New procedure for isolation of amino acids based on selective hydrolysis of trimethylsilyl derivatives

A rapid procedure for the isolation of amino acids from physiological fluids by class separation suitable for gas chromatographic and gas chromatographic—mass spectrometric analysis is described. A physiological fluid such as plasma is adjusted to pH 2 and extracted with diethyl ether to remove organic acids and neutrals. After precipitation of proteins with trichloroacetic acid, the aqueous plasma is dried and derivatized by trimethylsilylation. Organic compounds like sugars and amino acids are rendered soluble in petroleum ether leaving inorganic salts when the soluble layer is transferred. Separation of sugars from amino acids is achieved by taking advantage of the different rates of aqueous hydrolysis of the trimethylsilyl (TMS) derivatives. Mixing the petroleum ether extract with a small volume of water results in two phases. The petroleum ether layer contains TMS-sugar constituents of plasma and the aqueous layer contains free amino acids and amines. This procedure was used to isolate L-dopa, 3-O-methyldopa and tyrosine from human plasma in a quantitation assay using ¹⁵O-labelled amino acids and gas chromatography—mass spectrometry.     

Competitive protein binding assay of progesterone without chromatography

A competitive protein binding assay for the measurement of progesterone in human plasma without chromatographic separation of steroids and recovery evaluation in individual samples is described. It is based on the specificity of the progesterone binding plasma protein (PBP) of the pregnant guinea pig. A dried petroleum ether extract of plasma was incubated with ³H-progesterone and 1600 fold diluted pregnant guinea pig plasma. Bound radioactivity was measured with a dextran coated charcoal suspension technique. Plasma progesterone concentration was obtained by comparison with a standard curve and correction for extraction separately measured for each batch of petroleum ether. The sensitivity was 100 pg. Recovery experiments for progesterone and competing steroids added to plasma respectively showed the accuracy and the specificity of the method. However comparison of the results from assays with and without chromatographic separation of steroids, showed that in the latter-case the specificity was good only for plasmas containing more than 1ng/ml of progesterone. Concentration of progesterone in plasma from men was 0.46±0.14 ng/ml (mean ± S.D) and from post menopausal women 0.30± 0.13 ng/ml.Between days 1 and 13 and days 16 and 22 of the normal menstrualcycle the concentrations were respectively 0.81 ± 0.38 and 12.50 ± 2.96 ng/ml. The variations of the progesterone concentration during pregnancy are also shown.     

龙柏茎叶中抑菌活性成分的分离与鉴定

采用活性追踪和柱层析法,以苹果腐烂病菌、葡萄白腐病菌等为靶标菌,提取、分离、鉴定了龙柏茎叶中抑菌活性成分.结果表明,对龙柏茎叶乙醇提取物的石油醚、乙酸乙酯和正丁醇萃取物活性测定中,石油醚萃取物的活性最高.对石油醚萃取物进行硅胶柱层析,得到一种活性较高的化合物A,对苹果腐烂病菌的EC_(50)为0.862 4 mg/mL.经氢谱(1H-NMR)、碳谱(~(13)C-NMR)和红外等确定了化合物A的结构为4-松香酸.     

菹草提取物总黄酮、总酚酸和抗氧化活性含量研究

研究菹草提取物的有效成分,测定其总黄酮含量及总酚酸含量,并确定它的乙酸乙酯和石油醚部位的抗氧化活性成分。采用紫外可见分光光度法,以芦丁和没食子酸作为控制材料,测定菹草的石油醚部位及乙酸乙酯部位中总黄酮和总酚酸含量;采用清除DPPH自由基能力测定法、测定总还原能力铁氰化钾法和水杨酸捕捉羟基自由基法来对菹草各组分提取物的抗氧化能力进行研究。菹草中有一定量的黄酮类化合物和酚酸类化合物存在,不同溶剂提取的部位所含有的总黄酮和总酚酸含量是有差异的,石油醚部位的总黄酮含量要高于乙酸乙酯部位的总黄酮含量,并且总酚酸的含量亦是如此;石油醚部位和乙酸乙酯部位的提取物具有清除DPPH、羟基自由基和还原Fe^3+的能力,各部位清除DPPH、羟基自由基的能力和还原Fe^3+的能力随着样品浓度的增大而增大,且乙酸乙酯部位的测定结果均低于石油醚部位。     

七星瓢虫成虫石油醚提取物的化学成分及抑菌活性

【目的】确定七星瓢虫Coccinella septempunctata成虫石油醚提取物的化学成分及其抑菌活性。【方法】用甲醇室温提取, 石油醚、 乙酸乙酯和正丁醇梯度萃取, 利用气相色谱-质谱连用仪（Gas Chromatograph-Mass Spectrometer, GC-MS）对七星瓢虫成虫石油醚提取物成分进行分析, 通过硅胶、 凝胶Sephadex LH-20、 ODS-C₁₈柱层析法对正丁醇提取部分的成分进行分离, 采用质谱和核磁共振等手段鉴定结构。采用纸片扩散法对七星瓢虫成虫石油醚提取物的抑菌活性进行测试。【结果】经GC-MS联用技术从石油醚相中鉴定出14个化合物, 从正丁醇相中分离鉴定了7个化合物。石油醚提取物对肠道沙门氏菌Salmonella enterica有抑制作用, 在浓度为100和50 mg/mL时, 抑菌率分别为52.78%和27.05%, 在该浓度下对绿脓杆菌Pseudomonas aeruginosa、 玉米大斑病菌Exserohilum turcicum和水稻纹枯病菌Rhizoctonia solani没有表现出明显的抑制作用。【结论】七星瓢虫C. septempunctata成虫石油醚提取物对肠道沙门氏菌具有抑制作用。     

Pretreatment of milk thistle seed to increase the silymarin yield: an alternative to petroleum ether defatting

Milk thistle (Silybum marianum L.) seed meal is extracted for the flavonolignans, silychristin, silydianin, silybinin A, silybinin B, isosilybinin A and isosilybinin B, which are collectively known as the silymarin complex. To obtain the flavonolignans, the meal is usually treated with successive washes of petroleum ether to remove the lipids, followed by extraction of the flavonolignans with ethanol. This work examines the possible replacement of petroleum ether and ethanol by water or other aqueous solutions in these processes. To replace petroleum ether, pretreatments with 1.2% NaOH (w/w), 1.5% H2SO4 (w/w), 2% NaHCO3 (w/w), 0.14% cellulase and water were investigated. Of these pretreatments, 1.5% H2SO4 and water produced similar flavonolignan yields as petroleum ether. Results established that pretreating the milk thistle seed meal with 1.5% H2SO4 (w/w) at 50 degrees C for 18 h could replace the petroleum ether pretreatment. In addition, it was shown that similar amounts of flavonolignan could be recovered with a 1.5% H2SO4/water (100 degrees C) extraction as with a petroleum ether/ethanol extraction. Although cellulase pretreatment was not examined extensively, significant advances in cellulase effectiveness and cost have occurred in the past few years by companies such as Genencor International and Novozymes. These advances should help to make enzyme use for cellulose conversion, as well as extraction pretreatment, technically and economically feasible.     

Antifungal activity of crude extracts and fat-soluble constituents of Holotrichia diomphalia larvae

In this study, chemical compositions of fatty oils and bioactivity of crude extracts from Holotrichia diomphalia larvae as Chinese materia medica were investigated for the first time. The chemical compositions of the fatty oils were obtained by two different methods and determined by GC/MS. In total, the petroleum ether extract produced 21 compounds (96.3%) while the supercritical fluid extract produced six compounds (99.53%) for identification. The effect of petroleum ether and other crude extracts on Pyricularia oryzae was also examined. Results indicated that ethanol and petroleum ether extracts had excellent antifungal activities. These findings demonstrated that fatty oils from H. diomphalia larvae had great potential to be used as a source for natural health products.     

Simple extraction and bioassay of the female sex pheromone of the citrus mealybug, Planococcus citri

The sex pheromone released by the females of the citrus mealybug was extracted from virgin females by ethanol, diethyl ether or petroleum ether but not by toluene, w-hexane or w-pentane. Extracts in ethanol, diethyl ether or petroleum ether placed on filter paper and hydrophilized polymethylmethacrylate disks elicited high attraction and copulation responses in the male mealybugs.     

Antinociceptive activity of petroleum ether fraction from the MeOH extracts of Paederia scandens in mice

The petroleum ether fraction of MeOH extract from Paederia scandens was evaluated on anti-nociceptive activity in mice using chemical and thermal models of nociception. Given orally, the petroleum ether fraction (PEF) at doses of 20, 40 and 80 mg/kg produced significant inhibitions on chemical nociception induced by intraperitoneal acetic acid and subplantar formalin or capsaicin injections and on thermal nociception in the tail-flick test and in the hot plate test. More significant inhibition of nociception was observed at dose of 80 mg/kg of the petroleum ether fraction. In the pentobarbital sodium-induced sleeping time test and the open-field test, the petroleum ether fraction neither significantly enhanced the pentobarbital sodium-induced sleeping time nor impaired the motor performance, indicating that the observed anti-nociception was unlikely due to sedation or motor abnormality. Moreover, the petroleum ether fraction-induced anti-nociception in both capsaicin and formalin tests was insensitive to naloxone, but was significantly antagonized by glibenclamide. These results suggested that the petroleum ether fraction produced anti-nociception possibly related to glibenclamide-sensitive K⁺-ATP channels, which merited further studies regarding the precise site and mechanism of action. The major constituents of the petroleum ether fraction (PEF) determined by GC/MS analysis, are linoleic acid, the sterols and vitamin E. Therefore it can be suggested that they exert synergetic effects and are together responsible for the antinociceptive activity of the PEF-fraction.     

Lipids of Algae

A fresh cake of Scenedesmus. cells was extracted with hot acetone, and the extracts were saponified. The yield of the unsaponifiable matter was 4.5% of the dry matter. Hydrocarbons and alcohols were extracted with petroleum ether from this unsaponifiable matter. The petroleum ether extracts were fractionated by the vacuum distillation. Each of the fractions was further separated by column chromatography or/and by the solubility difference of the urea adducts. Phytol was the major component of the unsaponifiable matter. Small amounts of n-hexadecane, n-hexacosene and n-eicosanol were isolated. On the other hand, the residue of the petroleum ether extraction was extracted with ether to separate sterols, of which the major component was found to be chondrillasterol.     

Acaricidal activity of Juglans regia leaf extracts on Tetranychus viennensis and Tetranychus cinnabarinus (Acari: Tetranychidae)

Leaf extracts of the walnut, Juglans regia L., were evaluated under laboratory conditions to determine their acaricidal activity on the mites Tetranychus cinnabarinus (Boisduval) and Tetranychus viennensis Zacher (Acari: Tetranychidae). Extracts had both contact and systemic toxicity to these mites. The four solvents tested for preparing crude extracts were petroleum ether, chloroform, ethyl acetate, and methanol. Methanol was the most efficient solvent, with an extraction rate from 17.06 + 0.80 to 20.27 +/- 0.28%. Petroleum ether was the least effective solvent, with extraction rates from 2.30 +/- 0.13 to 2.71 +/- 0.13%. However, the crude extracts with petroleum ether resulted in the highest mite mortality (79.04 +/- 0.52%) in a slide dip bioassay. Mites mortalities from the concentrated extracts prepared by chloroform, ethyl acetate, methanol, or distilled water were significantly lower than petroleum ether. The mean lethal concentrations (LC50) of the extracts from petroleum ether, chloroform, ethyl acetate, methanol, and distilled water to the two mite species were 0.73 +/- 0.04, 1.66 +/- 0.28, 4.96 +/- 0.35, 7.45 +/- 0.67, and 9.91 +/- 0.32 mg/ml, respectively. After liquid chromatography and thin-layer chromatography, the concentrated extracts of petroleum ether were separated into eight fractions and tested for acaricidal activity. Fraction 6 produced significantly higher mite mortality rates than the other groups, killing approximately 90% of both species.     

寄生隐丛赤壳菌Cryphonectria parasitica致病毒素Cp-I的分离纯化和结构分析

寄生隐丛赤壳菌Cryphonectria parasitica菌株经液体培养,石油醚萃取其发酵液获得对板栗带叶嫩枝具有致萎活性的粗提物,以氯仿:石油醚:甲醇(6:2:2)作洗脱剂,粗提物经硅胶色谱分离,共得到3组纯组份,其中第1组份(Cp-I)对板栗幼苗致萎活性较高.质谱、核磁共振和红外光谱测定表明Cp-I分子量为278,化学式为C<,16>H<,22>O<,4>.     

山野木层孔菌子实体中抑制H22荷瘤小鼠肿瘤的活性成分研究

采用梯度提取法对山野木层孔菌子实体进行提取,得到石油醚层、甲醇层及水层3 种提取物及石油醚层中获得化合物4,6,8(14),22(23)-四烯-3-酮-麦角甾烷,并采用H22 荷瘤小鼠进行体内抗肿瘤活性研究,以抑瘤率、免疫器官指数、免疫因子为指标检测抗肿瘤活性。结果表明,石油醚高剂量组（100mg/kg）、单体化合物中剂量组（7.5mg/kg）抑制率分别为62.21%、57.67%;脾指数、胸腺指数均高于对照组和环磷酰胺（CTX）组,白介素-2（IL-2）的含量明显高于对照组和环磷酰胺（CTX）组（P＜0.01）;肿瘤坏死因子-α（TNF-α）含量明显低于对照组（P＜0.01）。因此认为上述石油醚提取物和单体化合物对H22荷瘤小鼠肿瘤有抑制作用,并且均能改善小鼠的免疫功能。     

Acaricidal activity of petroleum ether extract of neem (Azadirachta indica) oil and its four fractions separated by column chromatography against Sarcoptes scabiei var. cuniculi larvae in vitro

The petroleum ether extract of neem oil and its four fractions separated by column chromatography was diluted at different concentrations with liquid paraffin. The acaricidal bioassay was conducted using a dipping method. The results indicated that the median lethal concentration (LC50) of the petroleum ether extract (at the concentration of 500.0ml/l) was 70.9ml/l, 24h after treatment. At concentrations of 500.0, 250.0, 125.0, 62.5 and 31.2ml/l, the median lethal times (LT50) of the petroleum ether extract were 8.7, 8.8, 10.8, 11.5 and 13.1h, respectively. Thin-layer chromatography (TLC) showed that the petroleum ether extract of neem oil separated into four fractions (F1-F4). Acaricidal activity of 68.3% and 100.0% in the F2 and F4 was confirmed. These results suggest that petroleum ether extracts of neem oil and its four fractions possess useful acaricidal activity in vitro.     

不同溶剂对海边月见草种子油脂肪酸萃取的比较

分别用乙醚、石油醚及氯仿一甲醇等三种溶剂萃取海边月见草种子油,经KOH-Me0H室温酯化法或BF₃-Me0H酯化法将其脂肪酸转化成甲酯,而后采用毛细管气相色谱外标法进行定量测定,结果表明:三种溶剂萃取所获得的种子油脂肪酸中亚油酸、γ-亚麻酸的含量无显著差异。但若考虑到溶剂的残留,选择石油醚为溶剂更合适。此外,采用上述两种酯化法,γ-亚麻酸均能与其它脂肪酸,尤其是亚油酸较好分离。     

A rapid specific radioimmunoassay for unconjugated estriol in plasma.

A rapid, non-chromatographic radioimmunossaay for unconjugated estriol in pregnancy plasma has been developed which utilizes a commonly available antiestrogen antisera. Estradiol-17beta and estrone demonstrate 135% relative cross-reactivity with our antiserum, as compared with 100% for estriol. Specificity is achieved by purification of estriol with solvent partitioning using benzene: petroleum ether (1:1). The results obtained using this method are similar to a radioimmunoassay utilizing a highly specific, but commercially unavailable, antiestriol antiserum. The method is precise, with coefficients of variation ranging from 3.0 to 8.2%.     

寄生隐丛赤壳菌致病毒素Cp-I的分离纯化和结构分析

寄生隐丛赤壳菌Cryphonectria parasitica菌株经液体培养,石油醚萃取其发酵液获得对板栗带叶嫩枝具有致萎活性的粗提物,以氯仿:石油醚:甲醇(6:2:2)作洗脱剂,粗提物经硅胶色谱分离,共得到3组纯组份,其中第1组份(Cp-I)对板栗幼苗致萎活性较高。质谱、核磁共振和红外光谱测定表明Cp-I分子量为278,化学式为C16H22O4。     

裸花紫珠治疗烧烫伤模型大鼠活性部位的筛选及其作用机制研究

为筛选裸花紫珠治疗烧烫伤的活性部位,并初步研究其作用机制。实验采用90°C热水造成大鼠烫伤模型。以伤口面积大小,伤口性状,如颜色、软硬程度等为指标,通过宏观和微观检测筛选裸花紫珠治疗烧烫伤的活性部位。采用ELISA和Western blot等方法初步研究其作用机制。进入组织增生重塑期后,石油醚组相较其它各组,伤口愈合最为显著;在组织增生最后的成熟期,石油醚组的表皮和真皮恢复得最好,伤疤最小。石油醚组的VEGF浓度在早期出现了明显的上调;在第21天时,TGF-β1的表达水平最低。裸花紫珠治疗烧烫伤的活性部位初步推测在石油醚层,其治疗烧烫伤作用机制可能是早期裸花紫珠使VEGF上调,在后期下调了TGF-β1以达到。本研究为裸花紫珠治疗烧烫伤的进一步研究和应用提供依据。     

Analysis of bioactive composites and antiviral activity of Iresine herbstii extracts against Newcastle disease virus in ovo

The study was implemented to actuate the qualitative and quantitative phyto constituents of Iresine herbstii extracts and its antiviral efficacy against avian ND virus. Among four tested solvents, the ethanolic extract of Iresine herbstii revealed the presence of highest quantity of all tested phytochemicals while petroleum ether extract showed the least. Folin-Ciocalteu method assessed the range of TPC extended from 81.01 ± 0.67 to 126.35 ± 0.45 µg GAE/mg. Acetonic extract showed the highest amount among all extracts and petroleum ether possessed the lower quantity. TFC ranged from 54.37 ± 0.45 to 88.12 ± 0.26 µg QE/mg followed by colorimetric method. From all extract ethanolic extract showed highest quantity and petroleum ether revealed the lower. HPLC analysis of ethanolic extract of I. herbstii confesses the presence six bioactive components by using the HP5-MS column. To check the antiviral potential of plants, different prepared treatments of plant extract and live virus were inoculated at 9 days old SPF embryonated chicken eggs. Results exposed that all plant extracts produce antiviral activity against NDV in ovo according to their potential and phytochemical profile. The highest survival rate was observed in the ethanolic extract at 400 µg/mL and acetonic extract at 300 µg/mL as it controls the NDV activity completely, evidence of absence of embryo death and HA titre. Dichloromethane and petroleum ether could not inhibit the virus completely. 600 µg/mL concentration was proved as toxic in all extracts except petroleum ether extract which showed a dose dependent pattern.     

石香薷挥发油提取的比较研究

利用GC-MS对石香薷挥发油进行了定性、定量分析。采取超临界CO2萃取、水蒸气蒸馏和有机溶剂石油醚萃取这三种方法提取石香薷挥发油。这三种方法提取石香薷挥发油的主要成分基本相似,主要为含氧化合物(香薷酮、百里香酚和香荆芥酚)等,采取超临界CO2萃取和水蒸气蒸馏的石香薷挥发油品质较优。超临界CO2萃取法为提取石香薷挥发油的理想方法。