气相色谱法测定食品中植物甾醇含量研究

目的：建立气相色谱法测定食品中植物甾醇含量的方法。方法：对常见食品中的菜油甾醇、豆甾醇、β-谷甾醇含量进行定性定量分析。食品样品经皂化后提取,浓缩后用正己烷定容,上机测定。结果：本方法可有效分离食品中植物甾醇组分,各组分在0.005~0.2 mg/mL范围内线性关系良好,相关系数R2均在0.99以上;检出限为0.3 mg/100 g,其回收率在84.9%~106.2%,方法的重现性、精密度和稳定性良好,RSD均小于5%。结论：与液相色谱法相比,本方法能够有效分离和测定植物甾醇组分,且具有样品前处理简单、方法重现性好、准确度和灵敏度高等优点,可用于测定食品中植物甾醇组分含量。     

ASE快速溶剂萃取与测定青海十六种高寒植物中多糖含量

本研究采用快速溶剂萃取法(ASE)提取青海十六种高寒植物中的多糖,并建立了苯酚-硫酸法测定其多糖含量。使用ASE350通过在线净化过程加入乙醚除去样品中的脂类物质,加入75%乙醇在线除去游离单糖、低聚糖和糖醛酸,同时对样品进行脱色。继续加入蒸馏水用ASE在线提取出样品中的多糖。离心除去由淀粉溶胀糊化形成的胶体物质,上清液加入Sevag试剂除去蛋白质,试液依次加入5%苯酚溶液和浓硫酸显色。以蒸馏水做空白对照,在490 nm处测定吸光度,同时采用水提醇沉法进行多糖换算因子的测定。结果表明,各样品多糖在0.01~0.10 mg范围内具有良好的线性关系(r=0.9996),平均加样回收率为97.26%,RSD为0.53%(n=9)。该方法简便、准确、稳定性和重现性好,适用于测定植物样品中多糖的含量,可为青海高寒植物的质量控制提供依据。     

ASE快速溶剂萃取与测定青海十六种高寒植物中多糖含量北大核心CSCD

本研究采用快速溶剂萃取法（ASE）提取青海十六种高寒植物中的多糖,并建立了苯酚-硫酸法测定其多糖含量。使用ASE350通过在线净化过程加入乙醚除去样品中的脂类物质,加入75%乙醇在线除去游离单糖、低聚糖和糖醛酸,同时对样品进行脱色。继续加入蒸馏水用ASE在线提取出样品中的多糖。离心除去由淀粉溶胀糊化形成的胶体物质,上清液加入Sevag试剂除去蛋白质,试液依次加入5%苯酚溶液和浓硫酸显色。以蒸馏水做空白对照,在490 nm处测定吸光度,同时采用水提醇沉法进行多糖换算因子的测定。结果表明,各样品多糖在0.01-0.10 mg范围内具有良好的线性关系（r=0.9996）,平均加样回收率为97.26%,RSD为0.53%（n=9）。该方法简便、准确、稳定性和重现性好,适用于测定植物样品中多糖的含量,可为青海高寒植物的质量控制提供依据。     

植物络合素的测定方法

植物络合素是一类富含巯基,并能够与重金属离子相结合的多肽。植物络合素具有共同的结构：[γ-Glu—Cys]n Gly(n=2-11)。植物络合素是植物重金属抗性的要素之一。植物络合素的测定是一种植物重金属抗性机理的研究技术。介绍了植物络合素测定的理论,并综述了现有的植物络合素测定的方法：分光比色法、凝胶过滤法、液相色谱法和毛细管电泳法。其中,着重阐述了液相色谱法在植物络合素测定中的应用。     

植物组织内ACC含量测定的一些问题探讨

在许多植物组织中,乙烯生物合成的速率与组织中ACC含量密切相关,灵敏而准确地测定植物组织中ACC含量变化,对了解ACC含量对乙烯生成速率的影响十分必要。目前,测定ACC含量多数是应用LhaL和Yang［。j根据ACC可被Na0CI氧化产生乙烯的原理而提出的方法,此法的灵敏度和准确度取决于ACC转化为乙烯的效率,而ACC转化为乙烯的效率受植物样品内某些有机成分的影响,这些有机成分影响的大小与分析样品的前处理方法有密切关系。其前处理方法大致分为两类,一类是用5％横基水杨酸抽提样品中的ACC,再用阳离子交换树脂或离子交换纤维素…     

新型靶向化合物——植物大麻素的生物合成途径及 研究进展

植物大麻素是具有生物活性的一系列萜类化合物的总称,被认为是大麻的专有成分。具有主要药理活性的植物大麻素为Δ~9-四氢大麻酚(Δ~9-tetrahydrocannabinol,Δ~9-THC)和大麻二酚(Cannabidiol,CBD),均以内源性大麻素受体为靶点,通过激活内源性大麻素系统而参与人体许多生理病理过程,具有广泛的治疗潜力。目前,Δ~9-THC、CBD及其类似物或组合制剂,已用于治疗癫痫、癌症化疗患者的呕吐、多发性硬化症痉挛和缓解神经性疼痛以及晚期癌症患者的疼痛。随着对Δ~9-THC和CBD应用价值的深度发掘和药用标准化制剂需求量增加,Δ~9-THC和CBD在制药工业中实现规模化生产迫在眉睫。通过综述近年来植物大麻素的药理学研究进展,植物大麻素生物合成途径和关键酶的作用机制以及制药工业中植物大麻素的生产策略,旨在探索利用合成生物学技术解决植物大麻素药源问题的潜力,为合成大麻素的微生物工程研发提供理论基础,促进药用大麻素的规模化生产。     

脱除游离棉酚乳酸菌的分离鉴定及益生性能研究

从藜麦茎叶样品中筛选分离出乳酸菌株,测定菌株脱除游离棉酚能力,通过人工胃液、人工肠液耐受实验、抑菌实验、抗生素敏感实验及表面特性研究菌株各项性能,并通过生理生化及16S rDNA法对菌株进行鉴定。结果表明,筛选出1株高效游离棉酚脱除乳酸菌LR002,其游离棉酚脱除率为62.87%,该菌株具有高效的耐人工胃液、肠液效果,且具有广谱抑菌作用,对抗生素不敏感,经鉴定该菌株为植物乳杆菌（Lactobacillus plantarum）。本研究旨在筛选出高效游离棉酚脱除乳酸菌菌株,为棉粕在畜禽生产中的合理利用提供参考。     

微生物法测定食品中泛酸的含量

利用植物乳杆菌（ATCC8014）对泛酸的含量在一定条件下成线性关系,通过吸光度测定细菌繁殖浓度,可间接测定食物样品中泛酸的含量,并检测其回收率。结果表明标准曲线在10—100ng范围内线性关系良好,批内相对标准差（RSD）为2．1％-4．3％,批间测定RSD为2．7％-4．1％,回收率为88．4％～100．5％,达到了较好的分析效果。     

植物总DNA样品的快速制备

利用Ｑｉａｇｅｎ微量植物ＤＮＡ提取试剂盒,在１小时内即可从植物组织获得总ＤＮＡ,提取过程中勿需酚／氯仿和ＳＤＳ抽提,操作简便、快捷。所得ＤＮＡ样品的ＯＤ２６０／ＯＤ２８０值在１．７－１．９之间。样品纯度高;该样品不含ＰＣＲ反应抑制剂及其他酶反应抑制剂,可被各种限制性内酶完全降解,适合于ＰＣＲ、印迹、ＲＡＰＤ、ＡＦＬＰ和ＲＦＬＰ分析等各种下游应用。     

植物花粉漫谈

花粉是植物的雄性生殖细胞。它的形态各异,壁的纹饰在不同的种类有不同的图案,分类学家可以此作为植物分类的重要依据。花粉内所含的成份很复杂。经测定,花粉中所含水份为7％～12％,蛋白质和游离氨基酸24.9％,类脂物4.6％,糖39.02％,木质素16.2％,灰分2.98％,未知物12.3％。花粉含氨基酸近20种,所含糖类主要是人体易吸收的葡萄糖和果糖等,花粉中含有维生素如硫胺素(B_1)、核黄素(B_2)、泛酸、维生素PP、叶酸等,此外,还有芸香苷等。花粉中还含有15种微量元素,多种酶、辅酶、有机酸、黄酮类、激素等。因此,花粉的用途非常广泛。     

湖南八大公山的植物区系及其在植物地理学上的意义

湖南八大公山自然保护区是华中地区面积大且植物保存较完好的地点。土著种子植物计162科、709属、1775种。全部属按15个地理分布型进行分析,全部种按更细的分布型类别（中国特有分为10个亚型）进行分析。结果表明,本地热带分布的科属虽为数不少,但温带分布的科属更显优势,北温带科属比较集中,同时中国、东亚、及东亚一北美特有属也很集中。本山中国特有种计1120种,华中特有种为347种。植物区系的古老、残遗及特有性突出,可视为中国（华中）有典型代表性的山地。     

Quantification of isoflavones and lignans in urine using gas chromatography/mass spectrometry

Phytoestrogens (isoflavones and lignans) are of increasing interest due to their potential to prevent certain types of complex diseases. However, epidemiological evidence is needed on the levels of phytoestrogens and their metabolites in foods and biological fluids in relation to risk of these diseases. We report an assay for phytoestrogens which is sensitive, accurate, and uses low volumes of sample. Suitable for epidemiological studies, the assay consists of a simple sample preparation procedure and has been developed for the analysis of five isoflavones (daidzein, O-desmethylangolensin, equol, genistein, and glycitein) and two lignans (enterodiol and enterolactone), which requires only 200 microl of urine and utilizes one solid-phase extraction stage for sample preparation prior to derivatization for GC/MS analysis. Limits of detection were in the region 1.2 ng/ml (enterodiol) to 5.3ng/ml (enterolactone) and the method performed well in the UK Government's Food Standards Agency-sponsored quality assurance scheme for phytoestrogens. For the first time, average levels of all the above phytoestrogens were measured in samples of urine collected from a free living population sample of women. Results show a large range in both the amount and the type of phytoestrogens excreted.     

Thyroid gland function in ovariectomized ewes exposed to phytoestrogens

Phytoestrogens are by definition plant-derived substances that are able to activate the mammalian oestrogen receptors. We examined the possible effects of phytoestrogens on the secretion of thyroid hormones as well as on the immunoreactivity to oestrogen receptor alpha (ER alpha) in the thyroid glands of ovariectomized ewes. Eight ovariectomized ewes were fed 3.5 kg of 100% red clover silage for 14 days. Blood samples were collected before and on day 14 of exposure to phytoestrogens. After 5 months, four of the ewes were re-exposed to red clover silage as described above and the other four served as controls. Blood samples were collected as above. All ewes were slaughtered at the end of the experiment and the thyroid glands were weighed and examined for macroscopical changes. Tissue samples were taken for immunohistochemistry and image analysis. Ewes exposed to red clover silage had significantly higher plasma concentrations of total T(3) and free T(3) than ewes fed hay. The cross-section area of thyroid follicles tended to be larger in ewes fed red clover silage than in the control animals. ER alpha immunoreactivity was stronger in thyroid glands from ewes exposed to phytoestrogens than in ewes fed hay. In conclusion, daily ingestion of 81-95 mg phytoestrogens per kg body weight for 14 days stimulated secretion of thyroid hormones and tended to increase follicle size and ER alpha immunoreactivity of thyroid glands of ovariectomized ewes.     

Effect of dietary components, including lignans and phytoestrogens, on enterohepatic circulation and liver metabolism of estrogens and on sex hormone binding globulin (SHBG)

A brief account of our present knowledge on the enterohepatic metabolism of estrogens and on the origin, metabolism and biological effects of mammalian lignans and phytoestrogens is undertaken. Furthermore, recently published results on the effects of dietary fiber, fat and carbohydrates on estrogen metabolism are reviewed. New preliminary results are presented on quantitative assays of lignans and phytoestrogens in urine of women belonging to various dietary and population groups and in a group of chimpanzees. The highest values of lignans and phytoestrogens were found in the non-human primates, and in macrobiotic, lactovegetarian and Japanese women, all groups considered having a low risk for the development of breast and other hormone-dependent cancer. New results on correlations between intake of various fibers, lignan and phytoestrogen excretion and plasma levels of estrogens, free testosterone and SHBG in women are presented. There is a significant positive correlation between the intake of fiber and urinary excretion of lignans and phytoestrogens, and the concentration of plasma SHBG. Fiber intake and urinary excretion of lignans and equol correlated negatively with plasma percentage free estradiol. Enterolactone excretion correlated negatively with plasma free testosterone. It is concluded that dietary macro- and micronutrients seem to play an important role in estrogen metabolism.     

Synergistic inhibition of LDL oxidation by phytoestrogens and ascorbic acid

Increasing evidence indicates that oxidative modification of low-density lipoprotein (LDL) is an important determinant in atherogenesis, and following menopause, the incidence of coronary heart disease is as prevalent in women as it is in men. Estrogen has been demonstrated to inhibit the susceptibility of LDL to be oxidized, and more recently the use of phytoestrogens has been considered for estrogen replacement therapy. In this study the antioxidant activity of the three major phytoestrogens: genistein, daidzein, and equol were measured in terms of LDL oxidative susceptibility. Increasing levels of genistein, daidzein, and equol inhibited LDL oxidation, and this inhibitory effect was further enhanced in the presence of ascorbic acid. The synergism exhibited by these compounds is of clinical importance to phytoestrogen therapy since the efficacy of phytoestrogens as effective antioxidants is evident at concentration well within the range found in the plasma of subjects consuming soy products. However, this synergism, combined with the low reactivity of the phytoestrogens with peroxyl radicals, suggests that an antioxidant mechanism other then free radical scavenging reactions account for the phytoestrogen antioxidant effect. A structural basis for inhibition of LDL oxidation involving interaction of the phytoestrogens with apoB-100 is postulated.     

Insight into estrogenicity of phytoestrogens using in silico simulation

Phytoestrogens, including miroestrol and deoxymiroestrol, have the ability to act through competition with estrogen for binding to the estrogen receptor (ER). Here, we utilize manual ligand docking followed by molecular dynamics simulations and binding free energy calculations with the linear interaction energy method to predict the binding modes and the binding affinities of phytoestrogens on the ligand binding domain of ER (ERα-LBD). The calculations brought about the good correlation between the calculated binding free energy and the bioassays. Furthermore, consideration of Lennard-Jones and Coulomb interaction energies of miroestrol and deoxymiroestrol on ERα-LBD provided the information to develop the phytoestrogen derivatives as the preferred drug for ER positive breast cancer treatment.     

The effects of dietary phytoestrogens on aromatase activity in human endometrial stromal cells

Dietary phytoestrogens have been reported to inhibit aromatase activity in placental microsomes, but the effects in the human endometrium are unknown. Aromatase, the rate-limiting enzyme in the conversion of androgens to estrogens, has recently been shown to be expressed in the endometrium of women with endometriosis and is thought to play a role in the pathophysiology of this disease. Therefore, the objective of this study was to screen dietary phytoestrogens for their ability to inhibit aromatase activity in human endometrial stromal cells (ESC) and identify potential novel therapeutic agents for the treatment of endometriosis. The inhibition of aromatase activity by direct interaction with the dietary phytoestrogens genistein, daidzein, chrysin, and naringenin was tested in a cell free assay. Furthermore, test compound effects on aromatase activity in ESC cultures were also examined. Genistein and daidzein were inactive in the human recombinant aromatase assay whereas naringenin and chrysin inhibited aromatase activity. However, genistein (1 nM to 1 mM) stimulated aromatase activity in ESC whereas other phytoestrogens had no effect. Immunopositive aromatase cells were demonstrated in genistein-treated ESC but not in untreated control cultures. Taken together, our data suggest that genistein can increase aromatase activity in ESC likely via increased enzyme expression.     

Liquid chromatographic-photodiode array mass spectrometric analysis of dietary phytoestrogens from human urine and blood

Dietary phytoestrogens have been implicated in the prevention of chronic diseases. However, it is uncertain whether the phytoestrogens or the foods associated with phytoestrogens account for the observed effects. We report here a new liquid chromatography photodiode array mass spectrometry (LC-PDA-MS) assay for the determination of nanomolar amounts of the most prominent dietary phytoestrogens (genistein, dihydrogenistein, daidzein, dihydrodaidzein, glycitein, O-desmethylangolensin, hesperetin, naringenin, quercetin, enterodiol, enterolactone) in human plasma or serum and urine. This assay was found to be suitable for the assessment of quercetin exposure in an onion intervention study by measuring urinary quercetin levels. Other successful applications of this assay in clinical and epidemiologic studies validated the developed method and confirmed previous results on the negative association between urinary isoflavone excretion and breast cancer risk.     

Determination of phytoestrogens in traditional medicinal herbs using gas chromatography--mass spectrometry

Quantitative analytical methods for the 17 phytoestrogens containing isoflavonoids, lignans, and mycoestrogens in herbs were developed, and the amount of phytoestrogens was determined in 22 traditional medicinal herbs. The focus of this study was to simplify the purification procedure for removing many kinds of interferences in herbs, and to select adequate derivatization reagent for getting desirable selectivity and sensitivity in the quantitative determination of phytoestrogens. To satisfy these goals, we performed a solid-phase extraction with Oasis HLB cartridges following enzymatic and acidic hydrolysis, and we used the mixture of MSTFA/NH4I/DTE (1000:4:5, v/w/w) to form TMS derivatives of phytoestrogens. Overall recovery was more than 84% in all of the phytoestrogens, and the limit of quantification for phytoestrogens in herbs were set at 0.2 microg/g. Coefficient of variation percentages were in the range of 0.18-15.68% (within-day) and 0.23-16.61% (day-to-day), respectively. Most of the isoflavonoids and lignans were found in all of the herbs, but mycoestrogens were not detected at all. The Leguminosae family proved to be the richest source of isoflavonoids. Lignans such as enterodiol and enterolactone were detected at low concentration in most of the herbs. These results indicate that this assay is accurate and reliable for the determination of phytoestrogens in herbs. Also, information regarding the phytoestrogen contents in traditional medicinal herbs is useful in the prevention and treatment of chronic diseases such as cancer, osteoporosis, dementia, and cardiovascular disease.     

Phytoestrogen metabolites are much more active than phytoestrogens themselves in increasing prostaglandin F(2alpha) synthesis via prostaglanin F(2alpha) synthase-like 2 stimulation in bovine endometrium

Phytoestrogens have recently been suggested to be the cause of infertility by stimulating luteolytic prostaglandin (PG) F(2alpha) secretion from endometrium in cattle. The purpose of this study was to examine the enzymatic and molecular mechanisms involved in the preferential induction of PGF(2alpha) synthesis by phytoestrogens, and whether phytoestrogens influence endometrial cell viability. Cultured bovine endometrial epithelial and stromal cells were exposed to phytoestrogens (daidzein and genistein) and their metabolites (equol and p-ethyl phenol) for 24h. Prostaglandin F(2alpha) and PGE2 were stimulated by phytoestrogens in both stromal and epithelial cells, with a preference for PGF(2alpha) synthesis in epithelial cells (P<0.001). Although RT-PCR and Western Blot analyses did not reveal the influence of phytoestrogens on either gene expression or protein level of cyclooxygenase-2 (COX-2) and PGE2 synthase (PGES) in stromal and epithelial cells (P>0.05), the stimulative effects of equol and p-ethyl phenol on PGF(2alpha) synthase-like 2 (PGFSL2) gene expression and protein level were observed only in epithelial cells (P<0.05). The same compounds did not affect PGFSL2 gene expression and protein in stromal cells (P>0.05). Exposure to phytoestrogens and their metabolites decreased cell viability in both stromal and epithelial cells. Stromal cell viability decreased to 50% of the control and was more evident than that in epithelial cells (P<0.001). The overall results suggest that infertility in cattle, caused by phytoestrogen-dependent preferential stimulation of luteolytic PGF(2alpha) synthesis, is caused by increasing PGFSL2 in epithelial cells, and by decreasing stromal cell viability, which are the main source of luteotropic PGE2 production.