避蚊胺微球载药量的HPLC测定

避蚊胺(DEET,1)化学名为N,N-二乙基-间甲苯酰胺,是目前应用最广泛的广谱蚊虫驱避剂,通过自然挥发或借助载体挥发气味,使吸血昆虫产生忌避而难以接近人体[1,2].1具有驱避效果好、毒性小等优点,但现有制剂作用时间短、皮肤用量大、吸收较多,可能引发皮炎及心血管疾病等[3].我院采用溶剂挥发法,制备了用于皮肤给药的1乙基纤维素(EC)微球[4].本研究建立了HPLC法测定微球的载药量.     

茶树油乳液对水蛭驱避作用的研究

水蛭叮咬后通常流血较多,真皮组织被破坏形成终生疤痕,若水蛭进入人体腔道会更危险,因此亟需研发水蛭驱避剂。本研究以茶树油为主药,魔芋葡甘聚糖、乙基纤维素为主要辅料制备水包油型茶树油乳液。通过滤纸圈法、水中驱避法确定其驱避效率,评价茶树油乳液对水蛭的驱避作用。所有动物实验经军事科学院军事医学研究院伦理委员会批准且实验均按照相关指导原则和规定进行。以体内乙酰胆碱酯酶、谷胱甘肽S-转移酶和羧酸酯酶活性为评价指标,阐明茶树油乳液对水蛭的驱避机制。本研究成功制备了均一稳定的茶树油乳液,亲水性优良,可有效驱避水蛭,该乳液的驱避时间延长,可避免茶树油挥发,为类似挥发油用于长效、高效驱避提供了新思路。     

驱蚊宁(驱蚊剂42号)

【化学名】对-(艹孟)烯8(9)-二醇(1.2)。【结构式】【作用特点】本品为我国创制的新型广谱涂抹驱避剂,对蚊、蠓、山蚂蝗等吸血昆虫均有较好的驱避作用,对蚊有4～5小时驱避效果;对山蚂蝗亦有3个小时以上的效果。本品与国外广泛使用的DETA(N,N-二乙基-间-甲基苯甲酰胺)相比毒副作用较小。【适应症】涂抹于皮肤后,对蚊、蠓、山蚂蝗等吸血昆虫有驱避作用,可用作对人的个体防护。     

植物挥发油抗菌及驱虫作用研究进展

采用植物挥发油驱避灭杀病原生物效果显著,与化学驱避剂作用相当甚至更佳,此外,植物挥发油具有较明显的安全优势,被誉为天然驱避剂。本文归纳了植物挥发油对人体和动植物体的抗菌及驱虫作用,应用表明植物挥发油作为天然驱避剂具有广阔的开发应用前景。     

蚊虫驱避剂及其剂型的研究进展

植物源驱避剂,具有安全、环保、毒副作用小等特点,目前对其研究重点在于筛选高驱避活性成分。本文通过查阅近年来的文献资料,对蚊虫驱避剂剂型的研究进展进行综述,为蚊虫驱避剂剂型的研究开发提供科学依据。     

长效驱蚊霜野外现场驱避效果观察

目的:观察长效驱蚊霜野外驱避效果.方法:选择120名男性健康受试者,随机平均分为4组,第1组(空白对照组)、第2组(施美蚊不叮香露对照组)、第3组(长效驱蚊霜A型试验组)、第4组(长效驱蚊霜B型试验组).所有受试者左前臂裸露并分别涂抹驱蚊剂,每次间隔30 min,在蚊子最多的丛林中站立15 min,观察驱蚊剂的驱避效果.结果:在2 h内试验组驱避蚊虫的有效率为100%,对照组有效率为0.00%,两种驱蚊剂有非常显著的差异(P<0.001).结论:长效驱蚊霜对驱避蚊虫叮咬效果非常显著,驱避效果优于施美蚊不叮香露.     

Box-Behnken效应面法优化避蚊胺微囊的制备工艺研究

摘要：目的:优选避蚊胺（DEET）微囊的制备工艺。方法:以明胶作为囊材,采用复凝聚法制备微囊。以包封率为指标,在单因素试验的基础上,选取芯壁比、扩容体积、搅拌转速3个影响因素,采用Box-Behnken效应面法优化DEET微囊的制备工艺。应用Design-Expert 10软件分析试验结果。结果:DEET微囊的最佳制备工艺条件是芯壁比为1.15∶1,壁材在溶液中含量为3.1%,搅拌转速135 r·min-1。采用优选后的制备工艺制备的DEET微囊包封率可达52.65%,接近模型预测值52.71%。DEET微囊粒径为（36.17±3.42）μm,大小相对均匀,可观察到具有一定厚度的透明囊壁。结论:运用单因素考察法和Box-Behnken效应面法优化DEET微囊的制备工艺,结果准确、有效、可行。     

应用几丁聚糖提高避蚊胺驱蚊效果的实验观察

避蚊胺(delphene)是一种常用的高效、广谱、安全的驱避剂。30%避蚊胺酊剂在北方一次涂抹对蚊虫驱避时间仅为3～5h,对野外工作人员来说显得短了些。为延长避蚊胺对蚊虫的驱避时间,我们选用了大分子量的几丁聚糖(chitosan)作为避蚊胺的缓释成膜剂,将避蚊胺的驱蚊虫效果提高了一倍多。现将实验观察结果报告如下。 1 材料与方法 1.1 实验用药 1.1.1 避蚊胺酊剂由本队提供,用上皿天平称取避蚊胺结晶体1.2g,溶于95%乙醇10ml     

避蚊胺-海藻酸钙微球的制备及其体外透皮吸收研究

目的 制备海藻酸钙空白微球,减少避蚊胺(DEET)的透皮吸收,延长其作用时间.方法采用溶剂挥发法制备海藻酸钙微球,采用光学显微镜和激光粒度仪考察微球特性;吸附DEET后制成霜荆,通过Franz扩散池,用气相色谱法考察含药微球的透皮吸收.结果所得微球外观圆整,平均粒径42.5μm,吸附量为1.2177 g·g-1.透皮试验结果表明,海藻酸钙空白微球能延长DEET的释放,减少皮肤渗透.结论 海藻酸钙微球具有显著的缓释效果,并减少DEET对人体及皮肤的不良反应.     

南极适冷菌Pseudoalteromonas sp.S-15-13胞外多糖EPS-Ⅱ对小鼠S180肉瘤抑制作用的研究

目的 研究南极适冷菌Pseudoalteromonas sp．S-15—13胞外多糖EPS-Ⅱ的抑瘤作用。方法 不同浓度的EPS-Ⅱ腹腔注射于荷S180肉瘤小鼠，通过荷瘤小鼠的脾重和瘤重变化来测定EPS-Ⅱ的抑瘤效果；采用免疫组化方法，测定荷瘤小鼠增殖细胞核抗原（PCNA），三联组氨酸（FHIT）和S-100蛋白阳性表达的情况。结果 （1）不同剂量的EPS-Ⅱ均具有提高荷瘤小鼠脾脏重量，有效抑制肿瘤生长的作用，最高抑瘤率可达45．1％。（2）免疫组织化学结果表明，不同剂量的EPS-Ⅱ均能抑制PCNA表达，促进FHIT和S-100蛋白表达的作用。结论 南极适冷菌S-15—13胞外多糖EPS-Ⅱ可能通过调节PCNA，FHIT和S-100的表达来发挥抑瘤作用。     

Microsomal metabolism of N,N-diethyl-m-toluamide (DEET, DET): the extended network of metabolites.

1. The aim was to set out to establish the complete network of metabolites arising from the phenobarbital-treated rat liver microsomal oxidation of N,N-diethyl-m-toluamide (DEET). The products formed from DEET and all its subsequent metabolites were identified by HPLC retention times, UV spectroscopy, mass spectrometry and by comparison with authentic standards. 2. DEET (1a) produces three major metabolites, N-ethyl-m-toluamide (1b), N,N-diethyl-m-(hydroxymethyl)benzamide (2a) and N-ethyl-m-(hydroxymethyl)benzamide (2b), and, at low substrate concentrations or extended reaction times, two minor metabolites, toluamide (1c) and N,N-diethyl-m-formylbenzamide (3a). 1b and 2a are primary metabolites and their formation follows Michaelis-Menten-type kinetics. At low DEET concentrations, ring methyl group oxidation is favoured; at saturation concentrations, methyl group oxidation and N-deethylation proceed at similar rates. The rate of formation of 2b decreases with increasing DEET concentration; 2b is therefore a secondary metabolite of DEET and DEET acts as a competitive inhibitor of the metabolism of 1b and 2a. 3. Except for the primary amides, where N-dealkylation is impossible, metabolism of all subsequent compounds, 1b,c, 2a-c, 3a-c and 4a,b, involves an N-deethylation (NEt2 --> NHEt or NHEt --> NH2) competitive with a ring substituent oxidation (CH3 --> CH2OH, CH2OH --> CHO or CHO --> CO2H). Surprisingly, the aldehydes 3a-c are also reduced to the corresponding alcohols 2a-c (CHO --> CH2OH); CO inhibits the oxidative metabolism of 3a-c, but reduction to 2a-c continues uninhibited. 4. The outcomes of this work are that (1) previously unreported aldehydes 3b and 3c form part of the DEET network of metabolites, (2) the reduction of the aldehydes 3a-c has the potential to inhibit the formation of the more highly oxidized DEET metabolites, (3) amide hydrolysis was not observed for any substrate and (4) no evidence was obtained for N-(1-hydroxyethyl)amide intermediates.     

Rapid determination of N,N-diethyl-m-toluamide and permethrin in human plasma by gas chromatography-mass spectrometry and pyridostigmine bromide by high-performance liquid chromatography

A rapid and highly sensitive gas chromatography-mass spectrometry (GC-MS) method for simultaneous determination of N,N-diethyl-m-toluamide (DEET) and permethrin with (2)H(10)-phenanthrene (98 atom %) as an internal standard and a separate external standard high-performance liquid chromatography (HPLC) method for pyridostigmine bromide (PB) determination in human plasma were developed and validated. The GC-MS method for DEET and permethrin quantification utilizes a one-step extraction with tert-butylmethylether. The HPLC method for PB quantification involves a solid-phase extraction and UV detection. The range of the analytical method for DEET and permethrin was 1 ng/mL to 100 ng/mL and for PB was 5 ng/mL to 100 ng/mL. Recovery from plasma proved to be more than 80%. The intraday precision ranged from 1.3% to 8% for DEET, from 2.1% to 11.4% for permethrin, and from 3.0% to 4.8% for PB. The interday precision was 3% for DEET, ranged from 5% to 9% for permethrin, and from 5% to 9% for PB. The accuracy for the limit of quantification was 92% +/- 8% relative standard deviation (RSD) for DEET, 112% +/- 11% RSD for permethrin, and 109% +/- 5% RSD for PB. All 3 compounds were stable in human plasma at -80 degrees C for at least 12 months and after 2 freeze-thaw cycles with RSD values ranging from 7.1% (DEET, 80 ng/mL) to 8.1% (DEET, 8 ng/mL), from 2.3% (permethrin, 80 ng/mL) to 11.6 % (permethrin, 8 ng/mL), and from 0.2% (PB, 80 ng/mL) to 3.6% (PB, 8 ng/mL). Both methods were successfully applied to pharmacokinetic/ pharmacodynamic studies of combined exposure of DEET (skin application), permethrin (treated uniforms), and PB (30 mg orally three times/day for four doses) in healthy volunteers (n = 81).     

用NOE差谱和二维核磁共振技术研究3S-取代烷氧基-奎宁环烷的立体化学

用3S-奎宁环醇与环氧化合物反应,得到化合物(Ⅰ)的二个光学异构体(3S-1)和(3S-2)。它们的生物活性有明显差别。本文采用NOE差谱和二维NMR技术指定了各异构体的^{1H和13C信号的归属,确定了它们的溶液态构象,测定了其C-11的绝对构型,并且阐明了立体结构与生物活性间的关系。C-11绝对构型的测定结果经X-射线衍射测定进一步得以肯定。}     

Dermal absorption of the insect repellent DEET (N,N-diethyl-m-toluamide) in rats and monkeys: effect of anatomical site and multiple exposure

The dermal absorption of 14C-ring-labeled DEET (N,N-diethyl-m-toluamide) applied in acetone to the skin of Sprague-Dawley rats and rhesus monkeys for 24 h was determined. Absorption in rats dosed middorsally was 36 +/- 8% with a urinary excretion half-life (t1/2) of 20 h. Both the extent and rate of absorption in monkeys were highly dependent on anatomic site, with 14 +/- 5% (t1/2 = 4 h) penetrating the forearm, 33 +/- 11% (t1/2 = 6 h) the forehead, 27 +/- 3% (t1/2 = 7 h) the dorsal forepaw, and 68 +/- 9% (t1/2 = 8 h) the ventral forepaw. Since DEET is commonly applied frequently by the same individual, the effect of multiple exposure was investigated. No significant difference (p greater than or equal to .3) was obtained either between the total percentage absorbed dermally with single (36 +/- 8%; t1/2 = 20 h) as compared with three (31 +/- 5%; t1/2 = 16 h) DEET applications at 2-h intervals to rats, or between single (14 +/- 5%; t1/2 = 4 h) as compared with three (12 +/- 1%; t1/2 = 4 h) applications at 0.5-h intervals to monkey forearm. A DEET metabolite detected in urine 4 h following topical exposure in humans was extractable following either acid (HCl) hydrolysis or urine treatment with beta-glucuronidase and was identified as ethyltoluamide (parent ion 163; base ion 119) following HPLC purification and characterization by GC/MS.     

In vitro human metabolism and interactions of repellent N,N-diethyl-m-toluamide.

Oxidative metabolism of the insect repellent N,N-diethyl-m-toluamide (DEET) by pooled human liver microsomes (HLM), rat liver microsomes (RLM), and mouse liver microsomes (MLM) was investigated. DEET is metabolized by cytochromes P450 (P450s) leading to the production of a ring methyl oxidation product, N,N-diethyl-m-hydroxymethylbenzamide (BALC), and an N-deethylated product, N-ethyl-m-toluamide (ET). Both the affinities and intrinsic clearance of HLM for ring hydroxylation are greater than those for N-deethylation. Pooled HLM show significantly lower affinities (K(m)) than RLM for metabolism of DEET to either of the primary metabolites (BALC and ET). Among 15 cDNA-expressed P450 enzymes examined, CYP1A2, 2B6, 2D6*1 (Val(374)), and 2E1 metabolized DEET to the BALC metabolite, whereas CYP3A4, 3A5, 2A6, and 2C19 produced the ET metabolite. CYP2B6 is the principal cytochrome P450 involved in the metabolism of DEET to its major BALC metabolite, whereas CYP2C19 had the greatest activity for the formation of the ET metabolite. Use of phenotyped HLMs demonstrated that individuals with high levels of CYP2B6, 3A4, 2C19, and 2A6 have the greatest potential to metabolize DEET. Mice treated with DEET demonstrated induced levels of the CYP2B family, increased hydroxylation, and a 2.4-fold increase in the metabolism of chlorpyrifos to chlorpyrifos-oxon, a potent anticholinesterase. Preincubation of human CYP2B6 with chlorpyrifos completely inhibited the metabolism of DEET. Preincubation of human or rodent microsomes with chlorpyrifos, permethrin, and pyridostigmine bromide alone or in combination can lead to either stimulation or inhibition of DEET metabolism.     

圆果甘草三萜成分的研究

从河南产圆果甘草(Glycyrrhiza squamulosa Franch.)的根中分离鉴定了五个三萜类化合物,即白桦酯酸(betulinic acid,S-8)、马其顿酸甲酯(methyl ester of macedonic acid,S-9)、黄豆醇B(soyasapoge nol B,S-10)、齐墩果13(18)-烯-22α-氯-3β,24-二醇(olean-13(18)-ene-22α-Cl-3β,24-diol,S-12)和圆果皂甙元(squasapogenol.S-11),均为首次从该植物中获得,其中S-10为首次在甘草属中发现,S-12为酸水解转变物,S-11是一个新的五环三萜类皂甙元,经光谱解析确定其结构为齐墩果-11,13(18)-二烯-3β,22β-二醇(olean-11,13(18)-diene-3β,22β-diol)。     

Induction of urinary excretion of 3-nitrotyrosine, a marker of oxidative stress, following administration of pyridostigmine bromide, DEET (N,N-diethyl-m-toluamide) and permethrin, alone and in combination in rats

In this study, we determined levels of 3-nitrotyrosine in rat urine following administration of a single oral dose of 13 mg/kg pyridostigmine bromide (PB) (3-dimethylaminocarbonyloxy-N-methylpyridinum bromide), a single dermal dose of 400 mg/kg N,N-diethyl-m-toluamide (DEET) and a single dermal dose of 1.3 mg/kg permethrin, alone and in combination. Urine samples were collected from five treated and five control rats at 4, 8, 16, 24, 48, and 72 h following dosing. Solid-phase extraction coupled with high-performance liquid chromatography with ultraviolet detection at 274 nm was used for the determination of tyrosine and 3-nitrotyrosine. A single oral dose of PB and a single dermal dose of DEET or their combination significantly (P<0.05) increased levels of 3-nitrotyrosine starting 24 h after dosing compared with control urine samples. The maximum increase of 3-nitroytyrosine was detected 48 h after combined administration of PB and DEET. The ratio of 3-nitrotyrosine to tyrosine in urine excreted 48 h after dosing was 0.19+/-0.04, 0.20+/-0.05, 0.28+/-0.03, 0.32+/-0.04, 0.19+/-0.05, 0.42+/-0.04, 0.27+/-0.03, 0.36+/-0.04, and 0.48+/-0.04 following administration of water, ethanol, PB, DEET, permethrin, PB+DEET, PB+permethrin, DEET+permethrin, and PB+DEET+permethrin, respectively. The results indicate that an oral dose of PB and a dermal administration of DEET, alone and in combination, could generate free radical species, and thus increase levels of 3-nitrotyrosine in rat urine. Induction of 3-nitrotyrosine, a marker of oxidative stress, following exposure to these compounds could be significant in understanding the proposed enhanced toxicity following combined exposure to these compounds.     

Pyridostigmine bromide modulates topical irritant-induced cytokine release from human epidermal keratinocytes and isolated perfused porcine skin

Gulf War personnel were given pyridostigmine bromide (PB) as a prophylactic treatment against organophosphate nerve agent exposure, and were exposed to the insecticide permethrin and the insect repellent N,N-diethyl-m-toluamide (DEET). The purpose of this study was to assess the effects of PB to modulate release of inflammatory biomarkers after topical chemical exposure to chemical mixtures containing permethrin and DEET applied in ethanol or water vehicles. Treatments were topically applied to isolated perfused porcine skin flaps (IPPSFs). Concentrations of interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha) and prostaglandin E(2) (PGE(2)) were assayed in perfusate to probe for potential inflammatory effects after complex mixture application. IPPSFs (n=4/treatment) were topically dosed with mixtures of permethrin, DEET, and permethrin/DEET, in ethanol. Each treatment was repeated with perfusate spiked with 50 ng/ml of PB. Perfusate was also spiked with 30 ng/ml diisopropylfluorophosphate to simulate low level organophosphate nerve agent exposure. Timed IPPSF venous effluent samples (0.5,1,2,4, and 8 h) were assayed by ELISA for IL-8 and TNF-alpha and by EIA for PGE(2). Overall, PB infusion caused a decrease or IL-8 and PGE(2) release. Effects on TNF-alpha were vehicle dependent. To probe the potential mechanism of this PB effect, human epidermal keratinocyte HEK cell cultures were exposed to permethrin DEET permethrin/DEET, with and without PB in DMSO. IL-8 was assayed at 1, 2, 4, 8, 12 and 24 h. PB suppressed IL-8 in permethrin and ethanol treatment from 4 to 24 h confirming the IPPSF results. In conclusion, these studies suggest that systemic exposure to PB suppressed IL-8 release at multiple time points in two skin model systems. This interaction merits further study.     

Inverse dispersive liquid-liquid microextraction(I-DLLME) for the simultaneous and green preconcentration of DEET and permethrin from freshwater

Global warming can stimulate mosquitoes to proliferate and the consumption of repellents and insecticides. These formulations can have N,N-diethyl-m-toluamide (DEET) and permethrin, which were reported in water bodies, requiring sensitive methods for quantification. We propose changing the dispersive liquid-liquid microextraction (DLLME), a green extraction technique, by inverting the sequence of injection of the extractant-dispersant solvents to allow better interaction with samples, aiming to improve the reproducibility of analytical results. In the inverse dispersive liquid-liquid microextraction (I-DLLME), samples of deionized water or freshwater enriched with DEET and permethrin isomers (trans = PERM-1) and (cis = PERM-2) were injected over the extractant-dispersant solvents using a micro-syringe. Both DLLME and I-DLLME were optimized using a factorial design and multi-response optimization (desirability function) to establish the condition that provides higher enrichment factor (EF) and reproducibility, denoted by lower relative standard deviations (RSD). Preconcentrated samples were analyzed using a High-Performance Liquid Chromatography with Diode Array Detector (HPLC-DAD) method. Under the optimum I-DLLME-HPLC-DAD condition (50 μL of chloroform as dispersant, 600 μL of acetonitrile as extractant, 0.007 mol/L of NaCl, and pH = 3), the EF for DEET, PERM-1 and PERM-2 were respectively 67, 170 and 179 with considerable gains in reproducibility (RSDs≤12%, n = 6). Quantification limits for DEET, PERM-1 and PERM-2 for deionized water and freshwater were respectively 50.0, 4.42 and 2.88 μg/L, and 33.06, 12.62 and 6.45 μg/L, without matrix effect. The apparent recovery for freshwater varied between 93% and 103% (RSDs 2.3–12%). The I-DLLME-HPLC-DAD method scored 0.61, considering the 12 principles of green analytical chemistry from a unified 0–1 scale.     

Toxic encephalopathy associated with use of DEET insect repellents: a case analysis of its toxicity in children

(1) Respiratory distress and seizures developed in an 18-month-old boy following brief exposure to low-strength (17.6%) N,N-diethyl-m-toluamide (DEET). A review of the literature revealed 17 reports of DEET-induced encephalopathy in children. The objective of this study was to test the hypothesis that the potential toxicity of DEET is high and that available repellents containing DEET, irrespective of their strength, are not safe when applied to children's skin. (2) Although this is a case report, we used the features of published reports of DEET-induced encephalopathy in children to support the diagnosis, since the evidence that the child's illness was caused by DEET was circumstantial. In the following case analysis, clinical reports of children < 16 years old have been reviewed and analyzed in an effort to relate direct DEET toxicity to various clinical, demographic, and toxic compound exposure factors (Fisher's exacttest and logistic regression analysis). (3) DEET-induced encephalopathy in children (56% girls) followed not only ingestion or repeated and extensive application of repellents, but also a brief exposure to DEET (45%). Of those who reported a dermal exposure, 33% reported an exposure to a product containing DEET < 20%. Seizures, the most prominent symptom (72%), were significantly more frequent when DEET solutions were applied to the skin (P<0.01). Mortality (16.6%) did not correlate significantly with the concentration of the DEET liquid used, duration of skin exposure, pattern of use, age, or sex. (4) Data of this case analysis suggest that repellents containing DEET are not safe when applied to children's skin and should be avoided in children. Additionally, since the potential toxicity of DEET is high, less toxic preparations should be probably substituted for DEET-containing repellents, whenever possible.