng)(100∶0- 0∶100)梯度洗脫，最后用甲醇沖柱，將餾分經(jīng)TLC薄層層析后，合并TLC結(jié)果相近餾分，得S1～S10共10個組分.S6經(jīng)過硅膠(V氯仿∶V甲醇=5∶1)、凝膠Sephadex LH-20(V氯仿∶V甲醇=3∶1)柱色譜和半制備HPLC(V甲醇:V水=77∶23)分離，得化合物1(質(zhì)量10.2 mg)、2(質(zhì)量5.7 mg)、3(質(zhì)量4.5 mg).S7經(jīng)過凝膠Sephadex LH-20柱色譜(V氯仿∶V甲醇=3∶1)、硅膠柱色譜(V石油醚∶V乙酸乙酯=4∶1)得化合物4(質(zhì)量7.9 mg).S8經(jīng)過硅膠柱色譜(V氯仿∶V甲醇=3∶1)和半制備HPLC(V甲醇∶V水=70∶30)得化合物5(質(zhì)量3.5 mg)、6(質(zhì)量9.3 mg)、7(質(zhì)量3.1 mg)、8(質(zhì)量4.1 mg).S9經(jīng)硅膠(V氯仿∶V甲醇=5∶2)、凝膠Sephadex LH-20柱色譜(V氯仿∶V甲醇=2∶1)、半制備HPLC(V甲醇∶V水=72∶28)得化合物9(質(zhì)量6.1 mg)、10(質(zhì)量7.2 mg)、11(質(zhì)量3.1 mg).S10經(jīng)ODS反相硅膠柱(V甲醇∶V水=10∶100-100∶0)、凝膠Sephadex LH-20柱色譜(V氯仿∶V甲醇=5∶1)和半制備HPLC(V甲醇∶V水=65∶35)得化合物11(質(zhì)量2.9 mg)、12(質(zhì)量4.0 mg)、13(質(zhì)量3.6 mg)、14(質(zhì)量4.6 mg)、15(質(zhì)量6.7 mg).

2.5 細胞培養(yǎng)

SF-268、NCI-H460和 MCF-7 均培養(yǎng)于含有質(zhì)量分數(shù)為10% FBS和1%青-鏈霉素的 RPMI 1640 培養(yǎng)基中，放置于37 ℃、CO2體積分數(shù)為5%的細胞培養(yǎng)箱中.待細胞生長融合度至80%，棄培養(yǎng)液，用PBS洗滌2次，加適量的胰酶室溫靜置消化5 min，置細胞在顯微鏡下觀察呈現(xiàn)成圓形，用移液槍吹打細胞數(shù)次，使細胞充分分離成單細胞懸液.取適量單細胞懸液用于傳代培養(yǎng)，保證每次傳代培養(yǎng)與實驗用細胞都處于生長對數(shù)期.

2.6 活性化合物的篩選

運用MTT 法測定所分離化合物細胞毒活性.將處于對數(shù)生長期的腫瘤細胞接種到96孔板，細胞密度為8 × 104/mL，12 h培養(yǎng)，待細胞融合度至70%～80%開始加藥.各化合物和陽性藥 (順鉑) 均溶于DMSO中，配制成質(zhì)量濃度為10 μg/mL的母液.設(shè)順鉑組、空白組和樣品組，給藥的最大質(zhì)量濃度為100 μg/mL，隨后采用倍半稀釋的方法依次降低質(zhì)量濃度 (50、25、12.5、6.3、3.1、1.6 μg/mL).加藥后孵育2 d，然后加入30 μL MTT 溶液 (質(zhì)量濃度為5 μg/mL)再孵育2 d.孵育后，棄上清液，加入100 μL DMSO震蕩搖勻.使用酶標儀在570 nm 處測定樣品吸光度值，然后用軟件Origin 8 (OriginLab，Northampton，MA USA)計算出各個化合物的IC50值.

3 實驗結(jié)果

3.1 結(jié)構(gòu)鑒定

化合物1:黃色晶體; mp. 195～196 ℃; HR-ESI-MS:m/z146.060 0 [M+H]+(C9H8NO理論計算值, 146.060 6)；1H NMR (600 MHz, MeOH-d4)δ: 11.16 (1H, br. s, N-H), 10.05 (1H, s, H-9), 8.22 (1H, d,J=7.5 Hz, H- 4), 8.17 (1H, s, H-7), 7.52 (1H, d,J=7.9 Hz, H-1), 7.27 (1H, td,J=7.4, 1.2 Hz, H- 6), 7.24 (1H, td,J=7.4, 1.1 Hz, H-5);13C NMR (150 MHz, MeOH-d4)δ: 185.3 (C-9), 138.3 (C- 8), 138.0 (C-1), 125.5 (C-3), 124.5 (C- 6), 122.0 (C-5), 122.1 (C- 4), 120.1 (C-2), 112.9 (C-7).以上波譜數(shù)據(jù)與文獻[4]報道一致，故鑒定其為吲哚-3-甲醛.

化合物2:淡黃色油狀; mp. 79～81 ℃; HR-ESI-MS:m/z223.094 7 [M+Na]+(C10H16O4Na理論計算值, 223.094 7);1H NMR (400 MHz, DMSO-d6)δ: 5.92 (1H, br. s, H-5), 4.17 (1H, d,J=8.5 Hz, H-1′), 3.19 (1H, br. s, OH-1′), 2.01 (3H, s, CH3- 4), 1.78 (2H, m, H-2′,3′), 1.22 (1H, m, Hβ-3′), 0.92 (3H, t,J=7.5 Hz, CH3- 4′), 0.76 (3H, d,J=7.0 Hz, CH3-2′);13C NMR (100 MHz, CDCl3)δ: 172.2 (C-2), 158.3 (C- 4), 130.4 (C-3), 100.2 (C-5), 71.4 (C-1′), 40.3 (C-2′), 25.2 (C-3′), 15.3 (2′-CH3), 12.0 (4-CH3), 11.4 (C- 4′).以上波譜數(shù)據(jù)與文獻[5]報道一致，故鑒定其為5-羥基-3- (1-羥基-2-甲基丁基)-1-甲基-2 (5H)-呋喃.

化合物3:無色晶體; mp. 141～142 ℃; HR-ESI-MS:m/z211.144 2 [M+H]+(C11H19N2O2理論計算值, 211.144 2);1H NMR (600 MHz, CDCl3)δ: 6.08 (1H, br. s, H- 8), 4.12 (1H, dd,J=9.0, 7.8 Hz, H- 6), 4.01 (1H, dd,J=9.3, 3.6 Hz, H-9), 3.62 (1H, m, Hβ-3), 3.55 (1H, td,J=9.0, 3.8 Hz, Hα-3), 2.36 (1H, m, Hβ-5), 2.14 (1H, m, Hα-5), 2.09 (1H, m, Hβ-10), 2.03 (1H, m, Hα- 4), 1.92 (1H, m, Hβ- 4), 1.76 (1H, m, H-11), 1.51 (1H, ddd,J=14.4, 9.2, 5.1 Hz, Hα-10), 1.01 (3H, d,J=6.6 Hz, H-13), 0.96 (3H, d,J=6.0 Hz, H-12);13C NMR (150 MHz, CDCl3)δ: 170.5 (C-7), 166.4 (C-1), 59.2 (C- 6), 53.6 (C-9), 45.7 (C-3), 38.8 (C-10), 28.3 (C-5), 25.0 (C-11), 23.5 (C-13), 22.9 (C- 4), 21.3 (C-12).以上波譜數(shù)據(jù)與文獻[6]報道一致，故鑒定其為cyclo- (L-Pro-L-Leu).

化合物4:白色粉末; mp. 162～163 ℃; HR-ESI-MS:m/z223.131 3 [M-H]-(C15H15N2理論計算值, 223.123 6);1H NMR (400 MHz, CDCl3)δ: 8.07 (1H, br. s, NH-1), 7.52 (1H, d,J=8.3 Hz, H- 4), 7.19 (1H, s, H-7), 7.12 (1H, m, H-2), 7.02 (1H, dd,J=8.3, 1.4 Hz, H-5), 5.37 (1H, m,J=7.3 Hz，H-2′), 3.78 (2H, s, H-1″), 3.42 (2H, d,J=7.3 Hz, H-1′), 1.77 (6H, s, 2 × Me-3′);13C NMR (100 MHz, CDCl3)δ: 137.3 (C- 6), 136.8 (C-7α), 132.5 (C-3′), 124.1 (C-3α), 123.7 (C-2), 122.3 (C-2), 119.8 (C-5), 118.3 (C-3), 117.8 (C- 4), 110.7 (C-7), 104.6 (C-2″), 34.4 (C-1′), 25.8 (3′-CH3), 17.9 (3′-CH3), 14.5 (C-1′).以上波譜數(shù)據(jù)與文獻[7]報道一致，故鑒定其為6- (3-甲基-2-丁烯基)-3-吲哚乙腈.

化合物5:無色針晶; mp. 134～139 ℃; HR-ESI-MS:m/z577.215 5 [M+Na]+(C29H34N2O9Na理論計算值, 557.216 3);1H NMR (300 MHz, CDCl3)δ: 12.57 (1H, s, OH-2′), 8.55 (1H, d,J=8.0 Hz, H- 4′), 8.52 (1H, s, H-CHO), 7.96 (1H, s, NH-3′), 7.34 (2H, dd,J=7.6, 7.4 Hz, H-5″,7″), 7.29 (2H, d,J=7.4 Hz, H- 4″,8″), 7.28 (2H, d,J=7.4 Hz, H- 6″), 7.23 (1H, d,J=8.0 Hz, H- 6′), 7.05 (1H, d,J=7.7 Hz, NH-1′), 6.91 (1H, dd,J=8.1, 8.0 Hz, H- 4), 5.73 (1H, dd,J=7.7, 6.6 Hz, H-10), 5.27 (1H, dd,J=7.8, 7.7 Hz, H-3), 5.08 (1H, dd,J=10.0, 9.6 Hz, H- 8), 4.94 (1H, dd,J=9.6, 6.3 Hz, H-9), 3.68 (2H, s, H-2″), 2.49 (1H, dt,J=10.7, 10.7 Hz, H-7), 1.64 (2H, m, H-α), 1.30 (3H, d,J=6.6 Hz, CH3- 4), 1.16 (3H, d,J=6.3 Hz, CH3-9), 1.14 (1H, m, H-β), 1.05 (1H, m, H-γ), 0.80 (3H, t,J=7.0 Hz, H-δ);13C NMR (75 MHz, CDCl3)δ: 173.0 (C- 6), 170.3 (C-2), 170.3 (C-1″), 169.6 (1′-CO), 159.1 (CHO), 150.8 (C-2′), 133.3 (C-3″), 129.4 (C- 4″,8″), 129.0 (C-5″,7″), 127.7 (C-3′), 125.0 (C- 4′), 120.3 (C- 6′), 119.2 (C-5′), 112.8 (C-1′), 76.1 (C- 8), 75.0 (C-9), 71.1 (C- 4), 53.8 (C-3), 50.3 (C-7), 41.8 (C-2″), 29.4 (C-β), 28.1 (C-α), 22.5 (C-γ), 17.9 (9-CH3), 15.2 (4-CH3), 14.0 (C-δ).以上波譜數(shù)據(jù)與文獻[8]報道一致，故鑒定其為antimycin A9.

化合物6:無色針晶; mp. 130～133 ℃; HR-ESI-MS:m/z487.205 0 [M+Na]+(C23H32N2O8Na理論計算值, 487.205 8);1H NMR (600 MHz, CDCl3)δ: 12.68 (1H, br. s, 2′-OH), 8.55 (1H, d,J=8.5 Hz, H- 4′), 8.50 (1H, d,J=1.7 Hz, 3′-CHO), 7.92 (1H, s, NH-3′), 7.24 (1H, d,J=8.1 Hz, H- 6′), 7.09 (1H, d,J=7.6 Hz, 8-OH), 6.92 (1H, t,J=8.5 Hz, H-5′), 5.73 (1H, m, H- 4), 5.24 (1H, t,J=7.4 Hz, H-3), 4.87 (1H, m, H-9), 3.58 (1H, m, H- 8), 2.34 (1H, m, H-7), 1.58 (2H, m, H-1″), 1.47 (2H, d,J=6.2 Hz, H-2″), 1.30 (2H, m, H-3″), 1.29 (2H, m, H- 4″), 1.27 (2H, m, H-5″), 1.27 (3H, m, H- 6″);13C NMR (150 MHz, CDCl3)δ: 174.2 (C- 6), 170.4 (C-2), 169.6 (C-11), 159.0 (3′- CHO), 150.8 (C-2′), 127.6 (C-3′), 125.0 (C- 4′), 120.4 (C- 6′), 119.2 (C-5′), 112.8 (C-1′), 77.4 (C- 8), 76.5 (C-9), 70.9 (C- 4), 53.9 (C-3), 52.7 (C-7), 31.8 (C-δ), 29.3 (C-ε), 29.1 (C-α), 27.4 (C-γ), 22.7 (C-β), 18.6 (9-CH3), 15.2 (4-CH3), 14.2 (C-ζ).以上波譜數(shù)據(jù)與文獻[9]報道一致，故鑒定其為kitamycin A.

化合物7:紅色油狀; mp. 44～45 ℃; HR-ESI-MS:m/z817.609 8 [M+Na]+(C54H82O4Na理論計算值, 817.611 5);1H NMR (300 MHz, CDCl3)δ: 5.11 (8H, m, H- 6′,10′,12′), 4.93 (1H, m, H-2′), 4.01 (3H, s, H-2), 3.98 (3H, s, H-3), 3.18 (2H, d,J=7.0 Hz, H-1′), 2.06 (16H, m, H-3,5′,9′,17′,21′,25′,29′,33′), 2.01 (3H, s, H-5), 1.97 (16H, m, H- 4′,8′,12′,16′,20′,24′,28′,32′), 1.75 (3H, br. s, H-3′), 1.69 (3H, br. s, H-35′), 1.60 (3H, s, H-36′);13C NMR (75 MHz, CDCl3)δ: 184.8 (C-1), 183.9 (C- 4), 144.4 (C-3), 144.2 (C-2), 141.7 (C- 6), 138.9 (C-5), 137.7 (C-3′), 135.3 (C-7′), 135.0 (C-11′,15′), 134.9 (C-19′,23′,27′), 134.9 (C-31′), 131.3 (C-35′), 124.4 (C- 6′), 124.3 (C-10′,14′,18′,22′,26′), 124.2 (C-30′), 123.9 (C-34′), 118.8 (C-2′), 61.2 (C-2,3), 39.7 (C- 4′,8′,12′,16′,20′,C-24′,28′,32′), 26.8 (C-3′,5′,9′), 26.7 (C-17′,21′,25′), 26.5 (C-29′,33′), 25.7 (35′-CH3), 25.3 (C-1′), 17.7 (C-36′), 16.4 (3′-CH3), 16.0 (7′-CH3), 16.0 (31′-CH3), 12.0 (5-CH3).以上波譜數(shù)據(jù)與文獻[10]報道一致，故鑒定其為ubiquinone Q9.

化合物8:無色結(jié)晶; mp. 242～243 ℃; HR-ESI-MS:m/z431.352 0 [M+H]+(C28H47O3理論計算值, 431.352 7); 香草醛-濃硫酸溶液顯紫紅色，Liebermann-Burchard反應呈綠色，推測其為甾醇類化合物;1H NMR (600 MHz, MeOH-d4)δ: 5.26 (1H, m, H-7), 5.23 (2H, dd,J=12.0, 6.0 Hz, H-22,23), 3.98 (1H, m, H-3), 1.07 (3H, s, H-19), 1.06 (6H, d,J=12.0 Hz, H-26,27), 0.96 (3H, d,J=6.0 Hz, H-21), 0.63 (3H, s, H-18);13C NMR (150 MHz, MeOH-d4)δ: 143.9 (C- 8), 137.2 (C-22), 133.4 (C-23), 119.2 (C-7), 77.1 (C-5), 74.4 (C- 6), 68.6 (C-3), 57.6 (C-17), 56.1 (C-14), 44.9 (C-13), 44.5 (C-9,24), 42.0 (C-20), 40.9 (C- 4), 40.7 (C-12), 38.3 (C-10), 34.5 (C-25), 34.1 (C-2), 31.9 (C-1), 29.3 (C-16), 24.2 (C-15), 23.2 (C-11), 21.8 (C-21), 20.6 (C-27), 20.2 (C-26), 19.1 (C-19), 18.4 (C-28), 13.0 (C-18).以上波譜數(shù)據(jù)與文獻[11]報道一致，故鑒定其為5α-麥角甾-7,22-二烯-3β,5,6β-三醇.

化合物9:無色針晶; mp. 178～180 ℃; HR-ESI-MS:m/z447.346 9 [M+H]+(C28H47O4理論計算值, 447.347 6);1H NMR (600 MHz, CDCl3)δ: 5.22 (1H, dd,J=15.2, 7.5 Hz, H-23), 5.17 (1H, dd,J=15.2, 7.5 Hz, H-22), 4.40 (2H, d,J=2.5 Hz, H- 6), 3.92 (1H, m, H-3), 3.17 (1H, d,J=2.5 Hz, H-7), 2.61 (2H, m, H-15), 2.32 (1H, m, H-9), 2.10 (2H, m, H- 4), 1.94 (2H, m, H-2), 1.86 (1H, m, H-24), 1.76 (2H, m, H-16), 1.04 (1H, d,J=6.6 Hz, H-21), 0.91 (1H, m,J=6.8 Hz, H-25), 0.87 (6H, m, H-18,19), 0.82 (3H, d,J=6.8 Hz, H-28), 0.80 (3H, d,J=6.8 Hz, H-27);13C NMR (150 MHz, CDCl3)δ: 153.5 (C-14), 136.1 (C-22), 133.1 (C-23), 126.0 (C- 8), 69.5 (C-3), 68.7 (C-5), 65.9 (C- 6), 62.1 (C-7), 57.6 (C-17), 43.8 (C-24), 43.7 (C-13), 40.4 (C-20), 40.1 (C- 4), 39.6 (C-9), 37.4 (C-12), 36.7 (C-10), 33.9 (C-26), 33.0 (C-1), 31.9 (C-2), 28.0 (C-16), 25.8 (C-15), 22.1 (C-21), 20.8 (C-28), 20.5 (C-27), 19.8 (C-11), 18.9 (C-19), 18.5 (C-18), 17.3 (C-25).以上波譜數(shù)據(jù)與文獻[12]報道一致，故鑒定其為麥角甾- 8 (14),22E-二烯-3β,5α,6β,7α-四醇.

化合物10:無色針晶; mp. 227～232 ℃; HR-ESI-MS:m/z467.349 5 [M+Na]+(C29H48O3Na理論計算值, 467.350 4);1H NMR (600 MHz, DMSO-d6)δ: 5.22 (1H, dd,J=15.0, 7.2 Hz, H-22), 5.16 (1H, dd,J=15.0, 8.4 Hz, H-23), 5.08 (1H, d,J=2.4 Hz, H-7), 4.50 (1H, d,J=5.4 Hz, H- 6), 4.26 (1H, d,J=5.4 Hz, H-3), 3.74 (1H, m, H-3), 3.60 (1H, s, H-5), 3.38 (1H, br. s, H- 6), 2.01-1.83 (6H, m, H-2,9,12,20,24), 1.83-1.77 (1H, m, H-14), 1.69-1.61 (2H, m, H-16), 1.61-1.21 (11H, m, H-1,2,4,11,15,17,25), 0.99 (3H, d,J=6.0 Hz, H-21), 0.91 (3H, s, H-19), 0.88 (3H, d,J=6.6 Hz, H-27), 0.81 (3H, d,J=7.2 Hz, H-28), 0.80 (3H, d,J=7.2 Hz, H-26), 0.55 (3H, s, H-18);13C NMR (150 MHz, DMSO-d6)δ: 139.7 (C- 8), 135.5 (C-22), 131.5 (C-23), 119.5 (C-7), 74.5 (C-5), 72.3 (C- 6), 66.0 (C-3), 55.4 (C-17), 54.3 (C-14), 43.1 (C-13), 42.3 (C-9), 42.1 (C-24), 40.3 (C-2), 40.1 (C-20), 39.0 (C-12), 36.7 (C-10), 32.5 (C-11,25), 31.2 (C- 4), 27.8 (C-16), 22.7 (C-15), 21.4 (C-1), 21.1 (C-21), 19.8 (C-26), 19.5 (C-27), 17.8 (C-19), 17.3 (C-28), 12.1 (C-18).以上波譜數(shù)據(jù)與文獻[13]報道一致，故鑒定其為豆甾-7,22-二烯-3β,5α,6α-三醇.

化合物11:無色針晶;mp. 170～172 ℃; HR-ESI-MS:m/z451.318 2 [M+Na]+(C28H44O3Na理論計算值, 451.319 0);1H NMR (300 MHz, CDCl3)δ: 5.21 (1H, dd,J=16.0, 7.0 Hz, H-23), 5.16 (1H, dd,J=16.0, 7.5 Hz, H-22), 4.40 (1H, br. s, H-7), 3.97 (1H, m, H-3), 3.31 (1H, d,J=2.5 Hz, H- 6), 1.11 (3H, s, H-19), 1.01 (3H, d,J=7.0 Hz, H-21), 0.90 (3H, d,J=6.5 Hz, H-28), 0.84 (3H, d,J=7.0 Hz, H-27), 0.82 (3H, d,J=7.0 Hz, H-26), 0.60 (3H, s, H-18);13C NMR (75 MHz, CDCl3)δ: 136.4 (C-9), 135.3 (C-22), 132.8 (C-23), 127.8 (C- 8), 69.5 (C-3), 68.0 (C-5), 66.5 (C-7), 63.5 (C- 6), 54.5 (C-17), 50.5 (C-13), 43.7 (C-24), 42.9 (C-20), 41.3 (C- 4), 40.0 (C-14), 38.9 (C-12), 36.5 (C-10), 33.9 (C-25), 31.7 (C-1), 31.1 (C-2), 29.9 (C-16), 24.7 (C-15), 24.3 (C-21), 23.7 (C-27), 21.8 (C-26), 20.9 (C-11), 20.6 (C-28), 18.6 (C-18), 12.2 (C-19).以上波譜數(shù)據(jù)與文獻[14]報道一致，故鑒定其為5α,6α-環(huán)氧- (22E,24R)-麥角甾- 8,22-二烯-3β,7α-二醇.

化合物12:無色針晶; mp. 183～184 ℃; HR-ESI-MS:m/z451.318 1 [M+Na]+(C28H44O3Na理論計算值, 451.319 0);1H NMR (300 MHz, CDCl3)δ: 5.24 (2H, m, H-22,23), 4.76 (1H, m, H-7), 4.39 (1H, m, H-3), 3.47 (1H, d,J=2.8 Hz, H- 6), 2.60 (1H, d,J=12.5, 11.5 Hz, Hβ- 4), 1.55 (3H, s, H-19), 1.01 (3H, d,J=6.8 Hz, H-21), 0.94 (3H, d,J=6.8 Hz, H-28), 0.88 (6H, d,J=6.8 Hz, H-26,27), 0.85 (3H, s, H-18);13C NMR (75 MHz, CDCl3)δ: 137.2 (C-9), 135.7 (C-22), 132.4 (C-23), 126.7 (C- 8), 68.7 (C-3), 67.1 (C-7), 63.4 (C-5), 60.3 (C- 6), 54.5 (C-17), 51.3 (C-14), 43.0 (C-24), 42.1 (C-13), 40.7 (C-20), 39.2 (C- 4), 38.1 (C-10), 36.3 (C-12), 33.3 (C-25), 31.0 (C-1), 29.4 (C-16), 23.8 (C-15), 23.2 (C-11), 23.1 (C-19), 21.2 (C-21),20.2 (C-27), 19.8 (C-26), 17.8 (C-28), 11.7 (C-18).以上波譜數(shù)據(jù)與文獻[15]報道一致，故鑒定其為5α,6α-環(huán)氧- (22E,24R)-麥角甾- 8,22-二烯-3β,7β-二醇.

化合物13:無色針晶; mp. 195～197 ℃; HR-ESI-MS:m/z467.313 1 [M+Na]+(C28H44O4Na理論計算值, 467.313 9);1H NMR (600 MHz, CDCl3)δ: 5.49 (1H, d,J=1.8 Hz, H-7), 5.16 (1H, dd,J=15.2, 7.1 Hz, H-23), 5.11 (1H, dd,J=15.2, 7.7 Hz, H-22), 3.80 (1H, m, H-3), 0.94 (3H, d,J=4.0 Hz, H-21), 0.92 (3H, s, H-19), 0.85 (3H, d,J=8.0 Hz, H-28), 0.77 (3H, d,J=8.0 Hz, H-27), 0.74 (3H, d,J=8.0 Hz, H-26), 0.58 (3H, s, H-18);13C NMR (150 MHz, CDCl3)δ: 200.3 (C- 6), 165.1 (C- 8), 136.8 (C-22), 133.8 (C-23), 121.1 (C-7), 80.3 (C-5), 76.3 (C-9), 68.0 (C-3), 57.6 (C-17), 52.9 (C-14), 46.3 (C-13), 44.5 (C-24), 42.9 (C-10), 41.8 (C-20), 37.3 (C- 4), 36.3 (C-12), 34.5 (C-25), 31.1 (C-2), 29.5 (C-11), 29.3 (C-16), 26.8 (C-1), 23.6 (C-15), 21.8 (C-21), 20.7 (C-19), 20.6 (C-27), 20.3 (C-26), 18.4 (C-28), 12.75 (C-18).以上波譜數(shù)據(jù)與文獻[16]報道一致，故鑒定其為3β,5α,9α-三羥基- (22E,24R)-麥角甾-7,22-二烯- 6-酮.

化合物14:無色針晶; mp. 113～114 ℃；HR-ESI-MS:m/z393.315 0 [M+H]+(C28H41O理論計算值, 393.315 9);1H NMR (300 MHz, CDCl3)δ: 6.62 (1H, d,J=9.5 Hz, H-7), 6.05 (1H, d,J=9.5 Hz, H- 6), 5.71 (1H, s, H- 4), 5.28 (1H, dd,J=15.2, 7.3 Hz, H-23), 5.21 (1H, dd,J=15.2, 8.0 Hz, H-22), 1.08 (3H, d,J=6.7 Hz, H-21), 1.02 (3H, s, H-19), 0.98 (3H, s, H-18), 0.93 (3H, d,J=6.8 Hz, H-28), 0.85 (3H, d,J=6.8 Hz, H-27), 0.83 (3H, d,J=6.8 Hz, H-26);13C NMR (75 MHz, CDCl3)δ: 199.8 (C-3), 164.7 (C-5), 156.4 (C-14), 135.3 (C-22), 134.3 (C-7), 132.9 (C-23), 124.8 (C- 8), 124.7 (C- 6), 123.3 (C- 4), 56.1 (C-17), 44.7 (C-9), 44.3 (C-13), 43.2 (C-25), 39.7 (C-20), 37.0 (C-10), 35.9 (C-12), 34.4 (C-1,2), 33.4 (C-24), 28.1 (C-16), 25.6 (C-15), 21.5 (C-21), 20.3 (C-26), 20.1 (C-27), 19.3 (C-18), 19.3 (C-11), 18.0 (C-28), 17.1 (C-19).以上波譜數(shù)據(jù)與文獻[17]報道一致，故鑒定其為4,6,8 (14),22-麥角甾四烯-3-酮.

化合物15:無色針晶; mp. 131～132 ℃; HR-ESI-MS:m/z395.330 7 [M+H]+(C28H43O理論計算值, 395.331 6);1H NMR (300 MHz, CDCl3)δ: 5.80 (1H, d,J=2.1 Hz, H- 4), 5.23 (1H, dd,J=15.0, 7.0 Hz, H-23), 5.20 (1H, s, H-7), 5.16 (1H, d,J=15.0 Hz, H-22), 1.19 (3H, s, H-19), 1.03 (3H, d,J=6.5 Hz, H-21), 0.94 (3H, d,J=7.0 Hz, H-28), 0.84 (3H, d,J=7.0 Hz, H-27), 0.82 (3H, d,J=7.0 Hz, H-26), 0.56 (3H, s, H-18);13C NMR (75 MHz, CDCl3)δ: 199.5 (C-3), 169.2 (C-5), 139.7 (C- 8), 135.6 (C-22), 132.4 (C-23), 122.9 (C- 4), 115.8 (C-7), 56.0 (C-17), 55.2 (C-14), 46.0 (C-9), 43.6 (C-13), 43.0 (C-24), 40.6 (C-20), 39.2 (C-12), 38.2 (C-10), 34.3 (C-2), 33.3 (C-25), 33.1 (C- 6), 28.3 (C-16), 23.0 (C-15), 22.2 (C-11), 21.5 (C-19), 21.3 (C-21), 20.1 (C-26), 19.9 (C-27), 17.8 (C-28), 12.4 (C-18).以上波譜數(shù)據(jù)與文獻[18]報道一致，故鑒定其為 (22E,24R)-麥角甾- 4,7,22-三烯-3-酮.

3.2 細胞毒活性

體外抗腫瘤細胞活性篩選結(jié)果顯示，2、5、6、7和14對本實驗3種腫瘤細胞具有較好的抑制作用(表1).

表1 化合物1-15的體外細胞毒活性Table 1 Cytotoxic activities of compounds 1-15 in vitro (μg·mL-1)

4 討論

本研究從鏈霉菌H41-55發(fā)酵物中分離鑒定了15個化合物，主要為抗霉素類和甾醇類化合物.抗霉素類化合物具有多種生物活性，主要為抗真菌、抗蟲、抗炎及抗腫瘤等.抗腫瘤方面，抗霉素類化合物作為電子傳遞鏈的抑制劑， 能引起細胞內(nèi)活性氧(reactive oxygen species, ROS)水平上升和谷胱甘肽(glutathione, GSH)含量的損耗，誘導細胞凋亡，顯著地抑制人宮頸癌細胞HeLa的生長[19].甾醇類化合物也具有抗腫瘤活性，甾醇能夠抑制致癌物質(zhì)的產(chǎn)生，通過誘導非正常細胞周期的細胞進行細胞周期停滯，刺激腫瘤細胞脫噬來抑制腫瘤細胞的生長和繁殖.甾醇還能夠結(jié)合到細胞膜中，具有改變膜流動性和膜結(jié)合酶的能力，改變引起腫瘤生長的信號傳導路徑，抑制腫瘤血管的生長和轉(zhuǎn)移[20].本實驗從海洋放線H41-55發(fā)酵物中分離得到的抗霉素類化合物5和6以及甾醇類化合物14均具有一定的抗腫瘤作用，為進一步研究海洋放線菌抗霉素類和甾醇類化合物奠定了基礎(chǔ).