Occurrence of orally administered mulberry 1-deoxynojirimycin in rat plasma

1-deoxynojirimycin (DNJ), a potent glucosidase inhibitor, is a characteristic constituent of the mulberry leaf. Dietary mulberry DNJ may be beneficial for the suppression of abnormally high blood glucose levels, thereby preventing diabetes mellitus. Although there is considerable interest in the effects of mulberry DNJ, the intestinal absorption and pharmacokinetic profile of orally administered mulberry DNJ have never been characterized. In this study, we developed a method for determining the level of plasma DNJ by hydrophilic interaction chromatography coupled to a mass spectrometric detector (HILIC-MS) to investigate the absorption and metabolism of orally administered mulberry DNJ in rats. DNJ was separated from plasma extract on a TSK gel Amide-80 column, a representative column for HILIC. At postcolumn, DNJ was concurrently detected and identified by MS. The plasma DNJ concentration in fasted rats was below the detection limit [<1 microg (6 nmol)/mL]; however, the concentration reached a maximum [15 microg (92 nmol)/mL] 30 min after the administration of mulberry DNJ (110 mg/kg of body weight), and the DNJ concentration decreased rapidly thereafter. When the rats received different amounts of mulberry DNJ (1.1, 11, and 110 mg/kg of body weight), dose-dependent incorporation of DNJ into the plasma was confirmed. We did not detect any DNJ metabolites in the plasma. These findings indicate that orally administered mulberry DNJ is absorbed as an intact form from the alimentary tract and then is quickly excreted from the body. The developed HILIC-MS method could be applied in determining levels of DNJ in urine and tissues, and therefore, the method would be a powerful tool for studying the metabolic fate of mulberry DNJ as well as its bioavailability.     

Food-grade mulberry powder enriched with 1-deoxynojirimycin suppresses the elevation of postprandial blood glucose in humans

Mulberry 1-deoxynojirimycin (DNJ), a potent glucosidase inhibitor, has been hypothesized to be beneficial for the suppression of abnormally high blood glucose levels and thereby prevention of diabetes mellitus. However, DNJ contents in commercial mulberry products were as low as about 0.1% (100 mg/100 g of dry product), implying that the bioavailability of DNJ might not be expected. We carried out studies in two directions: (1) production of food-grade mulberry powder containing a maximally high DNJ content; (2) determination of the optimal dose of the DNJ-enriched powder for the suppression of the postprandial blood glucose through clinical trials. The following method was used: (1) DNJ concentrations in mulberry leaves from different cultivars, harvest seasons, and leaf locations were determined using hydrophilic interaction chromatography with evaporative light scattering detection. (2) Healthy volunteers received 0, 0.4, 0.8, and 1.2 g of DNJ-enriched powder (corresponding to 0, 6, 12, and 18 mg of DNJ, respectively), followed by 50 g of sucrose. Before and 30-180 min after the DNJ/sucrose administration, plasma glucose and insulin were determined. The following results were obtained: (1) Young mulberry leaves taken from the top part of the branches in summer contained the highest amount of DNJ. After optimization of the harvesting and drying processes for young mulberry leaves (Morus alba L. var. Shin ichinose), DNJ-enriched powder (1.5%) was produced. (2) A human study indicated that the single oral administration of 0.8 and 1.2 g of DNJ-enriched powder significantly suppressed the elevation of postprandial blood glucose and secretion of insulin, revealing the physiological impact of mulberry DNJ (effective dose and efficacy in humans). This study suggests that the newly developed DNJ-enriched powder can be used as a dietary supplement for preventing diabetes mellitus.     

Determination of D-chiro-Inositol in tartary buckwheat using high-performance liquid chromatography with an evaporative light-scattering detector

A simple method for rapid determination of d- chiro-inositol (DCI) (a potent mediator of insulin metabolism) contained in tartary buckwheat ( Fagopyrum tataricum L. Gaench) was developed on the basis of high-performance liquid chromatography coupled to an evaporative light scattering detector (ELSD). DCI was separated from an extract of tartary buckwheat on an Alltech Prevail Carbohydrates ES 5 mum column. During postcolumn detection, DCI was detected by ELSD. The detection limit was 100 ng. This method was sensitive enough for determining DCI in tartary buckwheat and its related products.     

Polyhydroxylated alkaloids isolated from mulberry trees (Morusalba L.) and silkworms (Bombyx mori L.)

New polyhydroxylated alkaloids, (2R,3R,4R)-2-hydroxymethyl-3,4-dihydroxypyrrolidine-N-propionamide from the root bark of Morus alba L., and 4-O-alpha-D-galactopyranosyl-calystegine B(2) and 3 beta,6 beta-dihydroxynortropane from the fruits, were isolated by column chromatography using a variety of ion-exchange resins. Fifteen other polyhydroxylated alkaloids were also isolated. 1-Deoxynojirimycin, a potent alpha-glucosidase inhibitor, was concentrated 2.7-fold by silkworms feeding on mulberry leaves. Some alkaloids contained in mulberry leaves were potent inhibitors of mammalian digestive glycosidases but not inhibitors of silkworm midgut glycosidases, suggesting that the silkworm has enzymes specially adapted to enable it to feed on mulberry leaves. The possibility of preventing the onset of diabetes and obesity using natural dietary supplements containing 1-deoxynojirimycin and other alpha-glucosidase inhibitors in high concentration is of great potential interest.     

Morphology and physiology of zinc‐stressed mulberry plants

The aim of this study was to induce symptoms of zinc deficiency and Zn excess and to relate the generation of reactive oxygen species (ROS) and the altered cellular redox environment to the effects of Zn stress in mulberry (Morus alba L.) cv. Kanva‐2 plants. The antioxidative responses of Zn‐stressed mulberry plants were studied by determining malondialdehyde content (MDA, a measure of lipid peroxidation) as indicator of oxidative damage and the ratio of dehydroascorbate (DHA) to ascorbic acid (AsA) as an index of the cellular redox state. The Zn‐deficiency effects appeared as faint paling and upward cupping of the young emerging leaves. The paling intensified with time, and affected leaves finally developed necrotic spots. At advanced stage of Zn deficiency, newly emerged leaves were spindle‐shaped, pale, and small in size. Apart from their stunted appearance, the plants supplied excess Zn did not show any specific visible symptom. Leaf water status of mulberry plant was affected in Zn‐stressed plants. Deficient leaves had decreased water potential (Ψ) and specific water content (SWC), contained less tissue Zn, less chloroplastic pigments, and high tissue Fe and Mn concentrations. However, excess supply of Zn was found to increase Ψ and decrease tissue Fe. Both hydrogen peroxide and MDA accumulated in leaves of Zn‐stressed plants. While the concentration of DHA did not vary in Zn‐deficient leaves, it was increased in leaves of plants supplied excess Zn. The ratio of the redox couple (DHA to AsA) was increased both in Zn‐deficient or Zn‐excess plants. The activities of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), peroxidase (EC 1.11.1.7), and ascorbate peroxidase (EC 1.11.1.11) increased in Zn‐stressed plants. The results suggest that deficiency or excess of Zn aggravates oxidative stress through enhanced generation of ROS and a disturbed redox homeostasis in mulberry plants.     

Evaporative light-scattering analysis of sulforaphane in broccoli samples: Quality of broccoli products regarding sulforaphane contents

Broccoli sulforaphane has received attention as a possible anticarcinogen. Sulforaphane analysis is difficult due to the lack of a chromophore for spectrometric detection. Hence, we developed a method for determining sulforaphane by using high-performance liquid chromatography (HPLC) coupled with an evaporative light-scattering detector (ELSD). Sulforaphane was extracted from acid-hydrolyzed broccoli samples, followed by solid-phase extraction and reversed-phase HPLC. Sulforaphane was detected by ELSD and concurrently identified by electrospray ionization time-of-flight mass spectrometry. The recovery of sulforaphane from broccoli samples was above 95%. The detection limit was 0.5 mug. The present method was sensitive enough to determine sulforaphane in mature broccoli, broccoli sprouts, and commercial broccoli products. Sulforaphane concentration in broccoli sprout (1153 mg/100 g dry weight) was about 10 times higher than that of mature broccoli (44-171 mg/100 g dry weight). Therefore, the broccoli sprout is recommended as a source of sulforaphane-rich products. In contrast, we found that sulforaphane could not be detected in most of broccoli products, suggesting present commercial broccoli products having low quality.     

Identification of anthocyanins in berries by narrow-bore high-performance liquid chromatography with electrospray ionization detection

Qualitative determination of anthocyanins in extracts of red fruits by narrow-bore HPLC/ESI-MS was carried out. This method was used to investigate anthocyanin contents of black bilberry (Vaccinium myrtillus L.), blackberry (Rubus sp.), and mulberry (Morus nigra). An ultraviolet diode array and a mass spectrometer with ESI source were used for detection. Anthocyanin identifications were made by using retention time data and UV-vis and mass spectra and comparing them with those of commercially available standard compounds. The method allowed the identification of fourteen anthocyanins in black bilberry extract, six anthocyanins in blackberry extract, and five anthocyanins in mulberry extract.     

Comparison of 1-deoxynojirimycin and aqueous mulberry leaf extract with emphasis on postprandial hypoglycemic effects: in vivo and in vitro studies

Carbohydrate digestion by α-glucosidase and subsequent glucose uptake at the brush border are critical for postprandial blood glucose control. Any specific inhibitors are useful as hyperglycemia modulating agents. In this study, it was postulated that an array of active components in mulberry leaf extract (MLE) may provide higher potency in inhibiting intestinal glucose absorption compared to the single component 1-deoxynojirimycin (DNJ), which is recognized as a promising inhibitor of intestinal glucose absorption. Both MLE and DNJ were active in inhibiting α-glucosidase. However, in Caco-2 cells, only MLE showed significant inhibition of 2-deoxyglucose uptake, whereas DNJ was ineffective. For glucose loading, co-administration of MLE resulted in potent inhibitions of glucose responses compared to those by DNJ in Sprague Dawley (SD) rats, but this was not found for maltose loading. These novel findings add evidence that the unabsorbed phytochemicals in MLE compete with glucose for intestinal glucose transporters, but DNJ itself does not. We also evaluated the timing of MLE consumption. By administering MLE for 30 min before glucose loading, the incremental area under the curve (IAUC) was significantly lowered in the rats, as compared to a simultaneously administered group. Similarly, cellular glucose uptake was significantly reduced in Caco-2 cells following pretreatment.     

Peroxyl radical scavenging capacity, polyphenolics, and lipophilic antioxidant profiles of mulberry fruits cultivated in southern China

Twenty-seven cultivars of mulberry fruits ( Morus atropurpurea Roxb) were analyzed for their total phenolic content, total anthocyanin content, and peroxyl radical scavenging capacities. The proanthocyanidin contents of the fruit were also quantified using 4-dimethylamino-cinnamaldehyde assay, and characterization was attempted using electrospray ionization mass spectra. The phenolic compounds of mulberry fruits were characterized using HPLC with ESI-MS and diode array detection. Results showed that the content of mulberry fruits varied with different cultivars with total phenolic content, total anthocyanin content, total proanthocyanidin content, and peroxyl radical scavenging capacities ranging from 0.060-0.244, 0.001-0.056, 0.001-0.015, and 0.301-1.728, respectively. Good correlations were observed among the phenolic, anthocyanin, and proanthocyanidin contents and the radical scavenging capacities of mulberry fruits. Mulberry fruits were found to contain low amount of proanthocyanidins. The high total phenolic content of mulberry fruits were mainly contributed by anthocyanins, rutin, and chlorogenic acids. The lipid soluble antioxidants are profiled by an HPLC method developed in-house, and the results of selected mulberry fruits revealed significant amounts of lutein and delta- and gamma-tocopherols but low alpha-tocopherol. Our results provide useful antioxidant nutritional information of a mulberry cultivar that has potential for large scale plantations.     

Mulberry nutrient management for silk production in Hubei Province of China

The silk industry is important for south China's rural economy. Leaves of mulberry (Morus spp.) are used for silkworm production. Hubei province is one of the main silk‐producing provinces in China. The objectives of this research were to survey the fertilization practices in the mulberry‐producing regions in the province and to determine the best nutrition‐management practice for mulberry plantations. A survey and a series of field experiments with N, P, K, and micronutrients were conducted from 2001 to 2002. In addition, a silkworm‐growth experiment was also conducted by feeding leaves harvested from various fertilization treatments. The results indicate that poor soil fertility and unbalanced fertilization were the main factors limiting mulberry‐leaf yield and quality in Hubei province. Nitrogen fertilization of mulberry has reached a high level (454 kg ha^–1 y^–1) in Hubei province, but P‐ and K‐fertilization rates have not been matched with N‐fertilization rates as farmers are not aware of the significance of P and K. Balanced fertilization showed positive nutrient interactions with respect to mulberry yield and quality. Potassium application increased yield and quality (protein and sugar concentration) of mulberry leaves. Silkworm growth and cocoon quality were improved when silkworms were fed with the leaves derived from K‐fertilized plants in comparison with those taken from control plots. Application of Mg, S, and B also significantly improved leaf sugar, essential and total amino acid concentrations, but did not increase leaf yield significantly. It is concluded that a fertilizer dose of 375 kg N ha^–1, 66 kg P ha^–1, and 125 kg K ha^–1 is suitable for the cultivation of mulberry in the Hubei province along with Mg, S, and B, wherever necessary, for the improvement of yield and quality of mulberry leaves.     

Assessment of Genetic Relationships between Wild and Cultivated Mulberry (Morus) Species using PCR Based Markers

In order to formulate appropriate strategies for the conservation and utilization of the wild mulberry genetic resources available in India, a study was undertaken with 20 mulberry genotypes from the four different species. Seventeen intersimple sequence repeat primers were used to generate a total of 114 markers, of which 98 (85.96%) were polymorphic. Seven unique bands for Morus serrata Roxb. and one for both M. serrata Roxb. and Morus macroura Miq. were identified, of which one fragment has been sequenced and deposited in the EMBL-GeneBank (AJ-585512). The genetic dissimilarity coefficients varied from 0.078 to 0.530 among these genotypes and from 0.168 to 0.465 among the species. The dendrograms realized from these markers clustered the genotypes into three groups. The outermost group was M. serrata Roxb., which was followed by the group of M. macroura Miq. and the innermost group contained genotypes of Morus indica L. and Morus alba L. This intermixing of genotypes of M. indica and M. alba supports the view that M. indica is merely a synonym of M. alba. Distribution of the genotypes on a two-dimensional figure upon multidimensional scaling with ALSCAL program, further, confirmed the genetic divergence between the cultivated and wild mulberry groups. On the basis of the results a few potential wild mulberry genotypes were identified for its conservation and utilization in breeding programs to confer the stress tolerance to the cultivated varieties of mulberry.     

Simultaneous liquid chromatographic screening of five coccidiostats in chicken liver

A reverse-phase liquid chromatographic (LC) method is described for simultaneously determining 5 coccidiostats--aklomide, dinsed, ethopabate, nitromide, and zoalene in chicken liver. The method entails blender extraction of 10 g liver with ethyl acetate, column chromatography through Sephadex LH-20 and neutral alumina, and LC analysis on a C18 column with UV detection at 260 nm. The drugs were eluted from Sephadex with methanol-benzene (10 + 90), from alumina with methanol-dichloromethane (10 + 90), and from C18 with acetonitrile-water (linear gradient: 25% acetonitrile for 10 min, increasing to 55% over 15 min; flow rate 1 mL/min). Liquid chromatography was completed in 40 min and calculations were based on peak height measurements. Average recoveries of the coccidiostats from fortified liver ranged from 72 to 97%, except for dinsed, which showed a relatively constant average recovery of 57%. The detection limit for the standards was 2.5 ng on column. Levels as low as 50 ng/g were detected in fortified liver samples.     

桑树中miR319的克隆及在雄花发育中表达分析

为探究miR319及靶基因在调控桑树雄花发育的分子机制,本研究通过石蜡切片技术判断桑树雄花芽发育时期及在线网站预测miR319的靶基因。结果表明,桑树雄花芽发育分为未分化期(A1)、分化初期(A2)、花序分化期(A3)和总苞形成期(A4)4个不同的阶段;桑树中miR319家族存在miR319a、miR319b和miR319c 3个成员,其中miR319b与miR319c位于同一条前体序列上,预测并获得了miR319 6个靶基因。运用5'-RACE技术验证了miR319a的4个靶基因,其切割位点主要集中在与miRNA互补位点的第10和第11位碱基之间。采用RT-qPCR技术检测miR319a及靶基因在雄花发育不同阶段的表达水平,发现miR319a在雄花发育A2-A3阶段表达水平迅速降低,靶基因Morus012208、Morus008229、Morus008100与Morus005893在此阶段表达水平迅速升高,茉莉酸(JA)含量也迅速升高。同时,JA合成关键基因PLDα1和LOX5在雄花发育A2-A3阶段表达上调,表明miR319通过调控靶基因影响JA合成来调控桑树花序分化。本研究为揭示桑树miR319在JA途径中调控花发育中的分子调控机制奠定了一定的理论基础。     

Improved cleanup for gas chromatographic determination of propiconazole residues in soil, wheat grain, straw, and leaves

A simple and sensitive method is described for determination of propiconazole, a new type of broad-spectrum systemic fungicide, in soil, wheat grain, straw, and leaves. Pesticide residues in or on grain and green plant materials are extracted with methanol (or a mixture of methanol and water (4 + 1), for soil), partitioned into methylene chloride, and cleaned up on an alumina column for grain and soil or an activated charcoal column for green plant materials. The amount of residue is quantitatively measured by gas chromatography using an alkali flame ionization detector in the nitrogen-sensitive mode. Recoveries from soil, grain, and green plant materials fortified at 0.1-5 mg/kg are better than 80%. The practical detection limits of this method are 0.01 mg/kg in grain and soil and 0.02 mg/kg in green plant materials.     

夏桑叶的体外抗氧化活性及其主要功能成分研究

利用体外清除自由基评价技术,测定并比较了夏桑叶与冬桑叶对3种氧中心自由基·OH、O 2-·和1O2的清除活性。结果显示,夏桑叶对·OH和O 2-·的清除活性与冬桑叶相近,而且清除^1O2能力较冬桑叶的高61%,提示夏桑叶也是一种极具研发应用前景的纯天然医药保健资源。进一步分别用干叶热水提取法、鲜叶热水提取法和鲜叶温水提...     

Determination of rutin, catechin, epicatechin, and epicatechin gallate in buckwheat Fagopyrum esculentum Moench by micro-high-performance liquid chromatography with electrochemical detection

A simple and sensitive method has been developed for determining rutin, catechin, epicatechin, and epicatechin gallate in buckwheat (Fagopyrum esculentum Moench) flour and seeds by micro-high-performance liquid chromatography with electrochemical detection. Chromatography was performed using an octadecylsilica column, acetonitrile-water-formic acid (13:87:1, v/v/v) as a mobile phase, and an applied potential at +0.5 V vs Ag/AgCl. We found that Japanese buckwheat flour contains rutin (12.7 mg/100 g), catechin (3.30 mg/100 g), epicatechin (20.5 mg/100 g), and epicatechin gallate (1.27 mg/100 g). The relative standard deviations for rutin, catechin, epicatechin, and epicatechin gallate peak heights were less than 0.86% (n = 5). The detection limit of rutin was 0.86 ng/mL. Moreover, the present method was applied to the distribution analysis of these compounds in buckwheat seed. The embryo proper and cotyledons of a mature buckwheat seed contained rutin with the highest concentration as compared to other parts. This method is useful in determining rutin, catechin, epicatechin, and epicatechin gallate in buckwheat with a small amount of sample for quality control in the food industry.     

氮钾肥配合施用对桑叶产量品质及蚕茧质量的影响

通过连续4年田间施肥试验和一季养蚕试验,研究了氮肥和钾肥配合施用对桑叶产量、品质及相应桑叶喂饲家蚕对蚕生长及蚕茧品质的影响。结果表明,桑园施用氮钾肥显著提高桑叶产量,在氮钾肥各养分配比中,以高氮高钾处理(N450K300)的产量最高,比N0K0处理年均增产35.2%,与其他各处理产量差异显著。桑叶品质分析结果表明,桑叶中必需氨基酸、氨基酸总量、粗蛋白、油脂含量随氮肥用量增加而提高,但单施氮肥会对蛋氨酸和油脂含量产生负面影响;施钾导致糖分含量下降,其他品质参数值随钾肥施用而提高,氮钾肥配合施用具有提高桑叶品质的作用。养蚕结果表明,桑园增施氮钾肥生产出的桑叶,有助于降低蚕茧的死笼率、具有提高全茧量和茧层率及提高蚕茧产量的作用。蚕茧上茧率、茧丝长、解舒率和茧丝净度随着钾肥用量的增加而提高,在施钾基础上增加氮肥用量有提高蚕茧品质的作用,但不施钾只施氮肥对上茧率有负面影响,说明氮钾肥配合施用能促进蚕茧质量的提高。     

Ion-pair liquid chromatographic determination of some penicillins in canine and equine sera

A sensitive liquid chromatographic method was developed for determining oxacillin, cloxacillin, and dicloxacillin (simultaneously), and penicillin G, amoxicillin, carbenicillin, and ticarcillin in canine and/or equine serum. The method involves filtering diluted serum through a 30,000 molecular weight cut-off filter and separating penicillins from other serum components by ion-pair liquid chromatography using a reverse-phase column eluted with acetonitrile-water solutions. The ultraviolet absorbance of the column effluent was monitored at 230 nm. Recoveries of oxacillin, cloxacillin, dicloxacillin, and penicillin G (spiked at 2.5 micrograms/mL), amoxicillin (spiked at 5 micrograms/mL), and carbenicillin and ticarcillin (spiked at 10 micrograms/mL) from canine and equine serums ranged from 78.3 to 104.4% with coefficients of variation ranging from 3.35 to 5.95%. The limit of detection for these penicillins was 0.02-0.05 microgram/mL.     

Capillary column gas chromatographic determination of dicamba in water, including mass spectrometric confirmation

A sensitive method is described for determining dicamba at low micrograms/L levels in ground waters by capillary column gas chromatography with electron-capture detection (GC-EC); compound identity is confirmed by gas chromatography-mass spectrometry (GC-MS) using selected ion monitoring. Dicamba residue is hydrolyzed in KOH to form the potassium salt. The sample is then extracted with ethyl ether which is discarded. The aqueous phase is acidified to pH less than 1 and extracted twice with ethyl ether. The combined ethyl ether extracts are concentrated, and the residue is methylated using diazomethane to form the corresponding dicamba ester. The derivatized sample is cleaned up on a deactivated silica gel column. The methylated dicamba is separated on an SE-30 capillary column and quantitated by electron-capture or mass spectrometric detection. Average recoveries (X +/- SD) for ground water samples fortified with 0.40 microgram/L of dicamba are 86 +/- 5% by GC-EC and 97 +/- 7% by GC-MS detections. The EDL (estimated detection limit) for this method is 0.1 microgram dicamba/L water (ppb).     

氟啶虫胺腈在棉花和土壤中的检测方法与残留动态研究

研究和建立了氟啶虫胺腈在土壤、棉籽和棉叶中的高效液相色谱检测方法,并在天津和杭州两地开展了氟啶虫胺腈在棉花中的田间残留试验研究。样品采用乙腈提取,正己烷萃取,氟罗里硅土柱层析净化,正己烷/丙酮(体积比6∶4)混合液洗脱,减压浓缩至干,甲醇定容,高效液相色谱配可变波长紫外检测器进行检测。当分别在空白土壤、棉籽和棉叶样品中添加浓度为0.05~2.5mg·kg-1的氟啶虫胺腈标准品时,其平均添加回收率在76.81%~94.43%之间,相对标准偏差(RSD)在0.54%~7.20%之间;氟啶虫胺腈的最小检出量为1 ng,在所有样品中的最低检出浓度均为0.05mg·kg-1。田间残留试验结果表明,氟啶虫胺腈在土壤和棉叶中的消解规律符合一级动力学模型Ct=C0e-kt,消解半衰期分别为1.36~5.10 d和6.13~9.37d。最终残留试验结果表明,在棉花田手动喷雾施用50%氟啶虫胺腈水分散粒剂,按推荐剂量和1.5倍推荐剂量施药,兑水喷雾处理2~3次,每次施药间隔7 d,在距最后1次施药7、14 d和21d时,氟啶虫胺腈在棉籽和土壤中的残留量均小于方法最低检出浓度0.05mg·kg-1。