微粒促进培养(MPEC)联合Triton X-100原位萃取竹红菌素

竹红菌素是具有抗肿瘤、抗病毒活性的光敏性苝醌类化合物,分离自我国传统的药用真菌——竹黄菌子实体和菌丝。竹红菌素作为非卟啉类新型光敏剂,已应用于肿瘤、真菌疾病的光动力治疗(photodynamic therapy, PDT)中。近年来,微粒促进培养(MPEC)技术在真菌发酵培养和生产中受到了广泛关注,笔者在竹黄菌种子培养期加入竹炭微粉(BCP)(2 g/L)处理,竹黄菌胞内竹红菌甲素(HA)含量和胞外HA产量在3 d后分别增加了63.0%～64.9%和63.5%～147.4%,发酵8 d后,HA总产量可达604.8 mg/L,是对照组的1.65倍。进一步在生产发酵阶段加入Triton X-100(25 g/L)进行萃取发酵,将胞内HA萃取到胶束溶液中,浊点系统的形成可简化HA的提取过程,提高生产效率。本研究为真菌苝醌类光敏药物生产提供了新型的萃取发酵技术。     

北醌化合物的光敏活性及其抗肿瘤机制

苝醌 (ｐｅｒｙｌｅｎｅｑｕｉｎｏｎｅ ,ＰＱ)化合物是一类分布于自然界生物中的光敏色素。ＰＱ类光敏色素是目前已知的在可见光区内优良的光敏剂 ,近年来的研究证明其具有良好的光敏杀伤肿瘤细胞和抑制艾滋病病毒 (ＨＩＶ 1 )的作用[1～ 4] 。1 .醌类化合物的光敏活性真菌能产生具有ＰＱ骨架的代谢产物 ,陈远滕先生报道竹红菌的光敏作用。安静仪报道竹红菌甲素 (ｈｙｐｏｃｒｅｌｌｉｎＡ ,ＨＡ)有产生1Ｏ2 的作用。张志义发现ＨＡ能产生Ｏ-2·、·ＯＨ和ＨＡ-,提出ＨＡ对生物系统的光敏损伤机制与上述多重作用有关[5 ] 。第五振军等报…     

用ESR方法研究光敏作用的动态过程

通过动力学方法建立了光敏作用中¹O₂被TEMPONE捕捉产生的TAN自由基对时间的函数关系式,据此可求出光敏作用Ⅰ,Ⅱ型机制产生各种活性中间体的相对速率常数.并应用上述公式,结合实验结果,具体求算了3种无酵类光敏剂HA,HB,CP在DMF-H₂O和DMSO-H₂O体系中产生¹O₂,O₂和PS的相对速率常数,进而探讨了苝醒光敏剂的构效关系和光敏机制与溶剂的关系.     

在胶束非均相体系中竹红菌乙素光敏作用特征的ESR研究

竹红菌乙素（ＨｙｐｏｃｒｅｌｌｉｎＢ,简称ＨＢ）属于脂溶性醌类光敏剂。本文采用近代ＥＳＲ实验技术,探讨了ＨＢ在ＴｒｉｔｏｎＸ—１００胶束非均相体系中光敏反应原初过程的特征。首先证明,在非均相体系中,ＨＢ仍具有产生１Ｏ２的作用,进而证实,ＨＢ通过电子传递亦能产生ＨＢ－,Ｏ２－和·ＯＨ自由基。结果发现,ＨＢ在非均相胶束体系中,上述活性中间体（１Ｏ２和自由基）的相对产额均显著高于均相体系值。这表明,在非均相体系中,ＨＢ光敏反应激发能转移和电子传递具有新的特点和规律。据此推测,ＨＢ在生物非均相体系中的光敏损伤作用将会有新的特征。     

竹红菌光敏作用的初步探讨*

用纸片法作为筛选光敏物质及光敏作用的研究方法。结果证实了(1)竹红菌Hypo crella bambusae中含有对革兰氏阳性细菌具光敏活性的成分;(2)从中筛选到一种红色的光敏色素,属苝醌的一个新的衍生物,取名竹红菌甲素(Hypocrellin A简称“甲素”);(3)揭示了“甲素”是对可见光敏感、作用光谱较宽的光动力学(photodynamic)物质。从化合物的类型、光敏特性、治疗方法、治疗对象及其效果看,可以认为甲素是一新型的光化学疗法药物。     

竹红菌光敏作用的初步探讨

用纸片法作为筛选光敏物质及光敏作用的研究方法。结果证实了(1)竹红菌Hypo crella bambusae中含有对革兰氏阳性细菌具光敏活性的成分;(2)从中筛选到一种红色的光敏色素,属苝醌的一个新的衍生物,取名竹红菌甲素(Hypocrellin A简称“甲素”);(3)揭示了“甲素”是对可见光敏感、作用光谱较宽的光动力学(photodynamic)物质。从化合物的类型、光敏特性、治疗方法、治疗对象及其效果看,可以认为甲素是一新型的光化学疗法药物。     

一株苝产醌类光敏剂丝状真菌AL18的液体发酵工艺研究

为研究一株丝状真菌AL18产苝醌类光敏剂的液体发酵工艺.以马铃薯综合培养基为发酵培养基,采用单次单因素实验法,研究了液体摇瓶培养条件对苝醌类光敏剂产量的影响.实验结果表明液体摇瓶最适培养条件为250ml三角瓶装液量40ml,接种量7.5%,接种种龄40h,初始pH5.75,摇床转数180r/min,30℃振荡培养48h.在此培养条件下,采用单因素法筛选了发酵培养基中的碳源、氮源和无机离子,选用L9(34)对筛选到的绵白糖(A)、蛋白胨(B)、蚕蛹粉(C)、CuSO4·5H2O(D)进行了正交试验.经优化后的发酵培养基配方为马铃薯200g/L,绵白糖30g/L,蛋白胨3g/L,蚕蛹粉12.5g/L,磷酸二氢钾1g/L,硫酸镁0.5g/L,硫酸铜0.05g/L,VB1 100mg/L.对此发酵培养基配方进行了5次验证实验,苝醌类光敏剂的平均产量为1.21g/L.     

Pheophorbide a在脂质体介质中的光敏反应特性

Pheophorbidea(PPa)———叶绿素a的一种分解产物 ,是一种光敏剂 .用自旋捕捉和消自旋等ESR技术及激光光解技术研究了PPa在PBS和DPPC脂质体介质中光敏反应的Ⅰ型和Ⅱ型机制 .通过比较发现 ,DPPC脂质体的加入显著增强了PPa光敏反应产生PPa ^- (Ⅰ型 )和¹O₂ (Ⅱ型 )的能力 .这种增强反应源于脂质体对PBS中的PPa聚集态的解聚作用和它对PPa三重激发态的保护作用 .这表明 ,PPa在非均相脂质体体系中的光敏反应有别于均相水体系 ,而表现出更高的光敏效率 .此结果对解释属于非均相生物体系的肿瘤的光动力治疗机理有重要意义 .     

竹红菌素光敏剂与微血管病变光动力治疗

光动力疗法已被用于临床治疗除实体肿瘤以外的一些微血管类疾病,包括鲜红斑痣(portwine stains,PWS)和老年视网膜黄斑变性(age-related macular degeneration,AMD)等。竹红菌素是一种苝醌类光敏剂,因为在光疗窗口(600～900 nm)的吸收较少,它不适用于实体肿瘤的光动力治疗,但对于治疗微血管类疾病却有其独特的优势。本文根据竹红菌素光物理特性提出其主要适应症范围,并根据其临床实用化问题提出应对策略。通过构造竹红菌素水溶性纳米制剂或具有优化脂水双亲性的衍生物,实现脂溶性光敏剂既可以安全给药又最大程度保持生物利用度和光动力活性。生物学实验证明竹红菌素类光敏剂对生物靶体具有超强的光动力活性。由此推测:竹红菌素类光敏剂在光动力治疗微血管疾病(如鲜红斑痣和老年黄斑变性)及其它浅表型疾病方面具有广阔的应用前景。     

铝酞菁:人癌细胞的吸收及光敏杀伤的研究

本文比较了光敏剂铝酞菁(AISPC)及血卟啉衍生物(HPD)在人癌细胞的吸收及光敏杀伤中的差异.探讨了两光敏剂在细胞中的分布、摄入量,血清的影响、及细胞对两光敏剂的吸收动力学过程,结果显示,除了类似的特征外,细胞对AISPC的吸收速度慢于HPD.光敏杀伤的结果表明,在红光照射下,两光敏剂对细胞有相近的杀伤效率;在室内自然光下,AISPC对细胞的损伤明显低于HPD,显示出其较低的光毒付作用,具有适合临床应用的优点.     

Anion of hypericin is crucial to understanding the photosensitive features of the pigment

Photosensitive behaviors of hypericin (HYP) have attracted much attention, because of HYP's great potential in photodynamic therapy. It has been found that HYP differs from homologous pigments, such as hypocrellin A (HA), in photosensitive features. For instance, despite the comparable triplet state quantum yields, HYP holds a much lower singlet oxygen yield than HA. To understand the unique photosensitive behaviors of HYP, time-dependent density functional theory is employed to calculate a series of excited-state properties of HYP and its anion (dominant in polar solvents), which are then compared with excited-state properties of HA. It is revealed that the stronger electron-donating power of HYP anion than that of HA is responsible for the HYP's photosensitive features.     

Internal heavy atom effect of Au(III) and Pt(IV) on hypocrellin A for enhanced in vitro photodynamic therapy of cancer

Hypocrellin A (HA), an a natural perylene quinine photosensitizers (PSs), can chelate with heavy metal ions, including Au(III) and Pt(IV), to form a 1:2 complex, which exhibits enhanced ¹O₂ generation quantum yield through the increased intersystem crossing efficiency mediated by internal heavy atom effect. Besides, the chelate process greatly improved the water solubility of HA. Comparative studies with HA and complexes have demonstrated that the heavy-atom effect on HA molecules enhances the efficiency of in vitro photodynamic (PDT) efficacy.     

Delivering a hydrophobic anticancer drug for photodynamic therapy by amorphous formulation

An amorphous formulation of hypocrellin A for photodynamic therapy is reported which can provide stable aqueous dispersion of such hydrophobic photosensitizers. In vitro studies have demonstrated the active uptake of amorphous formulation of hypocrellin A into the mitochondria of tumor cells. Compared with Tween-80 micelle embedded hypocrellin A, low dark-toxicity but similar light-toxicity of the amorphous one to drug impregnated tumor cells was observed. Thus, the potential of using amorphous formulation of hypocrellin A as drug delivery system for photodynamic therapy has been demonstrated.     

Assessment of tissue damaging effects of mixed micellar absorption enhancers on the intestinal mucosa of common carp (Cyprinus carpio), African catfish (Clarias gariepinus) and rainbow trout (Oncorhynchus mykiss) as a consequence of enhanced intestinal absorption of sGnRH-a

Non-ionic surfactant–polyoxyethylene sorbitan monooleate (Tween-80) and oleic acid are food and pharmaceutical ingredients for oral and parenteral delivery and generally regarded as safe (GRAS). They have the potential as an intestinal absorption enhancer for the development of oral drug delivery systems. However, their safety in terms of mucosal integrity has yet to be evaluated. Therefore, the purpose of the present work was to study the tissue damaging effects of Tween-80, oleic acid and of their mixed micellar formulation (oleic acid + Tween-80). This was investigated at 2 h and 24 h after rectal delivery and compared with the topical effect of polyoxyethylene 9 lauryl ether (Polydocanol). The same experiment was carried out on three species with distinct feeding habits: the common carp, Cyprinus carpio (L.); the African catfish, Clarias gariepinus (Burchell); and the rainbow trout, Oncorhynchus mykiss (Walb.). Based on the findings of this study it was concluded that Tween-80 (4%), or its mixed micelle with oleic acid (0.6%) can be considered as a safe formulation, inducing only a moderate alteration of the intestinal mucosa, comparable to the effect of an isotonic saline. By contrast, in the three species, the same dose of Polydocanol, or of its mixed micelle with oleic acid, induced severe tissue damage of the intestinal mucosa, still present after 24 h. Mixed micelles of oleic acid with Tween-80 were also demonstrated as increasing the intestinal absorption of the salmon gonadotropin releasing hormone analogue (sGnRH-a) in catfish, C. gariepinus , and consequently stimulating GtH II secretion. This effect was compared with the action of other drugs considered as intestinal absorption enhancers.     

Deactivation mechanisms of triplet excited state hypericin by β-carotene

The deactivation mechanisms of the triplet excited state hypericin (HYP) by β-carotene (CAR) were studied employing quantum chemical calculations in the present study. The results suggest that CAR may deactivate the triplet excited state HYP through the following two pathways on thermodynamic grounds: (1) direct energy transfer from the triplet excited state HYP to CAR; (2) electron transfer from the triplet excited state CAR, which was formed through direct energy transfer pathway, to the triplet excited state HYP.     

条斑紫菜抗高温和快速生长细胞株系HB的建立及栽培

本文主要应用单细胞培养技术培育紫菜新品系,以建立紫菜细胞工程快速育苗系统。通过酶解技术分离出紫菜叶状体细胞,并进行多克隆,从中获得4个细胞纯系植株(HA,HB,HC,HD)。对该4个细胞株系进行了纯系培养,并对其细胞苗和丝状体的生长速度和抗高温(19℃,21℃,23℃,25℃)性进行了测定。结果显示,船株系具有较高的抗高温性和最快生长速度。在1998年到2000年间,在江苏省启东县海丰海区对HB株系进行了海上栽培实验,结果显示,实验组的产量高于当地对照组产量,证明了条斑紫菜船株系不仅具有较高的抗高温性,而且也有较快生长速度。本实验说明应用细胞工程进行条斑紫菜育种是一条很好的快速育种途径。     

Tween类胶束体系中水杨酸铜络合物的SOD样活性

合成了水杨酸铜·乙醇络合物,提纯了Ｔｗｅｅｎ－２０、Ｔｗｅｅｎ－４０,Ｔｗｅｅｎ－６０．Ｔｗｅｅｎ－８０,并测定了它们在ｐＢ７．４的磷酸缓冲液中的ＣＭＣＯ。用Ｃｙｔ,ｃ－ＨＸ－ＸＯ法分别测定了水杨酸铜·乙醇络合物清除超氧阴离子自由基的活性,不同浓度的单纯表面活性剂对Ｏ２的影响,以及表面活性剂与水杨酸铜·乙醇络合物协同清除Ｏ２的作用。观察到在胶束体系中水杨酸铜·乙醇络合物的ＳＯＤ样活性明显高于缓冲液体系无表面活性剂时的ＳＯＤ样活性,表面活性剂本身也具有清除Ｏ２的功能。抗红细胞膜脂质过氧化实验也得到类似结果。     

The effect of surfactants on the phytase production and the reduction of the phytic acid content in canola meal byAspergillus carbonarius during a solid state fermentation process

Summary During the growth of A. carbonarius, the rates of biomass growth, phytase production and phytic acid content reduction in canola meal media during solid state fermentation were higher in the presence of Na-oleate or Tween-80 than in the control medium which was not supplemented with these surfactants. Addition of Triton X-100 had a negative effect on the studied processes.The optimum concentration of Na-oleate in solid state culture media was 1%.     

表面活性剂对大肠杆菌胞外生产α-环糊精葡萄糖基转移酶的影响

研究了不同浓度表面活性剂Tween-80,Triton X-100,SDS对大肠杆菌生产α-环糊精葡萄糖基转移酶（α-CGT酶）的影响。结果表明：发酵初始添加Tween-80和Triton X-100的最适浓度分别为2％,0.5％,最终胞外酶活分别达2.03U/ml和4.92U/ml,相对于未添加表面活性剂时提高4.6倍和12.67倍,且改变添加时间不能提高酶的产量;发酵36 h添加0.02％SDS对α-CGT酶产量促进最大,最终胞外酶活达5.31U/ml,较对照组提高12.75倍。表面活性剂对α-CGT酶生产的促进作用可能是由大肠杆菌细胞内外膜渗透性增加所致,使细胞周质空间中α-CGT酶能更加快速地渗透到胞外。     

Effect of oleic acid and Tween-80 on lysine synthesis by the culture Corynebacterium glutamicum]

The effect of oxidized and unoxidized oleic acid and Tween-80 on the growth and lysine synthesis by the producers C. glutamicum strains 95, 8, 28 was investigated. Surface active substances like oxidized and unoxidized oleic acid and Tween-80 during cultivation of the lysine producers on the glucose medium (the synthetic medium) and the medium with molasses and corn extract either inhibited the culture growth, thus reducing lysine yield, or accelerated the culture growth, thus increasing lysine yield. Oxidized and unoxidized oleic acid produced the greatest effect when added to the nutrient medium on the 48th cultivation hour. The increment of synthesized lysine was 120-150% of the control. Tween-80 proved to be very effective when added at early stages of fermentation (20 hours).