印楝素微波萃取技术研究

印楝素(Azadirachtin)是从印楝(Azadirachta indica A.Juss)种仁中分离得到的柠檬素类(limonoids)化合物,属四环三萜类化合物[1].生物活性试验证明低浓度的印楝素即对沙漠蝗(Schistocera gregaria)产生强烈的拒食活性,进一步的研究证明印楝素对多种农林业害虫具有较高的拒食、趋避及抑制生长发育等作用[2],且具有害虫不易产生抗性、对温血动物无害、对昆虫的天敌较安全、在自然界易降解等特点,其广泛应用有利于生态平衡及农林业的可持续发展,是一种有广阔应用前景的植物源生物农药.     

石蒜碱的全合成及结构修饰研究进展

石蒜碱是石蒜科生物碱中非常重要的一类异喹啉生物碱衍生物,近年来药物及有机化学工作者发现石蒜碱及其衍生物具有抗肿瘤、抗炎、抑制乙酰胆碱酯酶、抗疟疾、保护心血管等多种作用,由于其复杂而独特的分子结构和广泛的药理活性使得科研工作者们进行了大量的全合成尝试及结构修饰等研究.作者对石蒜碱的全合成及其结构修饰进行了综述.     

LC-MS法测定印楝油中印楝素含量

印楝(Azadirachta indica,A.Juss)是含有多种生物活性的植物,用于农药、医药、肥料、饲料、燃料、土壤改良、建筑材料和化工原料等行业,印楝是一种众所周知的三萜类化合物的来源,它对许多昆虫显示出拒食性和干扰生长规律性.这些杀虫活性物质以印楝植株各部位都含有的印楝素(Azadirchin)为主,而其种子中含量最高.印楝种仁中含有45%左右的印楝油,因此印楝油不仅具有杀虫活性,而且具有抗菌、抗病毒的活性,其中含有的活性物质具有一定的药用价值. 印楝籽油作为一种较好的医药、农药及日用化工原料, 是一种极具开发应用前景的高级植物油.本文运用色质联用技术,结合液相色谱法的高分离效能和质谱的高选择性、高灵敏度的特性,建立一种具有更高适用性的印楝油中印楝素的含量测定方法[1,2].     

天然活性吡咯烷-2,4-二酮类化合物研究进展

吡咯烷-2,4-二酮类化合物广泛存在于陆上及海洋生物中,其复杂多变的分子结构和广谱独特的生物活性引起人们的极大关注.结合本研究组在这一邻域的工作综述了天然吡咯烷-2,4-二酮类化合物的结构、生物活性,重点介绍此类化合物的全合成等方面的新进展.     

印楝素B的结构修饰研究

印楝素(Azadirachtin)是从印楝(Azadirachta indica A.Juss)种仁中分离得到的一系列柠檬素类(limonoids)化合物[1],主要含AZ-A至AZ-L等十余种活性组分.印楝素对多种农林业害虫具有拒食、趋避及抑制昆虫生长发育等作用,且具有害虫不易产生抗性,对天敌较安全、对温血动物无害、在自然界易降解、不污染环境等特点,其广泛应用有利于生态平衡及农林业的可持续发展[2,3],是一种有广阔应用前景的生物农药.     

苯并呋喃衍生物的合成和结构表征

天然化合物常表现出各种独特的生物活性,使其成为寻找和筛选各种生物活性物质如医药、农药等的天然宝库.楝酰胺(rocaglamide)(结构式如图式1)是从楝属植物中分离出的具有良好杀虫活性的化合物[1],对疆叶蛾的LC50为0.91μg/mL,与已知的天然杀虫剂苦楝素Azadirachtin(LC50为0.70μg/mL)的活性相当[2-4],这表明楝酰胺有可能成为一种极为重要的天然杀虫剂.     

藤黄科植物中具有桥环结构的天然产物全合成研究进展

藤黄科植物中含桥环的天然产物具有独特的化学结构和明显的生物活性. 综述了近年来这类天然产物的全合成研究进展.     

冬凌草甲素的结构修饰与生物活性研究进展

冬凌草甲素为对映贝壳杉烷类二萜化合物,主要分布在中草药冬凌草及相关植物中,具有多种生物活性如抗肿瘤、抗菌、抗炎等.主要综述了2000年以来冬凌草甲素的多功能结构修饰,表明冬凌草甲素结构修饰是提高冬凌草甲素药理活性的一种有效途径.     

亮点介绍

正Ir-催化不对称全合成吲哚生物碱(-)-Communesin F J.Am.Chem.Soc.2017,139,3364~3367吲哚生物碱Communesin家族是八个具有复杂七环系的天然产物.因具有独特的化学结构(四个氮原子构成罕见的上下两个aminal基团,外加两个连续手性季碳)和有趣的生物活性而成为明星分子,尤其是Communesin F的全合成备受化学家青睐,包括我国秦勇、马大为研究组在内有五个著名研究组报道过全合成.完成Communesin F这样高     

天然产物吲哚二酮哌嗪生物碱的结构及生物活性

吲哚二酮哌嗪生物碱是一种内生真菌次生代谢产物,多提取自曲霉属Aspergillus和青霉属Penicillium,是由不同氨基酸结合衍生而来的,在自然界分布广泛。该类化合物含有吲哚和二酮哌嗪两个母核,具有良好而广泛的生物活性,可为药物研发提供先导化合物,其新颖的结构和显著的生物活性也受到了化学全合成领域内学者的日益关注。本文主要从该类化合物的发现和化合物的结构以及化合物抗癌与抗肿瘤、抑菌、免疫调节、抗氧化及杀虫等生物活性方面对这类化合物的相关研究予以回顾,并对其结构与生物活性之间的活性构效关系进行了简要分析,为该类化合物的发现、合成及生物活性研究提供参考,并为药物研发提供借鉴。     

Estimation of Azadirachtin Content in Neem Extracts and Formulations

Abstract

A high performance liquid chromatographic reversed-phase procedure has been developed whereby azadirachtin content can be estimated in crude extracts of neem and in dust formulations of neem. An estimation of the azadirachtin content is achieved through the use of an external azadirachtin standard and valley-to-valley integration. Since azadirachtin seems to be the most potent insect feeding deterrent in these extracts and formulations, its content is a measurement of potency and represents an attempt at standardization.     

An HPLC method for determination of azadirachtin residues in bovine muscle

A high-performance liquid chromatography (HPLC) method for the determination of azadirachtin (A and B) residues in bovine muscle has been developed. Azadirachtin is a neutral triterpene and chemotherapeutic agent effective in controlling some pest flies in horses, stables, horns and fruit. The actual HPLC method uses an isocratic elution and UV detection. Liquid-liquid extraction and solid-phase purification was used for the clean-up of the biological matrix. The chromatographic determination of these components is achieved using a C18 analytical column with water-acetonitrile mixture (27.5:72.5, v/v) as mobile phase, 1 mL/min as flow rate, 45 °C column temperature and UV detector at 215 nm. The azadirachtin peaks are well resolved and free of interference from matrix components. The extraction and analytical method developed in this work allows the quantitation of azadirachtin with precision and accuracy, establishing a lower limit of quantitation of azadirachtin, extracted from the biological matrix.     

Purification of the seven tetranortriterpenoids in neem (Azadirachta indica) seed by counter-current chromatography sequentially followed by isocratic preparative reversed-phase high-performance liquid chromatography

Counter-current chromatography (CCC) sequentially followed by isocratic preparative reversed-phase high-performance liquid chromatography was used to isolate the seven bio-actives (azadirachtin A, azadirachtin B, azadirachtin H, desacetylnimbin, desacetylsalannin, nimbin and salannin) from the seed concentrate (NSC) of the neem tree (Azadirachta indica A. Juss). Reproducible, narrow polarity range, high purity fractions were obtained from repeated injections of the NSC (700 mg loadings/injection), on to a relatively small volume CCC coil (116 mL). The CCC biphasic solvent system chosen was hexane:butanol:methanol:water (1:0.9:1:0.9, v/v). A mass balance of injected material showed that 95+% were recovered.     

Development and evaluation of thin-layer chromatography-digital image-based analysis for the quantitation of the botanical pesticide azadirachtin in agricultural matrixes and commercial formulations: comparison with ELISA

A simple thin-layer chromatography-digital image-based analytical method has been developed for the quantitation of the botanical pesticide, azadirachtin. The method was validated by analyzing azadirachtin in the spiked food matrixes and processed commercial pesticide formulations, using acidified vanillin reagent as a postchromatographic derivatizing agent. The separated azadirachtin was clearly identified as a green spot. The Rf value was found to be 0.55, which was similar to that of a reference standard. A standard calibration plot was established using a reference standard, based on the linear regression analysis [r2 = 0.996; y = 371.43 + (634.82)x]. The sensitivity of the method was found to be 0.875 microg azadirachtin. Spiking studies conducted at the 1 ppm (microg/g) level in various agricultural matrixes, such as brinjal, tomato, coffee, and cotton seeds, revealed the recoveries of azadirachtin in the range of 67-92%. Azadirachtin content of commercial neem formulations analyzed by the method was in the range of 190-1825 ppm (microg/mL). Further, the present method was compared with an immunoanalytical method enzyme-linked immonosorbent assay developed earlier in our laboratory. Statistical comparison of the 2 methods, using Fischer's F-test, indicated no significant difference in variance, suggesting that both methods are comparable.     

In Vitro Azadirachtin Production by Hairy Root Cultivation of <Emphasis Type="Italic">Azadirachta indica</Emphasis> in Nutrient Mist Bioreactor

Azadirachtin, a well-known biopesticide is a secondary metabolite conventionally extracted from the seeds of Azadirachta indica. The present study involved in vitro azadirachtin production by developing hairy roots of A. indica via Agrobacterium rhizogenes-mediated transformation of A. indica explants. Liquid culture of hairy roots was established in shake flask to study the kinetics of growth and azadirachtin production. A biomass production of 13.3 g/L dry weight (specific growth rate of 0.7 day⁻¹) was obtained after 25 days of cultivation period with an azadirachtin yield of 3.3 mg/g root biomass. To overcome the mass transfer limitation in conventionally used liquid-phase reactors, batch cultivation of hairy roots was carried out in gas-phase reactors (nutrient spray and nutrient mist bioreactor) to investigate the possible scale-up of A. indica hairy root culture. The nano-size nutrient mist particles generated from the nozzle of the nutrient mist bioreactor could penetrate till the inner core of the inoculated root matrix, facilitating uniform growth during high-density cultivation of hairy roots. A biomass production of 9.8 g/L dry weight with azadirachtin accumulation of 2.8 mg/g biomass (27.4 mg/L) could be achieved in 25 days of batch cultivation period, which was equivalent to a volumetric productivity of 1.09 mg/L per day of azadirachtin.     

Effect of Elicitors and Precursors on Azadirachtin Production in Hairy Root Culture of Azadirachta indica

The present study involved strategies for enhancement in in vitro azadirachtin (commercially used biopesticide) production by hairy root cultivation of Azadirachta indica. Improvement in the azadirachtin production via triggering its biosynthetic pathway in plant cells was carried out by the exogenous addition of precursors and elicitors in the growth medium. Among the different abiotic stress inducers (Ag⁺, Hg⁺², Co⁺², Cu⁺²) and signal molecules (methyl jasmonate and salicylic acid) tested, salicylic acid at 15 mg l^?1 of concentration was found to enhance the azadirachtin yield in the hairy roots to the maximum (up to 4.95 mg g^?1). Similarly, among the different biotic elicitors tested (filter-sterilized fungal culture filtrates of Phoma herbarium, Alternaria alternata, Myrothecium sp., Fusarium solani, Curvularia lunata, and Sclerotium rolfsii; yeast extract; and yeast extract carbohydrate fraction), addition of filter-sterilized fungal culture filtrate of C. lunata (1 %?v/v) resulted in maximum azadirachtin yield enhancement in hairy root biomass (up to 7.1 mg g^?1) with respect to the control (3.3 mg g^?1). Among all the biosynthetic precursors studied (sodium acetate, cholesterol, squalene, isopentynyl pyrophosphate, mavalonic acid lactone, and geranyl pyrophosphate), the overall azadirachtin production (70.42 mg l^?1 in 25 days) was found to be the highest with cholesterol (50 mg l^?1) addition as an indirect precursor in the medium.     

Biological studies of azadirachtin and its derivatives against polyphagous pest, Spodoptera litura

Azadirachtin-A (Aza-A, figure 1) was subjected to a variety of synthetic transformations and the antifeedant activity and toxicity of the azadirachtin derivatives were assessed against second instar larvae Spodoptera litura on castor leaves in comparison with the crude material containing 9.14% Aza-A, as well as relatively pure 91% Aza-A. A probable mechanism for the base initiated fragmentation of the diketocarbonate into a decalin fragment and a spiroketal moiety has been proposed. The present study indicates that the combination of insect toxicity and antifeedancy of azadirachtin provides good crop protection.     

Highly selective entry to the azadirachtin skeleton via a Claisen rearrangement/radical cyclization sequence

[formula: see text] A highly diastereoselective, microwave-induced Claisen rearrangement of an appropriately substituted propargylic enol ether allows the formation of the sterically congested C8-C14 bond of azadirachtin. When combined with a radical-mediated cyclization of the corresponding allene, this sequence offers rapid entry to the framework of azadirachtin.     

Production of Biopesticides in an In Situ Cell Retention Bioreactor

The seeds of Azadirachta indica contain azadirachtin and other limonoids, which can be used as a biopesticide for crop protection. Significant variability and availability of seed only in arid zones has triggered biotechnological production of biopesticides to cope up with its huge requirement. Batch cultivation of A. indica suspension culture was carried out in statistically optimized media (25.0 g/l glucose, 5.7 g/l nitrate, 0.094 g/l phosphate and 5 g/l inoculum) in 3 l stirred tank bioreactor. This resulted in 15.5 g/l biomass and 0.05 g/l azadirachtin production in 10 days leading to productivity of 5 mg l(-1) day(-1). Possible inhibition by the limiting substrates (C, N, P) were also studied and maximum inhibitory concentrations identified. The batch kinetic/inhibitory data were then used to develop and identify an unstructured mathematical model. The batch model was extrapolated to simulate continuous cultivation with and without cell retention in the bioreactor. Several offline computer simulations were done to identify right nutrient feeding strategies (with respect to key limiting substrates; carbon, nitrate and phosphate) to maintain non-limiting and non-inhibitory substrate concentrations in bioreactor. One such continuous culture (with cell retention) simulation was experimentally implemented. In this cultivation, the cells were propagated batch-wise for 8 days. It was then converted to continuous cultivation by feeding MS salts with glucose (75 g/l), nitrate (10 g/l), and phosphate (0.5 g/l) at a feed rate of 500 ml/day and withdrawing the spent medium at the same rate. The above continuous cultivation (with cell retention) demonstrated an improvement in cell growth to 95.8 g/l and intracellular accumulation of 0.38 g/l azadirachtin in 40 days leading to an overall productivity of 9.5 mg l(-1) day(-1).