Produção in vitro, análise fitoquímica e avaliação da atividade antineoplásica de metabólitos de Hovenia dulcis Thunb. (Rhamnaceae) obtidos por diferentes estratégias biotecnológicas

Hovenia dulcis Thunberg, natural da Ásia Oriental, é cultivada no Brasil onde é conhecida como uva-do-japão. A espécie possui várias indicações na medicina popular e alguns estudos apontam o seu potencial antineoplásico, tripanocida e hepatoprotetor. Metabólitos secundários são substâncias não essenciais para a sobrevivência celular, mas que fornecem vantagens adaptativas aos vegetais, sendo atribuído, para algumas delas, atividades biológicas importantes. Substâncias de interesse medicinal têm sido obtidas por técnicas da cultura de tecidos vegetais, como a calogênese e a cultura de células em suspensão, que permitem a síntese de matéria-prima de forma contínua e homogênea, independentemente de fatores ambientais e sazonais. O presente estudo objetivou o estabelecimento de culturas in vitro de H. dulcis, visando à produção de metabólitos de interesse, com vistas à avaliação do seu potencial antineoplásico sobre células K562. Foram testados protocolos para o estabelecimento de diferentes sistemas, como culturas de calos, de células em suspensão (CCS) e compact callus clusters (CCC) e ainda a avaliação do uso de elicitores na otimização de metabólitos produzidos in vitro. Foi verificado que a adição dos fitorreguladores KIN e TDZ, substituindo o BAP, não foi capaz de induzir a formação de calos friáveis, bem como a manutenção das culturas em ausência de luz. O uso do nitrato de prata promoveu a friabilidade de calos em todas as concentrações testadas, considerando-se 2,0 mg.L-1 a melhor concentração. Foram alcançadas taxas de 100% de formação de CCS tanto na presença, quanto em ausência de AgNO3. O maior acúmulo de biomassa foi verificado na concentração mais baixa de PIC (0,625 mg.L-1). A análise dos espectros de RMN indicou a presença de (+)-dihidromiricetina, (+)-galocatequina, hovenitina II, hovenosideo G, hodulosideo III, hodulosideo IV, hodulosideo I e hovenidulciosideo B1 nas culturas de calos friáveis. No estabelecimento de culturas CCC, observou-se a formação de calos compactos verdes em todas as concentrações de ANA testadas. O aumento da velocidade de rotação para 135 rpm aumentou a dispersão das células com consequente formação dos agregados celulares desejados. A seleção de linhagens celulares demonstrou ser um método eficiente na uniformização do tamanho desses agregados e tal uniformidade se manteve estável por mais de cinco subcultivos em 100% das culturas. Uma fração rica em saponinas foi obtida a partir dos agregados celulares, correspondendo a 1,46% da massa seca. A análise por RMN sugeriu a presença das saponinas Hovenosideo G e dos hovenidulciosideos A2 e B2. O uso de elicitores em cultura de calos mostrou-se adequado à produção de metabólitos secundários, sem alterações morfológicas nos mesmos. A elicitação alterou o perfil cromatográfico analisado por HPLC. Na elicitação com 5,0 mg.L-1 de extrato de levedura foi verificado um aumento de quase três vezes (12,280 3,396 equivalentes de quercetina/mg de extrato) na síntese de flavonoides. Finalmente, os estudos de ação antitumoral in vitro demonstraram citotoxicidade dos extratos de calos não elicitados de H. dulcis sobre linhagem de leucemia mieloide crônica (IC50 de 74,05 μg.mL-1.) e inibição do crescimento de tais células (K562), sugerindo o potencial antineoplásico para um produto biotecnológio (calo) desta espécie.

染色体加倍对忍冬生物学性状及其生态适应性的影响

忍冬（Lonicera japonica Thunb.）属忍冬科忍冬属，是一种重要的药用植物，其花蕾称为金银花（Flos Lonicerae），在我国已有1000多年的药用历史，具有清热解毒、凉散风热等功效。我们利用秋水仙素诱导二倍体“大毛花”品种茎尖选育出同源四倍体“九丰一号”品种，并在生产中发现花蕾产量显著提高，而药效成份含量是否发生变化了呢？四倍体忍冬表现出典型的器官巨大性，这些变化是否与其较强生态适应性之间存在联系呢？染色体加倍增强了忍冬的生态适应性，其生态修复功能如何呢？在本研究中，主要从以下几个方面进行了探讨：（1）染色体加倍对其叶片，茎、花蕾和花蕾产量及其药效成份等生物学性状的影响;（2）染色体加倍后植物对水分胁迫的响应;（3）染色体加倍后植物对热胁迫的响应;（4）除上述两个品种外，增加一个变异品种“红银花”，探讨3个忍冬品种对退化生态系统的修复功能。主要结果如下： 1. 通过测定根尖细胞染色体数目和使用流式细胞仪分析茎尖细胞DNA含量，表明四倍体忍冬（2n = 4x = 36）确实来自二倍体忍冬（2n = 2x = 18）的染色体加倍。四倍体忍冬气孔细胞大小显著大于二倍体，而气孔密度显著低于二倍体。四倍体忍冬没有光合“午休”，而二倍体存在明显的光合“午休”，这可能与其存在抗高温的叶片解剖结构特性有关。四倍体忍冬叶片较二倍体变大、变厚、变浓绿（较高叶绿素含量）。与二倍体相比，四倍体忍冬单位叶面积重量显著增高，表明其具有较强的生态适应性。四倍体忍冬单个花蕾的鲜重和干重均显著大于二倍体。连续3年的花蕾产量调查表明，四倍体显著高于二倍体。染色体加倍使其茎干粗壮、节间变短、新梢上着生花蕾数目增多及单个花蕾变大，这是其花蕾高产的生物学基础。对金银花的药效成份而言，染色体加倍不影响绿原酸的含量，但增加了木犀草苷的含量。结果表明，染色体加倍能增加金银花的产量和药效成份的含量，建议四倍体忍冬在药材生产中推广应用。 2. 水分胁迫显著降低二倍体和四倍体忍冬叶片的净光合速率、气孔导度和蒸腾速率。水分胁迫也降低电子传递速率、光系统II实际量子产量和光化学猝灭，而增加非光化学猝灭、总可溶性糖、脯氨酸和丙二醛的含量。四倍体忍冬对水分胁迫的响应表现为其叶片水势、气体交换、叶绿素荧光和有关代谢物含量的变化程度低于二倍体，并且复水后其恢复能力快于二倍体，表明染色体加倍增强了忍冬的抗旱能力。分析其主要原因，可能是由于四倍体植株总叶面积减少、单位叶面积重量增加、叶片表皮细胞和栅栏组织增大以及叶片表皮毛较浓密等形态解剖结构特性有关。结果表明，染色体加倍能增加忍冬植物的抗旱能力，而使其具有较强的生态适应性。 3. 二倍体和四倍体忍冬受48 ºC热胁迫处理6 h和恢复10 h，以及离体叶片45 ºC，50 ºC，55 ºC 水浴热胁迫3 min，应用叶绿素荧光成像系统研究了它们对热胁迫的响应。热胁迫显著降低了两个品种叶片的最大光化学效率、电子传递速率、光系统II实际量子产量和光化学猝灭，降低了四倍体的非光化学猝灭，而增加了二倍体的非光化学猝灭。热胁迫增加了两个品种叶片的总可溶性糖、脯氨酸和丙二醛的含量。四倍体受热胁迫的叶绿素荧光参数和有关代谢物的响应程度低于二倍体，以及其恢复程度快于二倍体，表明染色体加倍提高了抗热性。此外，叶绿素荧光成像的异质性也表明，四倍体的抗热能力大于二倍体。进一步的叶片解剖结构分析表明，四倍体叶片的表皮细胞变大、栅栏组织增厚、表皮毛较浓密等特点，是其抗热性强的主要原因。 4.根据以上的研究结果，通过四倍体忍冬生态修复功能的野外试验证明染色体加倍后其生态适应性变化。在本研究中，针对北京市门头沟区大面积不同类型的废弃地急需恢复植被和景观的问题，在恢复生态学理念的指导下，综合运用集成技术，将3个忍冬品种植物用于这些退化生态系统的恢复。在王平镇的公路下边坡（以碎石和矿渣为主）、煤矿、石灰窑和采石场4种类型废弃地建立生态修复的试验示范区。结果表明，在4个废弃地类型上引进的3个金银花品种，具有使示范区快速复绿、当年成景和群落快速形成的潜力，并具有对不同退化迹地的适应能力和恢复效果，其中四倍体忍冬效果更好些，这主要与其形态解剖结构和较强的生态适应性有关。

青藏高原过牧草地上灌木种类和分布对小尺度植被格局的影响

植物种间作用是决定物种分布、种群动态、群落演变和生态系统功能的重要因子。长期以来，相关研究集中在竞争作用上。然而自1990年代以来，易化作用逐渐受到重视，被认为是植物群落中物种共存的主要推动力，尤其是在极端和受到干扰的环境中。很多研究都揭示出植物对其他特定的物种产生直接或间接的正向的作用。护卫植物主要通过两种机制对临近的物种产生正向作用，即对恶劣的自然环境的改善和对高强度干扰的阻止和减轻。灌木在很多条件下能够成为护卫植物，影响种群的分布、群落的多样性和生态系统的功能。 中国西南部的青藏高原的牧场上，非生物的环境胁迫（比如生长季短暂、辐射强度大和极端温度等）和生物的干扰作用（比如过度放牧等）都很强。有文献表明，随着过度放牧现象的加重和草地的不断退化，灌木的分布有逐渐扩大的趋势。针对灌木对过度放牧草地上小尺度植被格局的影响，我们在若尔盖地区展开了以下两方面的研究。 为了研究不同种类灌木的影响，我们在若尔盖高原上选择了既具有恶劣的自然环境又遭受过度放牧压力的草地，对三种灌木内外的相同大小面积上小尺度范围内的植被进行调查。三种灌木分别是高山绣线菊、窄叶鲜卑花和金露梅，它们在形态结构、高度和可食性上都存在差异。在不考虑灌木种类的基础上，我们发现物种丰度、总盖度、开花的物种数和花序数都是灌木内部高于外部，而均匀度则恰好相反，是外部要高于内部，香农多样性指数内外没有明显的差异。金露梅灌丛中内外物种丰度和均匀度的差异要明显高于窄叶鲜卑花，但是总盖度、开花的物种数和花序数的差异不随着灌木种类的不同而有明显的差异。大多数的物种（占总物种数的47-85%）并没有因为灌木的存在而在内外出现的频数上表现出差异，而相当一部分物种（占总物种数的13-39%）在灌木外部的频率要高，仅仅有很少的物种（占总物种数的3-13%）因为灌木的存在而受益，内部的频数要高于外部。这种对某些物种的保护作用没有表现在群落水平上，即提高群落的物种丰富度和多样性，因为同时发生的灌木和其他植物的竞争作用也会限制以致排除冠层下面某些物种的存在。灌木的可食性和形态结构（比如盖度、高度、冠层的紧实度等）能对它们跟其他植物的相互之间净作用的类型和强度产生影响。不同的灌木能保护不同种类、不同数量的植物，表明易化作用是物种特异性的，跟护卫植物有关，也跟受益者有关。通过植被的排序，金露梅和高山绣线菊内外的植被得到了很明显的分离，而窄叶鲜卑花的则没有。总体上来说，灌木的存在确实能够通过提高出现或开花的频率，保护了某些对放牧比较敏感的物种，而且这种易化作用是物种特异性的。内外植被的差异在放牧压力最大的金露梅灌丛中最大，表明过度放牧导致的大尺度的植被状况对于小尺度上灌丛内外的植被的差异所起的作用比灌木种类的作用更大。 为了研究同种灌木不同的分布类型对植被格局的影响，选择金露梅作为目标灌木。金露梅是当地常见灌木，有的植株孤立存在，有的则是很多株联合形成比较大的斑块。我们选择金露梅形成的斑块中央、外沿金露梅植株内部、外围和孤立的金露梅植株内部、外部五个位置，并在小尺度上进行了植被调查和比较，发现金露梅的存在确实保护了一些物种，但是种类有限，反而是大部分的物种在外部的频数要高与内部;从群落水平来看，金露梅冠层下的植被从丰富度、多样性上来讲并没有超过外部，仅仅是具有更高的均匀度和同质性;生殖方面的结果也是类似的，而且大部分物种跟其分布呈现出基本类似的格局。金露梅斑块的存在和自然围栏的产生没有起到很明显的保护作用，并没有使得斑块中央植被的丰富度、多样性、均匀度提高，而仅仅是提高了植物的长势（表现为具有较高的总盖度等）;在物种水平，没有发现什么特异种的存在，仅仅发现能够促进一些物种在内部的生长或开花，而且比例很低。在这种效应的影响下，斑块边缘的金露梅相对于孤立的金露梅而言，也没有表现出更好的保护植被的能力。几乎所有的群落水平的指标都仅仅是受到灌木内外这个因素的影响，而两种分布格局（即：在斑块边缘和孤立存在）的影响是微乎其微的。植被排序的结果也充分证明了这一点，灌木内外的植被被明显的分离开来，而不同灌木中相应位置的植被则是混杂在一起。并且在物种水平上，除了极个别的物种之外，绝大部分物种内外的差异是是一致的，没有受到灌木分布格局的影响。即使个别物种具有不同的格局，也没有证据表明是受到了斑块中央植被的影响而产生的。

中国药用牡丹资源评价

我国牡丹资源丰富、药用历史悠久，但长期以来，资源与药效关系不清严重阻碍着丹皮的产业化发展。建立一套综合评价体系，比较不同牡丹资源间的差异，科学评价、准确定位各地不同品种的利用价值，对稳定我国丹皮质量、可持续、集约化发展丹皮产业具有重要的意义。 本实验以我国丰富的药用牡丹资源为材料，从观赏和药用价值两方面首先探讨综合评价牡丹资源的体系。在观赏价值考察中重点比较了花型、花色、花期等性状;药用价值则从根的产量、重金属含量以及七种主要药效成分含量三方面来比较;重金属含量以原子分光光度法检测，药效成分则以高效液相色谱和质谱进行分离鉴定和定性、定量检测。利用建立的综合评价体系对野生种、各地药用品种进行了评价，对丹皮生产相关环节的问题进行了讨论，并初步探讨了药效成分的遗传规律。结果如下： 1、引种的野生牡丹药用价值不比栽培品种高，建议作为育种材料。 2、不同品种间药效成分含量存在显著差异，综合比较‘JSF’、‘CKL’、‘凤丹’适于药用栽培，‘赵粉’、‘朱砂垒’、‘首案红’等品种适宜观赏栽培，‘太平红’、‘JPH’等可花药兼用。 3、不同的土壤、气候环境对丹皮药效成分含量有明显的影响，丹皮中重金属含量主要与土壤环境关系密切。 4、根的粗细程度与药效成分含量关系不大，建议市场药材分级时充分考虑。 5、丹皮的药效成分含量与株龄、采收期、储藏时期关系密切;建议采收、应用根据各地的品种、气候条件及市场情况而定，在保证较高药效成分含量的前提下，尽量缩短生产、流通周期。 6、催花、嫁接对一些牡丹品种的药用价值影响不大，可合理开发利用。 7、药效成分的遗传有一定的规律，86%的子代丹皮酚含量高于父母本或亲本之一;丹皮酚原苷和没食子酰羟基芍药苷含量受父本影响较大，而芍药苷、苯甲酰芍药苷、丹皮酚新苷、没食子酰丹皮酚苷含量受母本影响较大。

药用牡丹资源与重金属污染问题的研究

我国牡丹资源丰富、药用历史悠久、药材出口量大。近年来国际上对进口植物药的重金属限量标准不断提高，已成为影响丹皮出口的最大贸易壁垒之一，严重阻碍了丹皮产业化进程。为了提高我国丹皮药材整体质量水平和国际竞争力，本试验探讨了不同产地、不同种质资源的丹皮与Cu、Cd、Pb、As四种重金属的关系，重点研究了两个代表性品种对重金属Cu的富集规律，分析了重金属Cu对牡丹生长、生理和药材品质的影响。实验中采用火焰原子吸收分光光度法（FAAS ）和电感耦合等离子体质谱法（ICP-MS）为基本检测手段来分析样品中重金属元素的含量，从植物与重金属相互关系的角度对牡丹药用、花药兼用以及生态修复可行性等方面进行了系统研究。结果如下： 1. 丹皮重金属含量与牡丹种质资源和栽培环境关系密切。野生种中滇牡丹丹皮中Cu、Cd含量相对较高，多数野生种丹皮中Pb、As含量较栽培种偏高；铜陵产区的丹皮重金属含量明显高于其它产区，铜尾矿上栽培出产的丹皮重金属Cu含量明显超标，建议其改变种植模式，可以考虑丰富牡丹品种，开发生态旅游、观赏栽培。 2. 综合比较同一地块的7个常用药用品种发现，‘JPH’和‘CKL’品种对重金属富集能力相对较弱。‘JPH’是传统的优良观赏品种，适宜花药兼用；结合前期研究的结果‘CKL’药效成分含量相对较高，药用价值较高，适宜药用栽培。 3. 对药用牡丹品种‘凤丹’和‘建始粉’外施不同浓度CuSO4溶液处理后发现，丹皮中重金属富集量与外施重金属浓度成显著正相关。当外施Cu 浓度超过150mg/kg 时，两个品种的丹皮中Cu含量均已超标。 4. 重金属Cu对牡丹生长的影响效应与重金属浓度和植物体自身部位有关。当外施Cu浓度小于600mg/kg 时，对牡丹生长有一定的促进作用，主要表现在地上部分；超过600mg/kg 时，对牡丹生长有抑制作用，随着浓度增大，毒害症状愈加明显。地下部主要表现出受抑制作用，抑制效应随外施浓度增大而加重。 5. 牡丹通过自身的生理生化调节机制抵抗Cu胁迫对细胞造成的氧化伤害：低浓度Cu可以刺激抗氧化酶系统SOD和POD 活性升高，高浓度下SOD和 POD活性则显著降低。相同处理条件下‘凤丹’品种的抗铜性要高于‘建始粉’。 6. 本试验未发现外施Cu处理对丹皮中药效成分丹皮酚、芍药苷产生影响。 7. ‘凤丹’及‘建始粉’根系铜富集质量分数均达到超富集植物的要求，因此牡丹可能在重金属Cu污染的环境修复方面存在巨大潜力。

National Agricultural Research Organisation. Fisheries Resources Research Institute Annual Report 2002/2003

One of the avenues through which the Government objective of poverty eradication in Uganda can be achieved is Fisheries development and management. Up to 20% of Uganda’s surface area is covered by aquatic systems i.e. lakes, rivers, streams and swamps and to a large extent, all these are interconnected. The large lakes: Victoria, Albert, Kyoga, George and Edward are sites of the more important commercial fisheries, but even the smaller water bodies, rivers (e.g. the Rivers Nile and Kagera) and the surrounding swamps provide sources of livelihood to rural areas. Fish is an important source of high quality food, employment revenue and is currently the second most important export commodity next to coffee generating approximately US $ 80 million annually. Fish exports to regional markets are worth at least US $ 20 million annually. Fish flesh is rich in proteins, which are superior to those of beef and poultry. Fish flesh contains an anticholesterol which assists in reducing heart diseases. Some fishes are of medicinal value e.g. haplochromines (Nkejje) are used to treat measles. Most of the fish in Uganda is got from lakes Victoria, Kyoga, Albert and Albert Nile, Edward and George production systems as well as from the 160 minor lakes and rivers and the associated wetland systems. Capture fisheries based in these systems contribute up to 99% of the fish production in Uganda but aquaculture is also picking up. The fishing industry employs up to one million Ugandans

National Agricultural Research Organisation. Fisheries Resources Research Institute Annual Report 2003/2004

About 18% of Uganda’s surface area is covered with water from which 300,000 metric tonnes of fish are produced. Fish are currently the second most important export commodity generating approximately US$100 million. Fish provides 50% of protein diet for the 20 million people translating into per capita consumption of 12 kg. Close to the production system, this figure rises to 50 – 100 kg. It is estimated that fishery-related activities employ at least one million people countrywide (i.e. 5% of the population). Fish is an important source of high quality food, employment, and revenue and it is currently the second most important export commodity next to coffee generating approximately US $ 80 million annually. Fish exports to regional markets are worth at least US $ 20 million annually. Fish flesh is rich in proteins, which are superior to those of beef and poultry. Fish flesh contains an anticholesterol which assists in reducing heart diseases. Some fishes are of medicinal value e.g. haplochromines (Nkejje) are used to treat measles. Most of the fish in Uganda is got from lakes Victoria, Kyoga, Albert and Albert Nile, Edward and George production systems as well as from the 160 minor lakes and rivers and the associated wetland systems. Capture fisheries based in these systems contribute up to 99% of the fish production in Uganda but aquaculture is also picking up. The fishing industry employs up to one million Ugandans.

National Agricultural Research Organisation. Fisheries Resources Research Institute Annual Report 2004/2005

About 18% of Uganda’s surface area is covered with water from which about 300,000 metric tonnes of fish are produced. Fish are currently the second most important export commodity generating approximately US$100 million annually. Fish provides 50% of protein diet for the 20 million people translating into per capita consumption of 12 kg. Close to the production system, this figure rises to 50 – 100 kg. It is estimated that fishery-related activities employ at least one million people countrywide (i.e. 5% of the population). Fish exports to regional markets are worth at least US $ 20 million annually. Fish flesh contains an anticholesterol which assists in reducing heart diseases. Some fishes are of medicinal value e.g. haplochromines (Nkejje) are used to treat measles. Most of the fish in Uganda is got from lakes Victoria, Kyoga, Albert and Albert Nile, Edward and George production systems as well as from the 160 minor lakes and rivers and the associated wetland systems. Capture fisheries based in these systems contribute up to 99% of the fish production in Uganda but aquaculture is also picking up. The fishing industry employs up to one million Ugandans

A new anti-HIV lupane acid from Gleditsia sinensis Lam.

A new lupane acid, 2 beta-carboxyl, 3 beta-hydroxyl-norlupA (1)-20 (29)-en-28-oic acid (1), together with five known lupane acid derivatives (2-6), were isolated from the stings of Gleditsia sinensis Lam.. Their structures were elucidated on the basis of

Lancifodilactone G: A unique nortriterpenoid isolated from Schisandra lancifolia and its anti-HIV activity

Lancifodilactone G (1), a novel, highly oxygenated nortriterpenoid featuring a partial enol structure and a spirocyclic moiety, was isolated from the medicinal plant Schisandra lancifolia. Its structure and stereochemistry were determined from extensive one- and two-dimensional NMR and mass spectral data, coupled with single-crystal X-ray analysis. Compound 1 exerted minimal cytotoxicity against C8166 cells (CC50 > 200 mu g/mL) and showed anti-HIV activity with EC50 = 95.47 +/- 14.19 mu g/mL and a selectivity index in the range of 1.82-2.46.

Halichondria sulfonic acid, a new HIV-1 inhibitory guanidino-sulflonic acid, and halistanol sulfate isolated from the marine sponge Halichondria rugosa Ridley & Dendy

A new sulfur-containing guanidino derivative, halichondria sulfonic acid (1) showing anti-HIV-1 activity, and halistanol trisulfate (2) with anti-tumor activity have been isolated from the marine sponge Halichondria rugosa Ridley & Dendy collected in the

Nortriterpenoids from Schisandra lancifolia

Six new nortriterpenoids, lancifodilactones 1-N (1-6), as well as nine known ones, were isolated from the leaves and stems of Schisandra lancifolia. Their structures were elucidated on the basis of spectroscopic methods including 2D NMR analysis, and the

Triterpenoids from Schisandra lancifolia with anti-HIV-1 activity

A new trinorcycloartane triterpenoid, lancifodilactone H (1), and a new A ring-secocycloartane triterpenoid, lancifoic acid A (2), together with a known compound, nigranoic acid (3), were isolated from the leaves and stems of Schisandra lancifolia. Their

Synthesis and biological evaluation of N-acetyl-beta-aryl-1,2-didehydroethylamines as new HIV-1 RT inhibitors in vitro

A variety of N-acetyl-o-aryl-1,2-didehydroethylamines were synthesized by direct reduction-acetylation of beta-aryl-nitroolefins and assayed as HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) for the first time. Compound 7a exhibited a TI v