A comprehensive review of azadirachtin: physicochemical properties, bioactivities, production, and biosynthesis

Azadirachtin,a complex tetratriterpenoid limonin with potent insecticidal properties,is the most widely used biological pesticide worldwide.Its versatile pharmacological applications include the inhibition of tumor growth and anti-malarial,anti-bacterial,and anti-inflammatory properties.Azadirachtin plays a pivotal role in pest control and novel drug development.The primary source of azadirachtin is the neem tree(Azadirachta indica A.Juss),with an azadirachtin content ranging from 0.3%to 0.5%.Despite the market demand for botanical pesticides reaching approximately 100,000 tons per year,the annual neem production in China is only 1.14 tons.Although azadirachtin can be obtained through plant extraction or chemical synthesis,the quantity obtained does not meet the market demand in China.The sluggish pace of azadirachtin biosynthesis results from the limited availability of genetic information and the complexity of the synthetic pathway.Recent advancements in azadirachtin biosynthesis hold promise as an efficient collection method.In this study,we explored the physicochemical properties,biological activities,mechanisms of action,and acquisition methods of azadirachtin.We also delved into recent progress in azadirachtin biosynthesis and assessed potential future usage challenges.This study aims to establish a theoretical foundation for the scientific application and efficient synthesis of azadirachtin,offering valuable reference information to the industry.

Variation in seed morphometric characters, oil content and azadirachtin content of seeds, in vitro shoot cultures and callus cultures among different populations of Azadirachta indica

We studied seed morphometric characters, oil content and azadirachtin content in a medicinally important and insect repellent tree species Azadirachta indica A.Juss. Ten populations were selected from two central Indian states of Madhya Pradesh and Chhattisgarh and twenty candidate plus trees were selected from each population based on tree attributes. Azadirachtin content was estimated in seeds, in vitro shoots and callus cultures through HPLC. Significant variation（p ／ 0.05） was observed in seed morphometric characters, oil content and azadirachtin content among the ten populations. Maximum value for 100 seed weight was obtained in seeds of Raigarh（30.51 g） which was on par with seeds of Bargi（28.37 g）and Chhatarpur（26.36 g）. The ratio of highest kernel to seed was also obtained in seeds of Raigarh（60.11%）.Kernels from Chhatarpur population recorded the highest oil content（24.20%）, which was equal to that in Sihore（23.75%） and Katni（21.75%）. We recorded highly significant variation in azadirachtin content in seed samples,in vitro shoot samples and callus cultures. Maximum azadirachtin was present in the seeds of Gwalior（0.882%）which was on par with azadirachtin content in seeds of Chhatarpur, Katni, Sihore, Shahdol and Chhindwara.Maximum azadirachtin in the in vitro shoots was present in the cultures of Gwalior（0.218%） which was statistically on par with Sihore（0.176%）. Similarly, in callus cultures of nodal segments maximum azadirachtin was recorded in the Gwalior population（0.033%） which was statistically on par with Sihore（0.028%）. The trees of Gwalior and Sihore populations can be produced in large numbers through vegetative propagation to obtain seeds with higher azadirachtin content.

Taste Adaptation of Helicoverpa armigera Larvae to Azadirachtin and Its Physiological Mechanism

Dual-choice behavioral assays and electrophysiological tip-recording technique were used to investigate the taste adaptation of different diet-experienced frith instar larvae of Helicoverpa armigera to azadirachtin. The behavior test showed that the larvae reared with normal artificial diets were highly deterred by aza- dirachtin. However, the larvae reared with the artificial diets containing low concentration of azadirachtin showed insensitivity to asadirachtin. The electrophysiological tests showed that there was a changed responsiveness in the deterrent neuron responding to asadirachtin in the lateral sensilla styloconica of different diet-experienced larvae. It indicated that there was a taste adaptation to azadiraehtin in different diet-experienced larvae of H. armigera and this taste adaptation was correlated with desensitization of related taste neurons.

STUDIES ON THE SYNTHESIS OF THE SPIROKETAL FRAGMENT OF AZADIRACHTIN(Ⅰ)

The stereoselective synthesis of substituted [2,2,1]-bicyclo- heptan-2-one 12,a supposed intermediate to the spiroketal fragment of azadi- rachtin was described.When 12 was treated with mCPBA in CH_2Cl_2 in the pres- ence of excess amount of solid sodium bicarbonate,only 13 was obtained.The spiroketal intermediate 20 was converted from 9 through 5 steps by an alterna- tive route.

Breakdown of Azadirachtin A in a Tropical Soil Amended with Neem Leaves and Animal Manures

A field investigation was conducted to assess the breakdown of azadirachtin A in a tropical coastal savanna soil amended with neem leaves (NL) combined with poultry manure (PM) or cow dung (CD) using gas chromatography. Samples in polythene bags 15 cm long and 4.8 cm in diameter were randomly placed to a depth of 14 cm in the soil, and azadirachtin A concentration was assessed on days 0, 14, 28, 42, 56, 70, and 84. Azadirachtin A degradation in the soil followed first-order reaction kinetics with different half-lives obtained for varying combinations of the amendments. Higher neem amendment levels of 100 g gave shorter half-lives of azadirachtin A than the lower levels of 50 g. Within the 50 g NL group the additions of the poultry manure and the cow dung gave significantly shorter (P < 0.05) half-lives of azadirachtin A than the sole neem amendment, whereas in the 100 g NL group only additions of 10 g CD and 10 g PM were significantly less (P < 0.05) than the sole neem amendment. Different changes resulting from the kind and quantity of animal manure added were observed in the half-lives of azadirachtin A. The 100 g NL group had significantly higher (P < 0.05) moisture content, which, coupled with the likely differences in microbial biomass, could be the major factor responsible for variations in the half-life of the compound. Therefore, the quantity of the neem leaves applied and the addition of animal manure affected the breakdown of azadirachtin A in the soil amended with neem leaves.

3种植物源化合物对亚洲小车蝗的生物活性及影响机制

亚洲小车蝗是草原上重要的害虫之一,本文研究了3种植物源化合物瑞香狼毒根石油醚提取物、百里香酚和印楝素对亚洲小车蝗3龄蝗蝻的生物活性及影响机制。结果表明,3种植物源化合物对亚洲小车蝗的毒性表现为:拒食作用>胃毒作用>触杀作用。48 h时瑞香狼毒根石油醚提取物、百里香酚和印楝素对亚洲小车蝗3龄蝗蝻拒食活性的拒食中浓度(AFC50)分别为433.97 mg/L、183.35 mg/L和86.47 mg/L,胃毒活性的致死中浓度(LC50)分别为7976.33 mg/L、269.65 mg/L、341.07 mg/L,触杀作用相对较小。3种植物源化合物对亚洲小车蝗3龄蝗蝻离体腺苷三磷酸酶(ATPase)和乙酰胆碱酯酶(ACh E)活性均有抑制作用,且随着药剂浓度增大抑制作用增强,抑制率不超过50%。3种化合物处理亚洲小车蝗3龄蝗蝻48 h后,亚洲小车蝗体内谷胱甘肽-S-转移酶(GSTs)和细胞色素P450活性升高,羧酸酯酶(CarE)活性受到抑制。研究结果表明,这3种植物源化合物对亚洲小车蝗都具有一定的杀灭作用,拒食作用最强,未来在亚洲小车蝗的防控上可进一步开发利用。

印楝素抑制基因转录导致棉铃虫幼虫蜕皮变态异常的分子机理

印楝素是一种对鳞翅目如棉铃虫Helicoverpa armigera有良好防治效果的植物源农药。本研究采用室内毒力测定方法,荧光定量PCR(qRT-PCR)和代谢产物差异分析等方法,分析了饲喂印楝素后导致棉铃虫幼虫死亡的分子机理。首先统计分析了5龄和6龄棉铃虫幼虫饲喂不同浓度的印楝素后的表型、体质量、死亡率和化蛹率,结果表明印楝素导致棉铃虫幼虫体质量降低、死亡率升高和化蛹率降低。随后,通过qRT-PCR、酶活和内源物质含量测定,明确了印楝素抑制海藻糖的生物合成和分解,抑制20E下游基因转录表达,并降低了ATP含量。本研究结果表明印楝素通过抑制棉铃虫海藻糖的生物合成和分解,抑制20E信号通路基因转录,降低ATP产量,导致幼虫蜕皮变态失败,最终导致幼虫死亡。本研究对进一步探索印楝素导致棉铃虫死亡的机制和防治有害生物具有重要意义。

印楝素对小菜蛾敏感种群和抗性种群的拒食活性

用叶片浸渍法测定了印楝素(Azadirachtin)对小菜蛾抗性及敏感种群拒食活性。实验结果表明,印楝素对田间抗性及敏感小菜蛾种群均具有较好的拒食作用,且拒食活性随其的浓度的增加而显著增强;相对于抗性种群,印楝素对小菜蛾敏感种群表现了更强的拒食活性,其抗性种群拒食中浓度AFC50(1.2591 mg/L)是敏感种群AFC50(0.2567 mg/L)的4.91倍。

超临界CO_2萃取印楝种子中印楝素的研究

应用超临界CO2萃取技术从印楝种子中萃取印楝素,研究了萃取温度、压力和夹带剂对印楝素萃取效果的影响。当萃取温度为35℃、压力为15MPa、甲醇用量为CO2体积的3%时,可将印楝种子中90%以上的印楝素A萃取出来,所得产品印楝素A的质量分数为20 3%。

川楝素和印楝素对家蝇生长发育及繁殖的影响

目的 :了解川楝素和印楝素 2种药物对家蝇生长发育及繁殖的影响。方法 :药物处理饲料 ,对家蝇进行实验室饲养观察。结果 :有效浓度的川楝素和印楝素均能显著抑制家蝇幼虫的生长发育 ,使其发育迟缓 ,发育历期延长 ,个体变小 ,活动性降低 ,延迟化蛹 ,甚至形成永久性幼虫不能化蛹。 2种药物均能使家蝇的一些蛹出现畸形 ,蝇蛹羽化率下降 ,并出现畸形成虫。川楝素和印楝素均降低家蝇成虫的产卵量 ,但前者的作用与对照比较差异无显著性 (P >0 .0 5 )。川楝素和印楝素对卵的孵化率无显著性影响 ,药物作用后羽化出的成虫平均寿命与对照比较差异无显著性(P >0 .0 5 )。结论 :川楝素和印楝素通过对家蝇生长发育和生殖能力的抑制 。

超声波用于强化有机溶剂提取印楝素

研究了用超声波强化有机溶剂提取印楝素的工艺过程,分别考察溶剂、料液比、超声波作用时间、超声波功率对提取率的影响。实验发现,较理想的提取条件为:13.3g印楝种仁粉,料液比1∶2,超声波功率200W;超声波作用时间与溶剂有关,甲醇15min、乙醇20min、乙酸乙酯10min;3种溶剂较佳提取率分别为0.3786%、0.3658%、0.2462%。提取样品利用高效液相色谱仪采用外标法测定提取物中印楝素的含量。实验结果表明,超声波强化提取与搅拌提取相比,不仅缩短了提取时间,而且提取率有所提高。

印楝细胞悬浮培养系的建立及悬浮培养

研究了印楝细胞悬浮培养系的建立及印楝细胞悬浮培养的条件。结果表明,印楝细胞悬浮系的建立以B5培养基,NAA2 0～4 0mg·L-1,采用直接倾倒法作为接种方法为好;基于生物量、印楝素含量和印楝素产率综合分析认为,印楝细胞悬浮培养的最佳条件为B5培养基,NAA2～4mg·L-1,蔗糖30g·L-1,培养温度25℃,及根据培养容器的大小选择合适的转速和装液量。

植物源农药印楝素的研究与应用

印楝素是目前世界上最优秀的植物源农药,田间登记应用已30多年,对全球作物保护和生态安全发挥了重要的作用。本文回顾了印楝素的发现和印楝在中国引种情况,总结了印楝素结构鉴定与全合成的历程,重点介绍印楝素拒食、抑制昆虫生长发育、诱导细胞凋亡和自噬的分子作用机理以及印楝分子生物学、印楝素生物合成研究的最新进展。1983年,赵善欢等成功地将印楝引种中国,随后我国许多学者开展印楝引种与繁育,种植面积曾超过6万hm^2,为我国印楝素杀虫剂的规模化生产提供了原料保障。1997年印楝素以新化合物结构作为新农药在我国登记,实现了商业化应用,2014年成为农业部推荐使用的低毒低残留农药主要品种,获得了显著的经济和生态效益。我国学者发现了印楝素诱导细胞自噬现象,阐明了印楝素诱导细胞凋亡和自噬的分子调控机理。随着强化农产品质量安全的源头控制,印楝素的研究和应用将会越来越深入普及。今后要进一步加强基础研究,明确印楝素的分子靶标,为印楝素的科学使用提供理论基础。

印楝素对水稻二化螟的生物活性及控制作用

系统地研究了印楝素对水稻二化螟的生物活性及作用方式。试验结果表明 ,印楝素乳油对二化螟有胃毒、拒食、忌避、内吸等多种作用方式。 0 .75和 0 .5 0mg/L印楝素乳油处理茭白饲喂 3龄幼虫 ,幼虫分别于 3、6d内全部死亡 ;0 .375、0 .30 0和 0 .2 5 0mg/L印楝素乳油低剂量处理茭白饲喂 3龄幼虫 ,幼虫拒食率分别达 94 .38%、88.74 %、80 .0 7% ;成虫产卵量分别下降 80 .70 %、6 2 .0 5 %、4 0 .86 %。6、3和 2mg/L印楝素乳油处理初孵蚁螟 ,2 4h内死亡率达 10 0 %。印楝素乳油对成虫产卵有显著的忌避作用。稻田喷施 0 .3%印楝素乳油 6 2 .5和 12 5mL/ 6 6 7m2 防治二化螟 ,2 1d保苗效果分别为 88.14 %和 94 .13% ,防治效果分别为 85 .36 %和 90 .76 %。表明印楝素对二化螟有较高的生物活性和理想的控制效果。

印楝素乳油对小菜蛾种群的控制作用

通过室内外系统试验探讨了印楝素乳油对小菜蛾Plutellaxylostella种群的控制作用。结果表明 ,其控制作用主要表现在对成虫产卵的显著忌避作用、对幼虫的拒食作用以及对生长发育的影响和减少幼虫危害力等方面。印楝素乳油 0 0 0 5mL L处理对成虫的忌避率为 78 3% ,对幼虫的选择性和非选择性拒食率分别为 71 8%和 6 2 9% ,幼虫总取食量是对照的5 9 0 % ,使小菜蛾种群趋势指数 (I值 )降为 2 4,干扰作用控制指数 (IIPC)为 0 10 79。采用状态空间分析法就印楝素乳油施用后小菜蛾种群数量动态的模拟分析亦表明 。

印楝素对SL-1的细胞凋亡诱导作用

本文以喜树碱（camptothecin）作对比,以二甲基亚砜（DMSO）作对照,系统研究了印楝素（azadirachtin）对斜纹夜蛾Spodoptera litura离体细胞系（SL-1）的凋亡诱导作用。印楝素0.75μg/mL处理后SL-1细胞后12-72h,倒置显微镜观察可见大量细胞皱缩,体积变小,胞膜气泡化,与邻周细胞脱落,胞浆浓缩,胞膜突起,细胞器密集,核染色质浓缩并凝聚在核膜周边,出现大量凋亡小体;AO染色荧光显微镜观察可见细胞核内致密明亮黄绿色荧光和亮绿色或橘黄色的凋亡小体;透射电镜观察可见细胞皱缩、微绒毛消失、染色质浓缩和边缘化、核膜皱缩界限模糊、部分线粒体嵴结构消失和数量明显增加的吞噬泡等细胞凋亡典型形态学特征;TUNEL实验可见大量被标记为小圆形或环形黄绿色或绿色荧光的阳性凋亡细胞。流式细胞仪测定表明,印楝素0.75μg/mL处理后48h凋亡率最高达11.45%,比对照提高381.3倍;而喜树碱以1.72μg/mL处理亦对SL-1具有相似的诱导作用,处理后36h凋亡率最高达到17.42%。扫描电镜观察表明,印楝素和喜树碱处理后,SL-1细胞表面没有形成明显的“孔”、“洞”、“门”之类的结构破坏。推测印楝素与喜树碱对SL-1的凋亡信号转导方式和途径不同,导致细胞凋亡时序性不同。

微波辅助法提取印楝素的研究

介绍了一种有效利用微波辅助从印楝(AzadirachtaindicaA.Juss.)种仁中提取印楝素的方法。通过考察辐射时间、溶剂、物料比以及辐射功率等因素对提取率的影响,得出结论为:预处理过的印楝种仁粉末6.65g(相当于印楝种仁10g),以甲醇做溶剂、微波功率210W、物料比1∶3(g/mL)、辐射时间为3min×3次,提取率最高。与传统的磁搅拌法相比,该方法不仅工艺简单,而且可以提高产率,缩短提取时间。用高压液相法定量地测定了所提取印楝素的含量,其提取率可达0.519%.

印楝愈伤组织形成及其印楝素含量测定

取印楝（AzadirachtaindicaA．Juss）不同器官作外植体，研究培养基和继代次数对愈伤组织生长及其印楝素（Azadirachtin）生物合成的影响．印楝的不同器官（根、叶、茎及皮）均能诱导出愈伤组织，这些愈伤组织均有合成印楝素的能力．其中以叶诱导的愈伤组织生长速率及印楝素含量为最高．含有较低按盐的B5培养基有利于细胞生长，含有较高铵盐的MS培养基有利于印楝素积累，不含铰盐的White培养基对两者均不利．愈伤组织继代2—3代，有利于愈伤组织生长和印楝素合成。

印楝素的合成、结构修饰及生物活性研究进展

自印楝素被分离鉴定以来,其广谱而独特的生物活性受到人们的广泛关注,已成为一种近乎完美的植物源杀虫剂;且在医药、保健、日化等领域都有广泛的应用.其复杂而独特的分子结构使得科研工作者进行了大量的全合成尝试、结构修饰及构效关系等研究.综述了印楝素A的片段合成、全合成和印楝素有关的结构修饰以及生物活性等方面的研究进展.

几种植物源农药对蚜虫的生物活性测定

分别采用浸叶法和浸渍法测定了苦参碱和印楝素对瓜蚜的室内毒力,结果表明:两药剂对瓜蚜有较好的毒杀作用,其毒力均明显高于对照药剂吡虫啉,苦参碱的毒力又显著高于印楝素(相同测定方法);苦参碱为正温度效应的杀虫剂,印楝素则为存在一定负温度效应的杀虫剂;测定了7.15%银杏酚(分别为80℃和100℃乙醇提取物)对桃蚜的生物活性,发现其对桃蚜有一定的触杀、拒食和内吸作用,LC50(AFC50)值分别为1 877和2 1324、675和3 027、1 838和2 835(96 h)a.i.mg/L,较常规杀虫剂毒力低。