栽培密度和施肥水平对黄花蒿生长特性和青蒿素的影响

采用大田试验,研究不同密度和施肥水平对黄花蒿生长、生物量分配和青蒿素含量的影响。试验设3个密度水平: 高密度（111111株/hm2）、中密度（55555株/hm2）和低密度（27778株/hm2）。 各密度设3个施肥水平(复合肥,N-P2O5-K2O为15-15-15): 不施肥、低肥（60 kg/hm2）和高肥（120 kg/hm2）。结果表明,黄花蒿对密度和养分条件变化的适应性较强,其中密度是植株大小、生物量分配和产量有关参数的主要决定因子,而青蒿素含量由施肥水平决定。相同施肥水平下,黄花蒿的基径与分枝数均随密度的降低而显著增大,其单株生物量也随密度的降低而显著增大; 中、高密度黄花蒿的支持结构生物量分数均显著大于低密度,低密度黄花蒿的根生物量分数和根/冠比显著大于中、高密度。相同密度下,施肥水平对黄花蒿的单株生物量影响不显著,但显著影响其生物量分配。低密度下,黄花蒿根生物量分数和根/冠比随施肥水平的增高而显著降低; 高密度下,黄花蒿叶生物量分数随施肥水平的增高而显著增大。所有处理中,低密度低施肥水平黄花蒿的青蒿素含量最高,中密度低施肥水平的叶产量和青蒿素产量最大。本试验条件下,黄花蒿栽培以密度55555株/hm2、 施肥60 kg/hm2为宜。     

Nutrient deficiency in the production of artemisinin, dihydroartemisinic acid, and artemisinic acid in Artemisia annua L

Artemisia annua became a valuable agricultural crop after the World Health Organization recommended artemisinin as a component of ACT (artemisinin-combination based therapies) for malaria in 2001. A cloned, greenhouse-grown, A. annua (Artemis) subjected to an acidic soil and macronutrient deficit was evaluated for artemisinin production. Lack of lime (L) and macronutrients (N, P, and K) reduced leaf biomass accumulation. When L was provided (pH 5.1), the highest average leaf biomass was achieved with the "complete" (+N, +P, +K, and +L) treatment (70.3 g/plant), and the least biomass was achieved with the untreated (-N, -P, -K, and -L) treatment (6.18 g/plant). The nutrient least required for biomass accumulation per plant (g) was K (49.0 g), followed by P (36.5 g) and N (14.3 g). The artemisinin concentration (g/100 g) was significantly higher (75.5%) in -K plants when compared to plants under the complete treatment (1.62 vs 0.93%). Although the artemisinin total yield (g/plant) was 21% higher in -K plants, it was not significantly different from plants under the complete treatment (0.80 vs 0.66 g/plant). There were no marked treatment effects for concentration (g/100 g) or yield (g/plant) of both dihydroartemisinic acid and artemisinic acid, although higher levels were achieved in plants under the complete or -K treatments. There was a positive and significant correlation between artemisinin and both artemisinic acid and dihydroartemisin acid, in g/100 g and g/plant. This is the first report where potassium deficiency significantly increases the concentration (g/100 g) of artemisinin. Thus, under a mild potassium deficiency, A. annua farmers could achieve similar gains in artemisinin/ha, while saving on potassium fertilization, increasing the profitability of artemisinin production.     

Synergistic effect of Glomus mosseae and nitrogen fixing Bacillus subtilis strain Daz26 on artemisinin content in Artemisia annua L.

Artemisia annua L. (Asteraceae) is an important medicinal plant whose secondary metabolite artemisinin is used for the treatment of cerebral malaria. A study was undertaken to determine the effect of arbuscular mycorrhizal (AM) fungi, Glomus mosseae, Glomus aggregatum, Glomus fasciculatum, Glomus intraradices and two free living nitrogen fixing bacteria (NFB) (identified as Bacillus subtilis and Stenotrophomonas spp. through 16S rRNA gene sequence analysis), inoculated alone or in combinations on the biomass, nutrient uptake, and content of artemisinin in A. annua under glass house conditions. Various parameters like plant height, total plant biomass, NPK content, leaf yield, mycorrhizal colonization, bacterial population in rhizosphere, phosphatase activity and artemisinin content were determined and found to vary with different treatments. Among all the treatments, plants inoculated with G. mosseae + B. subtilis performed better than any other treatment or uninoculated control plants. The results of the experiment clearly indicated the compatibility and synergy between G. mosseae and B. subtilis and suggested the use of this microbial consortium in A. annua for enhancing growth, biomass yield, and the content and yield of artemisinin.     

Analysis of polyphenolic antioxidants from the fruits of three pouteria species by selected ion monitoring liquid chromatography-mass spectrometry

Pouteria campechiana, Pouteria sapota, and Pouteria viridis are tropical plants in the Sapotaceae family that bear edible fruits. The fresh fruits of these three Pouteria species were each extracted, and activity-guided fractionations were performed to identify the antioxidant constituents. Seven polyphenolic antioxidants, gallic acid (1), (+)-gallocatechin (2), (+)-catechin (3), (-)-epicatechin (4), dihydromyricetin (5), (+)-catechin-3-O-gallate (6), and myricitrin (7), were isolated and identified. Extracts of the three Pouteria fruits were analyzed by a selected ion monitoring liquid chromatography-mass spectrometry method to quantify their polyphenolic antioxidants. The highest level of the seven measured polyphenols was found in P. sapota, the second highest in P. viridis, and the lowest in P. campechiana. The levels of the seven polyphenols corresponded with the results of the 1,1-diphenyl-2-picrylhydrazyl assay, by which P. sapota had the highest antioxidant activity, P. viridis the second highest, and P. campechiana the lowest.     

Multiresidue determination of pesticides in malt beverages by capillary gas chromatography with mass spectrometry and selected ion monitoring

A method was developed to determine pesticides in malt beverages using solid phase extraction on a polymeric cartridge and sample cleanup with a MgSO4-topped aminopropyl cartridge, followed by capillary gas chromatography with electron impact mass spectrometry in the selected ion monitoring mode [GC-MS(SIM)]. Three GC injections were required to analyze and identify organophosphate, organohalogen, and organonitrogen pesticides. The pesticides were identified by the retention times of peaks of the target ion and qualifier-to-target ion ratios. GC detection limits for most of the pesticides were 5-10 ng/mL, and linearity was determined from 50 to 5000 ng/mL. Fortification studies were performed at 10 ng/mL for three malt beverages that differ in properties such as alcohol content, solids, and appearance. The recoveries from the three malt beverages were greater than 70% for 85 of the 142 pesticides (including isomers) studied. The data showed that the different malt beverage matrixes had no significant effect on the recoveries. This method was then applied to the screening and analysis of malt beverages for pesticides, resulting in the detection of the insectide carbaryl and the fungicide dimethomorph in real samples. The study indicates that pesticide levels in malt beverages are significantly lower than the tolerance levels set by the United States Environmental Protection Agency for malt beverage starting ingredients. The use of the extraction/cleanup procedure and analysis by GC-MS(SIM) proved effective in screening malt beverages for a wide variety of pesticides.     

Quantitation of flavor-related alkenylbenzenes in tobacco smoke particulate by selected ion monitoring gas chromatography-mass spectrometry

Little is known about the possible health effects associated with inhaling alkenylbenzenes through cigarette smoking, even though these flavor-related compounds have known toxic effects in animals. We developed a rapid and sensitive solid-phase extraction (SPE) method to quantify seven alkenylbenzenes and piperonal in mainstream cigarette smoke particulate. The smoke particulate fraction of a single cigarette was collected on Cambridge filter pads, solvent extracted, concentrated, purified with SPE, and analyzed by selected ion monitoring gas chromatography-mass spectrometry. We positively identified and quantified five alkenylbenzenes compounds (eugenol, isoeugenol, methyleugenol myristicin, and elemicin) and piperonal in the smoke particulate from eight U.S. brands with mean levels (measured in triplicate) ranging from 6.6 to 4210 ng per cigarette. Additionally, complete blocking of nearly invisible ventilation holes in the cigarette filter increased 2- to 7-fold the percent transfer of alkenylbenzenes from tobacco to the particulate fraction of mainstream smoke.     

转青蒿反义鲨烯合酶基因对烟草鲨烯合酶基因表达的影响

利用反义技术，通过根癌农杆菌（Agrobacterium tumefaciens）介导，将青蒿（Artemisia annua）的反义鲨烯合酶基因导入烟草（Nicotiana tabacum）株系W38中，存转基因烟草中，鲨烯合酶的表达成功地得到抑制，其mRNA的水平降低。对鲨烯合酶的下游产物之一胆同醇的检测显示，转基因烟草的胆固醇含量低于对照，同时以法呢基焦磷酸为前体的二萜支路产物之一的GA，含量得到提高。实验结果表明，反义抑制烟草鲨烯合酶可以降低植物甾醇的生物合成，并促进类异戊二烯途径中以法呢基焦磷酸为前体的其它代谢支路的生物合成。     

Analysis of bromide ion in wine by ion selective electrode.

The use of the bromide ion selective electrode for the determination of bromide ion in wine has been found to be rapid and reliable. The method has been used for still wines and carbonated wines and is applicable to all wines regardless of their country of origin. The method consists of treating a 50 ml aliquot of wine with 2 ml each of 3.75M H3PO4, saturated KNO3, and 1M CuSO4. After 10 min the electrodes are immersed in the samples and a millivolt reading is obtained. One hundred mul 500 ppm bromide ion standard is added and the millivolt reading is taken. Bromide ion concentration in the wine equals (Cdelta x 1)/((antilog deltaE/S)-1) where Cdelta equals 1, deltaE equals the change in potential expressed in millivolts, and S equals the electrode slope.     

Evaluation of the botanical authenticity and phytochemical profile of black cohosh products by high-performance liquid chromatography with selected ion monitoring liquid chromatography-mass spectrometry

Black cohosh (Actaea racemosa L., syn. Cimicifuga racemosa L.) has become increasingly popular as a dietary supplement in the United States for the treatment of symptoms related to menopause, but the botanical authenticity of most products containing black cohosh has not been evaluated, nor is manufacturing highly regulated in the United States. In this study, 11 black cohosh products were analyzed for triterpene glycosides, phenolic constituents, and formononetin by high-performance liquid chromatography-photodiode array detection and a new selected ion monitoring liquid chromatography-mass spectrometry method. Three of the 11 products were found to contain the marker compound cimifugin and not cimiracemoside C, thereby indicating that these plants contain Asian Actaea instead of black cohosh. One product contained both black cohosh and an Asian Actaea species. For the products containing only black cohosh, there was significant product-to-product variability in the amounts of the selected triterpene glycosides and phenolic constituents, and as expected, no formononetin was detected.     

Antifungal constituents of the essential oil fraction of Artemisia dracunculus L. Var. dracunculus

The isolation and structure elucidation of antifungal constituents of the steam-distilled essential oil fraction of Artemisia dracunculus are described. Antifungal activities of 5-phenyl-1,3-pentadiyne and capillarin against Colletrotichum fragariae, Colletrotichum gloeosporioides, and Colletrotichum acutatum are reported for the first time. The relative abundance of 5-phenyl-1,3-pentadiyne is about 11% of the steam-distilled oil, as determined by GC-MS. Methyleugenol was also isolated and identified as an antifungal constituent of the oil.     

Characterization of flavonol conjugates in immature leaves of pak choi [Brassica rapa L. Ssp. chinensis L. (Hanelt.)] by HPLC-DAD and LC-MS/MS

The flavonoid composition of immature leaves of pak choi [Brassica rapa L. ssp. chinensis L. (Hanelt.)] was investigated. Flavonol aglycone content was measured in 11 pak choi varieties, indicating significant differences (P < 0.05) in content between varieties and relatively high contents of kaempferol and isorhamnetin. Levels of quercetin ranged from 3.2 to 6.1 mg/100 g of dry weight (DW), whereas levels of isorhamnetin and kaempferol were significantly higher (8.1-35.1 and 36.0-102.6 mg/100 g of DW, respectively). A large number of glycoside and hydroxycinnamic acid derivatives of quercetin, kaempferol, and isorhamnetin were identified in cv. 'Shanghai' by LC/UV-DAD/ESI-MS/MS. The UV-DAD data allowed identification of hydroxycinnamic acid derivatives, but detailed MS/MS fragmentations were required for the structure elucidation. Pak choi could be a potentially important source of dietary flavonols, in particular, kaempferol and isorhamnetin.     

低温处理对青蒿叶片脯氨酸、SOD和CAT的含量及蛋白质的影响

青蒿是一种重要的抗痢疾药物的原材料,低温是影响青蒿的青蒿素含量的重要因素。本试验以2个不同产地的青蒿为材料,研究低温处理对青蒿叶片脯氨酸、SOD和CAT的含量及蛋白质的影响。结果表明,低温处理条件下,青蒿叶片脯氨酸、超氧化物歧化酶活性(SOD)和过氧化氢酶活性(CAT)与室温相比均增加,且差异显著。SOD含量的增加,会增强青蒿植株抵抗低温的能力;CAT含量的增加,有利于缓解过量的过氧化物对青蒿叶片细胞的伤害;脯氨酸含量的增加,有利于调节细胞渗透压,促使细胞正常生长。进一步研究表明,在低温和室温条件下,青蒿叶片蛋白质电泳图谱存在强弱差异条带,说明低温处理对青蒿叶片中蛋白质产生影响。本文结果将为今后青蒿的栽培生产提供有价值的参考。     

Determination of olive oil oxidative status by selected ion flow tube mass spectrometry

The emergence of primary and secondary oxidation products in New Zealand extra virgin olive oil during accelerated thermal oxidation was measured and correlated with the concentrations of 13 headspace volatile compounds measured by selected ion flow tube mass spectrometry (SIFT-MS). SIFT-MS is a mass spectrometric technique that permits qualitative and absolute quantitative measurements to be made from whole air, headspace, or breath samples in real-time down to several parts per billion (ppb). It is well-suited to high-throughput analysis of headspace samples. Propanal, hexanal, and acetone were found at high concentrations in a rancid standard oil, while propanal, acetone, and acetic acid showed marked increases with oxidation time for the oils used in this study. A partial least-squares (PLS) regression model was constructed, which allowed the prediction of peroxide values (PV) for three separate oxidized oils. Sensory rancidity was also measured, although the correlations of headspace volatile compounds with sensory rancidity score were less satisfactory, and too few results were available for the construction of a PLS regression model. A fast (approximately 1 min), reliable method for prediction of olive oil PVs by SIFT-MS was developed.     

Determination of halogenated phenols in raw and potable water by selected ion gas chromatography-mass spectrometry

Pentafluorobenzylation and in situ acetylation are compared in the determination of phenol and halogenated phenols in water samples. The latter technique is considered superior to the former for determining phenols at the ng/L level because of less background interference and better recoveries (80% or better except for pentachlorophenol and trichloroguaiacol which had recoveries of about 60%). Further evaluation of the in situ technique by electron capture gas chromatography and gas chromatography-mass spectrometry shows that the latter, in the selected ion monitoring mode, is more suitable because, unlike GC-ECD, it can confirm and quantitate all phenols. In particular, GC-ECD could not detect even high levels of phenol and the monohalogenated phenols. Phenols at 5-473 ng/L were detected in some Canadian drinking water supplies by the in situ acetylation technique combined with GC-MS.     

Rise and fall of Stachys annua (L.) L. in the Carpathian Basin: a historical review and prospects for its revival

Genetic Resources and Crop Evolution - Stachys annua (L.) L., a melliferous archaeophyte plant became a dominant weed of the cereal stubbles of the Carpathian Basin in the medieval three-field...     

Monitoring of artemisinin, dihydroartemisinin, and artemether in environmental matrices using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS)

The area cultivated with Artemisia annua for the extraction of the antimalarial compound artemisinin is increasing, but the environmental impact of this cultivation has not yet been studied. A sensitive and robust method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the determination of artemisinin in soil. Dihydroartemisinin and artemether were included in the method, and performance on analytical columns of both traditional C(18) phenyl-hexyl and porous shell particles-based Kinetex types was characterized. The versatility of the method was demonstrated on surface water and groundwater samples and plant extracts. The limit of detection was 55, 30 (25 ng/g soil), and 4 ng/mL for dihydroartemisinin, artemisinin, and artemether, respectively. Method performance was demonstrated using naturally contaminated soil samples from A. annua fields in Kenya. The highest observed concentrations were above EC(10) for lettuce growth. Monitoring of artemisinin in soil with A. annua crop production seems necessary to further understand the impact in the environment.     

Analysis of coffee for the presence of acrylamide by LC-MS/MS

A variety of popular instant, ground, and brewed coffees were analyzed using a modified liquid chromatography-tandem mass spectrometry (LC-MS/MS) method specifically developed for the determination of acrylamide in foods. Coffee test portions were spiked with 13C3-labeled acrylamide as an internal standard prior to their extraction and cleanup. Ground coffees (1 g) and instant coffees (0.5 g) were extracted by shaking with 9 mL of water for 20 min. Brewed coffee test portions (9 mL) were taken through the cleanup procedure without further dilution with extraction solvent. Coffee test portions were cleaned up by passing 1.5 mL first through an Oasis HLB (hydrophilic/lipophilic copolymer sorbent) solid phase extraction (SPE) cartridge and then a Bond Elut-Accucat (cation and anion exchange sorbent) SPE cartridge. The cleaned up extracts were analyzed by positive ion electrospray LC-MS/MS. The MS/MS data was used to detect, confirm, and quantitate acrylamide. The limit of quantitation of the method was 10 ng/g for ground and instant coffees and 1.0 ng/mL for brewed coffee. The levels of acrylamide ranged from 45 to 374 ng/g in unbrewed coffee grounds, from 172 to 539 ng/g in instant coffee crystals, and from 6 to 16 ng/mL in brewed coffee.     

Characterization of phytoecdysteroid glycosides in Meadowfoam (Limnanthes alba) seed meal by positive and negative ion LC-MS/MS

Meadowfoam ( Limnanthes alba) is an oilseed crop grown in western Oregon. The seed meal has potential value as a biopesticide due to glucosinolate degradation products and phytoecdysteroids, a group of polyhydroxylated triterpenoids with potent activities as arthropod molting hormones. Liquid chromatography in combination with tandem mass spectrometry operated in the precursor ion mode revealed the presence of four ecdysteroid glycosides in meadowfoam seed meal. The carbohydrate sequence and the identity of the ecdysteroid aglycones, ponasterone A and 20-hydroxyecdysone, were determined by product ion scanning. Ecdysteroids were detected in the negative ion mode as [M + formate] (-) ions, which yielded [M - H] (-) and alpha-cleavage fragments with retention of hydroxyl groups in MS/MS experiments (not seen in the positive ion mode), allowing the determination of the number of hydroxyl groups in the side chain and in the steroid ring system. MS/MS of glycoside ions ([MH] (+) or [M + formate] (-)) provided carbohydrate sequence information.     

Changes in the enantiomeric distribution of selected volatile constituents of Mentha pulegium L. powders caused by hot water treatment

The variation, in general, of the composition of the aromatic fraction and, in particular, of the enantiomeric composition of certain chiral volatile compounds of commercial Mentha pulegium L. powders caused by boiling water was evaluated. A comparison between the volatile profile of the studied herbs demonstrated that most M. pulegium L. samples contained high proportions of Mentha piperita L., even when this information was not specified on the label. Likewise, substantial changes in the volatile fraction of the infusions with respect to the composition of the original plant used in their preparation were found. The enantiomeric composition of some chiral compounds of the dried plant material, particularly limonene, was modified by adding hot water, whereas others were kept invariable. The results shown in this work reflect the need for the control of the composition of commercial powders and brews of M. pulegium L. to ensure their correct application.     

Multiresidue pesticide analysis in wines by solid-phase extraction and capillary gas chromatography-mass spectrometric detection with selective ion monitoring

A method was developed to determine pesticides in wines. The pesticides were extracted from the wine using solid-phase extraction on a polymeric cartridge, and the coextractives were removed with an aminopropyl-MgSO(4) cartridge. Analysis was performed using capillary gas chromatography with electron impact mass spectrometric detection in selective ion monitoring mode (GC-MSD/SIM). Three injections are required to analyze all 153 organohalogen, organonitrogen, organophosphate, and organosulfur pesticides and residues. Pesticides were confirmed by retention times of the target ions and three qualifier-to-target ion ratios. Detection limits for most of the pesticides were less than 0.005 mg/L, and quantitation was determined from approximately 0.01 to 5 mg/L. Spike recoveries were performed by fortifying red and white wines at 0.01 and 0.10 mg/L. At the 0.01 ppm level, the spike recoveries were greater than 70% for 116 and 124 pesticides (out of 153) in red and white wines, respectively, whereas at the higher spike concentration of 0.10 mg/L, the recoveries were greater than 70% for 123 and 128 pesticides in red and white wines, respectively. The recoveries of less than 70% were most likely from pesticide polarity or lability, resulting in the inefficient adsorption of the pesticide to the polymeric sorbent, ineffective elution of the pesticide from the sorbent, or thermal degradation of the pesticide under GC-MSD conditions.