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红树植物角果木的化学成分及其防污活性研究
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摘要
海洋污损生物附着在人造设备上,造成巨大的经济损失,是海洋技术面临的重大问题。有机锡和氧化亚铜被广泛的用来控制污损生物,但这些防污涂料会污染海洋环境。随着人们对环境问题的日益关注,人类迫切需要寻找高效环保型的天然防污涂料,而天然防污产物是研究天然防污涂料的基础。角果木(Ceriopstagal Perr.)是红树科(Rhizophoraceae)一种重要的红树植物,富含萜类化合物包括一系列新的萜类化合物,而新的海洋萜类化合物是海洋防污涂料开发的重要源泉。本论文综合运用硅胶柱色谱、凝胶柱色谱、HPLC等多种色谱分离技术,对角果木根的乙醇提取物进行系统分离纯化。同时依靠红外光谱(IR)、质谱(包括ESIMS、HRESIMS)、旋光和核磁共振波谱法(包括~1H-NMR、~(13)C-NMR、DEPT、HMQC、HMBC、~1H-~1H COSY、NOESY)等多种波谱技术确定它们的结构和相对立体构型,在此基础上利用白脊藤壶(Balanus albicostatus Pilsbry)金星幼体附着抑制实验模型对其防污活性进行了系统地研究,主要研究结果如下:
     (1)从角果木根的乙醇提取物中得到4个新骨架的新化合物,这些化合物均为二萜二聚体,分别命名为tagalsin L(1)、tagalsin M(2)、tagalsin N(3)和tagalsin P(4)。并得到二萜类新化合物1个,命名为tagalsin O(5)。
     (2)从角果木根的乙醇提取物中得到13个已知的化合物单体,这些化合物中二萜类化合物3个,包括ent-8(14)-pimarene-15R,16-diol(6)、ent-8(14)-pimarene-15,16-O-isopropylidene(7)和tagalsin C(8);二萜二聚体1个,tagalsin I(9);三萜类化合物5个,包括betulin(10)、lup-20(29)-en-3-oxo-oic acid(11)、betulin acid(12)、lupane-20(29)-en-3-oxo-28-diol(13)和lup-20(29)-en-3β-24-diol(14);甾体2个,包括stigmasterol(15)和β-sitosterol(16);芳香类化合物2个,包括coffeic acid methyl ester(17)和coffeic acid(18)。其中化合物6、7、14和17为首次从该植物中分离得到。
     (3)利用白脊藤壶(B.albicostatus)金星幼体附着抑制实验模型对红树植物角果木根的防污活性进行研究。以国内外已有数项防污专利的辣椒素为参照物(EC_(50)为1.32±0.02μg/cm~2),把防污活性设为以下5个等级:防污活性很强(EC_(50)<0.1μg/cm~2)、防污活性强(0.1μg/cm~2<EC_(50)<1μg/cm~2)、防污活性中等(1μg/cm~2<EC_(50)<10μg/cm~2)、防污活性弱(10μg/cm~2<EC_(50)<50μg/cm~2)、无防污活性(EC_(50)>50μg/cm~2)。筛选出13个有防污活性的化合物,其中防污活性很强的天然防污产物1个即二萜类化合物ent-8(14)-pimarene-15R,16-diol(EC_(50)为0.04±0.00μg/cm~2);防污活性强的天然防污产物有2个,包括新化合物tagalsin O(EC_(50)为0.32±0.01μg/cm~2)和化合物tagalsin C(EC_(50)为0.65±0.02μg/cm~2);防污活性中等的天然防污产物有6个,包括新骨架化合物tagalsin N(EC_(50)为8.79±0.36μg/cm~2)和化合物betulin(EC_(50)为9.27±0.30μg/cm~2)、lupane-20(29)-en-3-oxo-28-diol(EC_(50)为8.73±0.43μg/cm~2)、ent-8(14)-pimarene-15,16-O-isopropylidene(EC_(50)为4.02±0.06μg/cm~2)、stigmasterol(EC_(50)为4.05±0.15μg/cm~2)、lup-20(29)-en-3-oxo-oicacid(EC_(50)为3.20±0.17μg/cm~2),另外还有4个防污活性较低的天然防污产物,包括tagalsin L(EC_(50)为26.05±0.26μg/cm~2)、tagalsin M(EC_(50)为22.19±0.21μg/cm~2)、tagalsin I(EC_(50)为11.67±0.47μg/cm~2)和β-sitosterol(EC_(50)为18.47±0.40μg/cm~2)。所有天然产物都是低毒的天然防污产物。这些天然防污产物极在开发防污涂料中潜力很大。
     (4)对萜类化合物ent-8(14)-pimarene-15R,16-diol进行结构修饰,合成了5个化合物,其中防污活性很强的化合物1个,即化合物MOT-3-4-3(EC_(50)为0.05±0.00μg/cm~2):防污活性强的化合物3个,包括MOT-1-2-2(EC_(50)为0.14±0.01μg/cm~2)、MOT-1-1(EC_(50)为0.30±0.01μg/cm~2)和MOT-2(EC_(50)为0.57±0.01μg/cm~2);防污活性中等的化合物1个,即MOT-3-3-4(EC_(50)为7.47±0.13μg/cm~2)。
     (5)通过对pimarene型的二萜骨架的结构一活性关系进行研究,总结了该类型化合物的一些规律:
     a.支链上的双羟基是该类型化合物的活性基团,对化合物的防污活性起到了关键的作用,但是双羟基对化合物的毒性没影响。
     b.支链上的取代基对活性的影响:双羟基>环氧>乙酰基>甲氧基>甲磺酰基团。
Marine fouling organisms often cause technical and economic problems by settling on artificial surfaces submerged in seawaters.Although organotin compounds and booster biocides have been widely used for controlling these fouling organisms, they may also pollute the aquatic environments.Along with the increasing concern about environment,effective and environmentally friendly antifoulants are urgently needed.Therefore,an important source of such effective and environmentally friendly antifoulants is based on the research of natural product antifoulants.Ceriops tagal Perr.(Rhizophoraceae) is an important mangrove species.This species is rich in terpenoids(including a series of new terpenoids),while marine new terpenoids are an important source of marine antifoulants.The chemical constituents and antifouling activities of the mangrove plant C.tagal were studied in this paper.The EtOH extracts from the roots of C.tagal were chromatographed over Silica gel,Sephadex LH-20 and HPLC respectively to yield compounds.The structure and relative stereochemistry were elucidated by means of extensive NMR(including ~1H-NMR, ~(13)C-NMR,DEPT,HMQC,HMBC,~1H-~1H COSY,NOESY),IR and MS(including ESIMS and HRESIMS) analyses.By using the settlement inhibition assay with cyprids larvae of barnacle Balanus albicostatus Pilsbry,the roots of C.tagal was investigated for testing antifouling activity.The main results were shown as followed:
     (1) Four novel backbone compounds were isolated from the roots of C.tagal. These compounds are all dimeric diterpenes,namely,tagalsin L(1),tagalsin M(2), tagalsin N(3) and tagalsin P(4).In addition,one new diterpene was isolated from the the roots of C.tagal and determined as tagalsin O(5).
     (2) Thirteen known compounds were isolated from the roots of C.tagal.Among these compounds,three diterpenes are ent-8(14)-pimarene- 15R,16-diol(6),ent-8(14) -pimarene-15,16-O- isopropylidene(7) and tagalsin C(8);one dimeric diterpene is tagalsinⅠ(9);five triterpenes are betulin(10),lup-20(29)-en-3-oxo-oic acid(11), betulin acid(12),lupane-20(29)-en-3-oxo-28-diol(13) and lup-20(29)-en-3β-24-diol (14);two phytosterins are stigmasterol(15) andβ-sitosterol;two aromatic compounds are coffeic acid methyl ester(17) and coffeic acid(18).Among these compounds, compounds 6,7,14 and 17 were isolated from the roots ofC.tagal for the first time.
     (3) Using the settlement inhibition assay with cyprids larvae of bamacle Balanus albicostatus,the compounds from the roots of C.tagal was investigated for testing antifouling activity.Capsaicin,as a widely used antifoulant,was chosen as the reference.According to the antifouling activity,five grades were set:very strong (EC_(50)<0.1μg/cm~2),strong(0.1μg/cm~2<EC_(50)<1μg/cm~2),medium(1μg/cm~2<EC_(50)<10μg/cm~2),feeble(10μg/cm~2<EC_(50)<50μg/cm~2) and non-antifouling(EC_(50)>50μg/cm~2).Comparing with the antifouling activity of capsaicin(EC_(50)= 1.32±0.02μg/cm~2),thirteen compounds were approved to be active.Among these compounds,one natural product antifoulant with very strong antifouling activity is ent-8(14)-pimarene-15R,16-diol(EC_(50) = 0.04±0.00μg/cm~2);two natural product antifoulants with strong antifouling activity are tagalsin O(EC_(50) = 0.32±0.01μg/cm~2) and tagalsin C(EC_(50) = 0.65±0.02μg/cm~2);six natural product antifoulants with medium antifouling activity are tagalsin N(EC_(50) = 8.79±0.36μg/cm~2),betulin (EC_(50) = 9.27±0.30μg/cm~2),lupane-20(29)-en-3-oxo-28-diol(EC_(50) = 8.73±0.43μg/cm~2),ent-8(14) -pimarene-15,16-O-isopropylidene(EC_(50)= 4.02±0.06μg/cm~2), stigmasterol(EC_(50) = 4.05±0.15μg/cm~2) and lup-20(29)-en-3-oxo-oic acid(EC_(50) = 3.20±0.17μg/cm~2);four natural product antifoulants with feeble antifouling activity are tagalsin L(EC_(50) = 26.05±0.26μg/cm~2),tagalsin M(EC_(50) = 22.19±0.21μg/cm~2),tagalsinⅠ(EC_(50) = 11.67±0.47μg/cm~2) andβ-sitosterol(EC_(50) = 18.47±0.40μg/cm~2).On the other hand,their toxicities to cyprids were quite low and they all inhibited cyprid settlement in a non-toxic way.These compounds are great potential marine environmentally friendly antifoulants.
     (4) The structure of ent-8(14)-pimarene-15R,16-diol was modified and five compounds was synthesized.Among these compounds,one compound with very strong antifouling activity is MOT-3-4-3(EC_(50)= 0.05±0.00μg/cm~2);three compounds with strong antifouling activity are MOT-1-2-2(EC_(50) = 0.14±0.01 μg/cm~2),MOT-1-1(EC_(50) = 0.30±0.01μg/cm~2) and MOT-2(EC_(50) = 0.57±0.01μg/cm~2);one compound with medium antifouling activity is MOT-3-3-4(EC_(50) = 7.47±0.13μg/cm~2).
     5.Our structure-activity relationship analysis of ent-pimarane diterpenoid is summarized as follows:
     a.The antifouling activity might be due to the number of free hydroxyl group in side chain of pimarane diterpenoid.However,there was no great effect of the number of free hydroxyl group on the toxicity of pimarane diterpenoids.
     b.Effect of substituent group in side chain on the activity of ent-pimarane diterpenoids:hydroxyl group>epoxy>acetyl group>methoxyl group>mesyl group.
引文
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