灵芝三萜提取工艺研究

研究灵芝中三萜的提取工艺.方法:采用单因素试验法和正交试验法,以灵芝三萜的提取率为考察指标,考察溶剂浓度、溶剂用量、温度、时间等因素对提取率的影响.结果:浸提温度是影响三萜提取率的主要因素.最佳工艺条件:溶剂为无水乙醇,溶剂用量为18倍量,时间2 h,温度80℃,此条件下三萜提取率达94.47%.结论:该工艺的三萜提取率高,可用于灵芝中三萜的提取.     

Neurotrophic and Immunomodulatory Lanostane Triterpenoids from Wood-Inhabiting Basidiomycota

Neurotrophins such as nerve growth factor (ngf) and brain-derived neurotrophic factor (bdnf) play important roles in the central nervous system. They are potential therapeutic drugs for the treatment of neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. In this study, we investigated the neurotrophic properties of triterpenes isolated from fruiting bodies of Laetiporus sulphureus and a mycelial culture of Antrodia sp. MUCL 56049. The structures of the isolated compounds were elucidated based on nuclear magnetic resonance (NMR) spectroscopy in combination with high-resolution electrospray mass spectrometry (HR-ESIMS). The secondary metabolites were tested for neurotrophin (ngf and bdnf) expression levels on human astrocytoma 1321N1 cells. Neurite outgrowth activity using rat pheochromocytoma (PC-12) cells was also determined. Twelve triterpenoids were isolated, of which several potently stimulated the expression of neurotrophic factors, namely, ngf (sulphurenic acid, 15α-dehydroxytrametenolic acid, fomefficinic acid D, and 16α-hydroxyeburicoic acid) and bdnf (sulphurenic acid and 15α-dehydroxytrametenolic acid), respectively. The triterpenes also potentiated ngf-induced neurite outgrowth in PC-12 cells. This is, to the best of our knowledge, the first report on the compound class of lanostanes in direct relation to bdnf and ngf enhancement. These compounds are widespread in medicinal mushrooms; hence, they appear promising as a starting point for the development of drugs and mycopharmaceuticals to combat neurodegenerative diseases. Interestingly, they do not show any pronounced cytotoxicity and may, therefore, be better suited for therapy than many other neurotrophic compounds that were previously reported.     

齐墩果酸药理作用研究进展

五环三萜类化合物齐墩果酸广泛分布于多种天然植物中,具有多种生物活性。本文综述了近年来对齐墩果酸药理方面的研究进展并对其研究前景进行了展望。     

Enhanced Water Solubility and Anti-Tumor Activity of Oleanolic Acid through Chemical Structure Modification

Cancer has been a major health problem in the world in the past decades. It is urgent to develop new, effective and safe drugs for the treatment of cancer. There are many pentacyclic triterpenoids with positive anti-tumor activity and safety in nature. Oleanolic acid (OA), as one of the pentacyclic triterpenoids, also has broad biological activities including liver protection, anti-inflammatory, hypoglycemic, antiviral and anti-tumor. Therefore, to investigate its anti-tumor activity and mechanism, many OA derivatives have been developed. Some derivatives are less toxic to normal hepatocytes, which may be due to the strong liver protection ability of OA. However, the poor water solubility of OA is one of the main reasons for the weak anti-tumor activity. It is reported that some OA derivatives could enhance solubility by chemically linking some hydrophilic groups to improve anti-tumor activity. This review not only summarizes the highly water-soluble OA derivatives that can improve anti-tumor activity reported in recent years, but also introduces their possible anti-tumor mechanisms.     

Synthesis and SAR of Tetracyclic Inhibitors of Protein Kinase CK2 Derived from Furocarbazole W16

The serine/threonine kinase CK2 modulates the activity of more than 300 proteins and thus plays a crucial role in various physiological and pathophysiological processes including neurodegenerative disorders of the central nervous system and cancer. The enzymatic activity of CK2 is controlled by the equilibrium between the heterotetrameric holoenzyme CK2α₂β₂ and its monomeric subunits CK2α and CK2β. A series of analogues of W16 ((3aR,4S,10S,10aS)-4-{[(S)-4-benzyl-2-oxo-1,3-oxazolidin-3-yl]carbonyl}-10-(3,4,5-trimethoxyphenyl)-4,5,10,10a-tetrahydrofuro[3,4-b]carbazole-1,3(3aH)-dione ((+)- 3 a )) was prepared in an one-pot, three-component Levy reaction. The stereochemistry of the tetracyclic compounds was analyzed. Additionally, the chemically labile anhydride structure of the furocarbazoles 3 was replaced by a more stable imide ( 9 ) and N-methylimide ( 10 ) substructure. The enantiomer (−)- 3 a (K_i=4.9 μM) of the lead compound (+)- 3 a (K_i=31 μM) showed a more than sixfold increased inhibition of the CK2α/CK2β interaction (protein-protein interaction inhibition, PPII) in a microscale thermophoresis (MST) assay. However, (−)- 3 a did not show an increased enzyme inhibition of the CK2α₂β₂ holoenzyme, the CK2α subunit or the mutated CK2α′ ^C336S subunit in the capillary electrophoresis assay. In the pyrrolocarbazole series, the imide (−)- 9 a (K_i=3.6 μM) and the N-methylimide (+)- 10 a (K_i=2.8 μM) represent the most promising inhibitors of the CK2α/CK2β interaction. However, neither compound could inhibit enzymatic activity. Unexpectedly, the racemic tetracyclic pyrrolocarbazole (±)- 12 , with a carboxy moiety in the 4-position, displays the highest CK2α/CK2β interaction inhibition (K_i=1.8 μM) of this series of compounds.     

Cloning and Characterization of Farnesyl Diphosphate Synthase Gene Involved in Triterpenoids Biosynthesis from Poria cocos

Poria cocos (P. cocos) has long been used as traditional Chinese medicine and triterpenoids are the most important pharmacologically active constituents of this fungus. Farnesyl pyrophosphate synthase (FPS) is a key enzyme of triterpenoids biosynthesis. The gene encoding FPS was cloned from P. cocos by degenerate PCR, inverse PCR and cassette PCR. The open reading frame of the gene is 1086 bp in length, corresponding to a predicted polypeptide of 361 amino acid residues with a molecular weight of 41.2 kDa. Comparison of the P. cocos FPS deduced amino acid sequence with other species showed the highest identity with Ganoderma lucidum (74%). The predicted P. cocos FPS shares at least four conserved regions involved in the enzymatic activity with the FPSs of varied species. The recombinant protein was expressed in Pichia pastoris and purified. Gas chromatography analysis showed that the recombinant FPS could catalyze the formation of farnesyl diphosphate (FPP) from geranyl diphosphate (GPP) and isopentenyl diphosphate (IPP). Furthermore, the expression profile of the FPS gene and content of total triterpenoids under different stages of development and methyl jasmonate treatments were determined. The results indicated that there is a positive correlation between the activity of FPS and the amount of total triterpenoids produced in P. cocos.     

High-Performance Multifunctional Fuel Additives Derived from Renewable Fatty Acids and Phosphazene

The increasing need for high-quality diesel fuel can be fulfilled by using environmental friendly, high-performance additives. We have synthesized novel fatty acid-derived multifunctional additives coupled with phosphazene moiety having the backbone of alternative nitrogen and phosphorous atoms which provided stability, while the additives possessed affinity for the metal surface to form a surface-complex film leading to the hindered metal contact and providing friction-reducing and antiwear properties. In addition, the applicability of the synthesized additives was investigated by testing the anti-oxidant and anticorrosion properties in polyethylene glycol (PEG200) which was taken as the reference base fluid. A rotating bomb oxidation test was used to evaluate the anti-oxidant characteristics, and a four-ball test for the tribological properties. Among the synthesized additives, phosphazene ester of erucic acid exhibited the best antiwear and antifriction properties; however, phosphazene ester of oleic acid was found to be the best anti-oxidant additive.     

Triterpenoids from the Roots of Rhaphiolepis indica var. tashiroi and Their Anti-Inflammatory Activity

Two new triterpenoids, 2α,3β-dihydroxyolean-11,13(18)-dien-19β,28-olide (1) and 3β,5β-dihydroxyglutinol (2), together with eight known compounds (3–10) were isolated from the roots of Rhaphiolepis indica var. tashiroi (Rosaceae). The structures of 1–10 were determined by spectroscopic techniques. Among these isolates, 2α,3β-dihydroxyolean-13(18)-en-28-oic acid (9) exhibited inhibitory effect on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide production, with an IC₅₀ value of 16.50 μM.     

固定化Gibberella intermedia转化3-氰基吡啶制备烟酸

研究不同复合包埋材料对含腈水解酶的Gibberella intermedia CA3-1进行固定化,选择出最为适合的壳聚糖-聚乙烯醇作为复合包埋材料,其优化后的浓度分别为1.6%和2%。实验结果表明该固定化细胞的最适转化条件为：3-氰基吡啶初始浓度300mmol/L,转化温度30℃,pH值7.0,转化时间60min。固定化细胞可以稳定进行26个批次转化,每克干细胞可转化产生283.5g烟酸,为游离细胞的3倍。此外,固定化细胞对高浓度3-氰基吡啶的耐受性大幅提高,温度稳定性有一定增强。连续补料19次,固定化细胞酶活保留约30%,经折算产率为每克干细胞转化生成191.3g烟酸。     

中华苦荬菜总黄酮和总三萜提取工艺优化研究

以中华苦荬菜的总黄酮和总三萜为筛选指标,分别对索氏提取、超声波提取、微波提取设计正交试验,比较3种方法对有效成分的提取效率。每种方法得到优化整合方案,最后进行得率比较,优选最佳提取工艺。结果表明超声波提取法对总黄酮和总三萜提取得率最高,单次提取即可同时达到2种物质提取率的较好方法。其最佳条件为80%乙醇,在45℃条件下提取40 min。该法简便,提取率高,可用于快速提取中华苦荬菜中的有效成分。     

Formation of Triterpenoids throughout <Emphasis Type="Italic">Olea europaea</Emphasis> Fruit Ontogeny

Drupes were handpicked from olive (Olea europaea L.) trees, cv chemlali, at 13 distinct stages of fruit development, referred to as weeks after flowering (WAF), and analyzed for their free and esterified sterols and triterpenoids content. These two classes of compounds are synthesized via the acetate/mevalonate pathway and share common precursors up to oxidosqualene (OS). Cyclization of OS in either cycloartenol or beta-amyrin constitutes a branch point between primary (sterol pathway) and secondary (triterpenoid pathway) metabolisms. At the onset of fruit development, i.e., between 12 and 18 WAF, drupes were found to contain high amounts of alpha- and beta-amyrins as well as more-oxygenated compounds such as triterpenic diols (erythrodiol and uvaol) and acids (oleanolic, ursolic and maslinic acids). Concomitantly, sterol precursors were barely detectable. From 21 WAF, when the olive fruit reached its final size and began to turn from green to purple, alpha- and beta-amyrins were no longer present, while 4,4-dimethyl- and 4alpha-methylsterols started to be formed, indicating a redirection of the carbon flux from the triterpenoid pathway towards the sterol pathway. Between 21 and 30 WAF, sterol end products, mainly represented by sitosterol, progressively accumulated and triterpenic diols were replaced by triterpenic acids, essentially maslinic acid. Interestingly, the developing olive fruit was found to accumulate significant amounts of parkeol as an ester conjugate. Whatever the stage of development, triterpenoids represent the major triterpenic compounds of the olive fruit.     

The Influence of Exogenous Jasmonic Acid on the Biosynthesis of Steroids and Triterpenoids in Calendula officinalis Plants and Hairy Root Culture

The interplay between steroids and triterpenoids, compounds sharing the same biosynthetic pathway but exerting distinctive functions, is an important part of the defense strategy of plants, and includes metabolic modifications triggered by stress hormones such as jasmonic acid. Two experimental models, Calendula officinalis hairy root cultures and greenhouse cultivated plants (pot plants), were applied for the investigation of the effects of exogenously applied jasmonic acid on the biosynthesis and accumulation of steroids and triterpenoids, characterized by targeted GC-MS (gas chromatography-mass spectroscopy) metabolomic profiling. Jasmonic acid elicitation strongly increased triterpenoid saponin production in hairy root cultures (up to 86-fold) and their release to the medium (up to 533-fold), whereas the effect observed in pot plants was less remarkable (two-fold enhancement of saponin biosynthesis after a single foliar application). In both models, the increase of triterpenoid biosynthesis was coupled with hampering the biomass formation and modifying the sterol content, involving stigmasterol-to-sitosterol ratio, and the proportions between ester and glycoside conjugates. The study revealed that various organs in the same plant can react differently to jasmonic acid elicitation; hairy root cultures are a useful in vitro model to track metabolic changes, and enhanced glycosylation (of both triterpenoids and sterols) seems to be important strategy in plant defense response.     

Total Synthesis of Capsaicin Analogues from Lignin‐Derived Compounds by Combined Heterogeneous Metal,Organocatalytic and Enzymatic Cascades in One Pot

The total synthesis of capsaicin analogues was performed in one pot, starting from compounds that can be derived from lignin. Heterogeneous palladium nanoparticles were used to oxidise alcohols to aldehydes, which were further converted to amines by an enzyme cascade system, including an amine transaminase. It was shown that the palladium catalyst and the enzyme cascade system could be successfully combined in the same pot for conversion of alcohols to amines without any purification of intermediates. The intermediate vanillylamine, prepared with the enzyme cascade system, could be further converted to capsaicin analogues without any purification using either fatty acids and a lipase, or Schotten–Baumann conditions, in the same pot. An aldol compound (a simple lignin model) could also be used as starting material for the synthesis of capsaicin analogues. Using L ‐alanine as organocatalyst, vanillin could be obtained by a retro‐aldol reaction. This could be combined with the enzyme cascade system to convert the aldol compound to vanillylamine in a one‐step one‐pot reaction.

     

Supercritical CO2 extraction of triterpenes from rosemary leaves: Kinetics and modelling

The pentacyclic triterpene α,β-amyrin is a promising bioactive natural product. Supercritical fluid extraction and fractionation are used to obtain pentacyclic triterpenes compounds from dried rosemary leaves. Experiments were conducted at 50°C and 180 bar, and the fractionation of the extract was accomplished in two separators. The shift to a diffusion-controlled regime occurred when 65% of the total oil was extracted. The moments analysis of kinetic curves indicated that the contribution of the axial dispersion, the external transfer and the internal transfer were 9.16%, 80.24% and 10.58%, respectively. The overall extraction curve was represented using three mathematical models.     

新技术在甾体药物微生物转化中的应用

主要综述了固定化细胞和原生质体转化、双水相转化、超临界流体技术、有机介质中微生物转化以及磁场和超声处理等新技术应用于甾体药物微生物转化的研究进展。简要介绍了相应的反应器研究开发趋势。     

柿叶三萜类化合物提取工艺条件研究

采用单因素实验和正交设计实验法对柿叶中三萜类化合物的提取工艺进行了研究。结果表明,以70%乙醇为溶剂的回流提取效果最佳;提取的最佳条件为料液比1：20,提取次数为4次,提取时间为2 h,三萜化合物的提取率可达2.16%。     

人参皂苷的定向生物转化研究进展

稀有人参皂苷是人参的重要活性成分,但其含量极低或需经过肠道转化才能产生,工业生产中常通过去糖基、脱水等方法将常见人参皂苷转化为稀有人参皂苷。基于此,本文概述了人参皂苷的构效关系及常用转化方法,总结了微生物、酶法定向转化人参皂苷的最新进展,着重介绍了益生菌、食药用真菌转化人参皂苷制备功能食品,以及糖苷酶筛选和组合在提高人参总皂苷转化效率及产率等方面的研究进展;同时探讨了基因工程、溶剂工程、固定化酶等技术对人参皂苷转化效率、产率的影响,展望了蛋白质工程、合成生物学等方法在人参皂苷转化及合成方面的潜在应用价值,为稀有人参皂苷的规模化生产提供了基础。     

极端微生物与工业生物催化剂开发

工业催化领域对生物催化剂提出了苛刻的要求,特别是高浓度底物或产物、高离子强度、酸碱条件及其非水相催化,如何获得高效的生物催化剂是催化技术发展的关键。极端微生物是高效工业生物催化剂的重要来源,本文主要介绍从嗜热菌、嗜盐菌或耐盐菌、耐辐射微生物、耐有机溶剂等极端微生物出发发现并开发高效工业生物催化剂,可望为生物技术的进一步发展提供一个更广阔的资源。     

分枝杆菌转化甾醇产9α-羟基雄甾烯酮促进剂的筛选

以甾醇为原料、利用分枝杆菌等微生物转化生产甾体医药中间体9α-羟基雄甾烯酮,是当下甾体医药工业体系的一条重要工艺路线.甾醇作为一种非极性化合物,在水中的溶解度极低,传质效率低,其生物可利用度不高,这严重制约了分枝杆菌对甾醇底物的摄取利用效率.就PEG200、PPG2000和多种硅油对分枝杆菌转化甾醇产9α-羟基雄甾烯酮的促进效果进行了比较分析.结果表明：PEG200、PPG2000和聚醚改性硅油的添加均能有效改善菌体的甾醇转化能力,产物的积累量大幅提高,升幅分别约为50％、85％和300％.随后,重点考察了多种硅油对甾醇转化的影响,从中筛选出效果最好的甾醇转化促进剂并就添加量和添加方式进行了优化,结果显示,最佳甾醇转化促进剂是聚醚改性硅油,其最佳添加方式是在发酵起始时与甾醇同时添加、10 g·L-1甾醇投料量下的最佳添加量为15％.     

Substrate Profiling of Anion Methyltransferases for Promiscuous Synthesis of S-Adenosylmethionine Analogs from Haloalkanes

Biocatalytic alkylation reactions can be performed with high chemo-, regio- and stereoselectivity using S-adenosyl-l -methionine (SAM)-dependent methyltransferases (MTs) and SAM analogs. Currently, however, this methodology is limited in application due to the rather laborious protocols to access SAM analogs. It has recently been shown that halide methyltransferases (HMTs) enable synthesis and recycling of SAM analogs with readily available haloalkanes as starting material. Here we expand this work by using substrate profiling of the anion MT enzyme family to explore promiscuous SAM analog synthesis. Our study shows that anion MTs are in general very promiscuous with respect to the alkyl chain as well as the halide leaving group. Substrate profiling further suggests that promiscuous anion MTs cluster in sequence space. Next to iodoalkanes, cheaper, less toxic, and more available bromoalkanes have been converted and several haloalkanes bearing short alkyl groups, alkyl rings, and functional groups such as alkene, alkyne and aromatic moieties are accepted as substrates. Further, we applied the SAM analogs as electrophiles in enzyme-catalyzed regioselective pyrazole allylation with 3-bromopropene as starting material.