Solid-phase extraction and LC-MS analysis of pyrrolizidine alkaloids in honeys

Strong-cation-exchange, solid-phase extraction of pyrrolizidine alkaloids and their N-oxides from honey samples was followed by reduction of the N-oxides and subsequent analysis of total pyrrolizidine alkaloids using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. A limited survey of 63 preprocessing samples of honey, purposefully biased toward honeys attributed to floral sources known to produce pyrrolizidine alkaloids, demonstrated levels of pyrrolizidine alkaloids up to approximately 2000 parts per billion (ppb) in a sample attributed to Echium plantagineum. Up to 800 ppb pyrrolizidine alkaloids was detected in some honeys not attributed by the collector to any pyrrolizidine alkaloid-producing floral source. No pyrrolizidine alkaloids were detected in approximately 30% of the samples in this limited study, while some honeys showed the copresence of pyrrolizidine alkaloids from multiple floral sources such as E. plantagineum and Heliotropium europaeum. In addition, retail samples of blended honeys (with no labeling to suggest that pyrrolizidine alkaloid-producing floral sources were used in the blends) have been shown to contain up to approximately 250 ppb pyrrolizidine alkaloids.     

Pyrrolizidine alkaloids of Echium vulgare honey found in pure pollen

The pyrrolizidine alkaloids previously identified in floral honey attributed to Echium vulgare (Boraginaceae) have been detected (8000-14 000 ppm) in pure pollen collected from the anthers of Echium vulgare. Pyrrolizidine alkaloids and/or their N-oxides were isolated from the aqueous acid extracts of pollen by use of strong cation-exchange, solid-phase extraction and identified by liquid chromatographic/mass spectrometric (LCMS) analysis. The pyrrolizidine alkaloids in the pollen are present mainly as the N-oxides. In addition to seven previously described pyrrolizidine alkaloids and/or their N-oxides (echimidine, acetylechimidine, uplandicine, 9-O-angelylretronecine, echiuplatine, leptanthine, and echimiplatine), one unidentified (echivulgarine), but previously found in honey, and two previously undescribed (vulgarine and 7-O-acetylvulgarine) pyrrolizidine alkaloids and/or their N-oxides were identified in the pollen. Tentative structures for these unidentified pyrrolizidine alkaloids are proposed on the basis of the mass spectrometric data and biogenetic considerations. The implications of these results for identifying the source and subsequent concentrations of pyrrolizidine alkaloids in honeys and commercial bee pollen are briefly discussed.     

Improved method for extraction and LC-MS analysis of pyrrolizidine alkaloids and their N-oxides in honey: application to Echium vulgare honeys

A method for analyzing honey samples was developed that enabled the simultaneous detection and identification of pyrrolizidine alkaloids and their N-oxides. Honey samples were treated with methanol or dilute sulfuric acid and then centrifuged to remove insoluble material. Subsequent strong cation exchange, solid-phase extraction of the supernatant provided a fraction that was analyzed for the presence of pyrrolizidine alkaloids and their N-oxides using high-pressure liquid chromatography coupled to electrospray ionization mass spectrometry. The procedure was validated using extracts of Echium plantagineum and authenticated standards of pyrrolizidine alkaloids and their N-oxides from other plant sources. Of several variations of the solid-phase extraction method assessed in this study, the best combination for generic use involved the dilution of honey with 0.05 M sulfuric acid and the subsequent application of the centrifuged solution to solid-phase extraction columns at the rate of a maximum of 10 g of honey per solid-phase extraction column. The method was applied to the analysis of nine floral honeys, five of which were attributed by the apiarist to Echium vulgare. Seven of the honey samples were positive for pyrrolizidine alkaloids and N-oxides characteristic of E. vulgare.     

Analysis of herbal teas made from the leaves of comfrey (Symphytum officinale): reduction of N-oxides results in order of magnitude increases in the measurable concentration of pyrrolizidine alkaloids

OBJECTIVES: To determine the relative quantities of two hepatotoxic pyrrolizidine alkaloids, symphytine and echimidine, in teas prepared from comfrey leaves (Symphytum officinale), and to determine the potential contribution of the N-oxide forms of these alkaloids to levels of the parent alkaloids. DESIGN: Comfrey leaves were purchased from three commercial sources and used to prepare tea in a manner consistent with the methods used by consumers. An extraction scheme was devised for extraction of the alkaloids, and a gas chromatographic method was developed to quantify the two major alkaloids, symphytine and echimidine. Recognising that the N-oxide derivatives of these alkaloids have also been identified in comfrey preparations, chemical reduction was applied to determine the total quantities of the alkaloids as free bases and as N-oxide derivatives. RESULTS: The concentration of symphytine and echimidine varied considerably between teas prepared from leaves purchased from the different vendors of plant material. Moreover, a much higher concentration of symphytine was found in the tea when steps were included to reduce N-oxides prior to analysis. The treatment of pure symphytine with hot water did not generate the N-oxide derivative de novo. CONCLUSIONS: Since the pyrrolizidine alkaloids are known to be hepatotoxic, consumption of herbal teas made from comfrey leaves may be ill-advised. The concentration of pyrrolizidine alkaloids in such teas may be underestimated substantially unless the concentration of N-oxides is taken into consideration.     

Development of enzyme-linked immunosorbent assays for the hepatotoxic alkaloids riddelliine and riddelliine N-oxide

Pyrrolizidine alkaloid-containing plants are widely distributed throughout the world and are particularly common in the genus Senecio. The structural types and concentrations of the alkaloids vary among plant species. In addition, within a species of plant, concentrations vary with environment and location. Many pyrrolizidine alkaloids are toxic and cause poisoning in livestock and in humans. Rapid, sensitive, and specific diagnostic techniques are needed to identify poisoned animals and to determine the particular plants and conditions under which livestock are likely to be poisoned. In this study, two competitive inhibition enzyme-linked immunosorbent assays for riddelliine, riddelliine N-oxide, and other closely related pyrrolizidine alkaloids were developed using polyclonal antibodies. One assay is class specific toward the free base forms of the pyrrolizidine alkaloids; the other assay showed cross-reactivity to both the free base and N-oxide forms of the alkaloids. The assay with the lowest limit of detection had an I(50) of 803.9 pg with a limit of detection of 47.5 pg for riddelliine. Spike and recovery studies for riddelliine in bovine blood ranged from 45 to 74%. The assay that showed cross-reactivity between the N-oxide and free base forms of the pyrrolizidine alkaloids allowed estimation of the total pyrrolizidine alkaloid content in Senecio riddellii in admixture with alfalfa. These findings suggest that these techniques will be excellent tools to diagnose poisoned animals and identify highly toxic plants.     

Hepatotoxic pyrrolizidine alkaloids in pollen and drying-related implications for commercial processing of bee pollen

Using HPLC-ESI-MS, several saturated and 1,2-dehydropyrrolizidine alkaloids were detected, mainly as their N-oxides, in fresh pollen collected from flowers of the pyrrolizidine alkaloid-producing plants Echium vulgare, E. plantagineum, Senecio jacobaea, S. ovatus, and Eupatorium cannabinum, and/or pollen loads from bees (bee pollen) that foraged on those plants. A major alkaloidal metabolite in S. ovatus was tentatively identified, using its mass spectrometric data and biogenic considerations, as the previously unreported, saturated alkaloid, 2-hydroxysarracine. Heating had very little effect on the 1,2-dehydropyrrolizidine alkaloids and their N-oxides from a variety of sources. Considered in conjunction with international concerns about the adverse effects of these alkaloids, the results strongly indicate a need for monitoring pollen supplies intended for human consumption, at least until conditions for processing and/or selection are clearly defined such as to significantly reduce the hepatotoxic (and potentially carcinogenic and genotoxic) pyrrolizidine alkaloid content of bee pollen.     

无线射频所有权转移协议在中药材溯源中的应用

针对现有的所有权转移协议,大多只涉及到单个标签的所有权转移过程,普遍存在隐私数据泄露、所有权转移过程不稳定等问题,该文在轻量级加密算法的基础上,提出一种改进的共享所有权转移协议(TSOTP,TTP model shared ownership transfer protocol),采用基于可信第三方(TTP,trusted third party)的对称加密机制,在完成初始标签认证后,通过TTP授权认证,使用对称加密算法,产生群组对称密钥,新所有者利用共享群组密钥对标签身份进行认证,然后为标签分配新的密钥,从而最终获得授权,读取标签中包含的药材敏感数据.TSOTP协议能够提高标签在所有权转移过程中的稳定性,很好地实现所有权在共享用户之间的安全转移,保证标签的数据安全,减少隐私数据泄露、Dos攻击、重放攻击等风险,提高前向与后向安全性,同时可以避免所有权重复转移,简化了标签认证计算量.经过试验证明,TSOTP协议与群组所有权转移协议(GOT,group ownership transfer)协议相比,标签数据库认证消耗时间节省57％,标签计算量消耗时间节省38％,能够成功阻止重放攻击和异步攻击等,具备较好的稳定性和认证效率,可以满足中药材质量溯源系统的研究需要,研究结果为建立中药材质量溯源系统的标签安全机制提供了技术参考.     

无线射频所有权转移协议在中药材溯源中的应用

针对现有的所有权转移协议,大多只涉及到单个标签的所有权转移过程,普遍存在隐私数据泄露、所有权转移过程不稳定等问题,该文在轻量级加密算法的基础上,提出一种改进的共享所有权转移协议（TSOTP,TTP model shared ownership transfer protocol）,采用基于可信第三方（TTP,trusted third party）的对称加密机制,在完成初始标签认证后,通过TTP授权认证,使用对称加密算法,产生群组对称密钥,新所有者利用共享群组密钥对标签身份进行认证,然后为标签分配新的密钥,从而最终获得授权,读取标签中包含的药材敏感数据。TSOTP协议能够提高标签在所有权转移过程中的稳定性,很好地实现所有权在共享用户之间的安全转移,保证标签的数据安全,减少隐私数据泄露、Dos攻击、重放攻击等风险,提高前向与后向安全性,同时可以避免所有权重复转移,简化了标签认证计算量。经过试验证明,TSOTP协议与群组所有权转移协议（GOT,group ownership transfer）协议相比,标签数据库认证消耗时间节省57%,标签计算量消耗时间节省38%,能够成功阻止重放攻击和异步攻击等,具备较好的稳定性和认证效率,可以满足中药材质量溯源系统的研究需要,研究结果为建立中药材质量溯源系统的标签安全机制提供了技术参考。     

Chemical comparison of goldenseal (Hydrastis canadensis L.) root powder from three commercial suppliers

The characterization of herbal materials is a significant challenge to analytical chemists. Goldenseal (Hydrastis canadensis L.), which has been chosen for toxicity evaluation by NIEHS, is among the top 15 herbal supplements currently on the market and contains a complex mixture of indigenous components ranging from carbohydrates and amino acids to isoquinoline alkaloids. One key component of herbal supplement production is botanical authentication, which is also recommended prior to initiation of efficacy or toxicological studies. To evaluate material available to consumers, goldenseal root powder was obtained from three commercial suppliers and a strategy was developed for characterization and comparison that included Soxhlet extraction, HPLC, GC-MS, and LC-MS analyses. HPLC was used to determine the weight percentages of the goldenseal alkaloids berberine, hydrastine, and canadine in the various extract residues. Palmatine, an isoquinoline alkaloid native to Coptis spp. and other common goldenseal adulterants, was also quantitated using HPLC. GC-MS was used to identify non-alkaloid constituents in goldenseal root powder, whereas LC-MS was used to identify alkaloid components. After review of the characterization data, it was determined that alkaloid content was the best biomarker for goldenseal. A 20-min ambient extraction method for the determination of alkaloid content was also developed and used to analyze the commercial material. All three lots of purchased material contained goldenseal alkaloids hydrastinine, berberastine, tetrahydroberberastine, canadaline, berberine, hydrastine, and canadine. Material from a single supplier also contained palmatine, coptisine, and jatrorrhizine, thus indicating that the material was not pure goldenseal. Comparative data for three commercial sources of goldenseal root powder are presented.     

Profiling glucosinolates, flavonoids, alkaloids, and other secondary metabolites in tissues of Azima tetracantha L. (Salvadoraceae)

Azima tetracantha L. (needle bush; bee sting bush; Salvadoraceae) is used as a food and for various herbal medicines in Africa, India, and Madagascar, but there is very little information on the secondary metabolites in this species. High concentrations of N-methoxy-3-indolylmethyl-glucosinolate, a common glucosinolate of Brassica crops such as Brussels sprouts and broccoli, were found in the roots and seeds of A. tetracantha. Lower concentrations were detected in the stems and young leaves. The roots also contained another indole glucosinolate that was provisionally identified, from MS data and comparison with indole glucosinolate standards, as N-hydroxy-3-indolymethyl-glucosinolate. The roots, stems, and leaves contained neoascorbigen (the condensation product of N-methoxy-indole-3-carbinol and ascorbic acid). The seeds of A. tetracantha contained a complex mixture of 26 flavonoids predominantly as glycosides and acyl-glycosides, with traces of aglycones. The core aglycones of these flavonoids were identified as quercetin, isorhamnetin (3'-O-methylquercetin), rhamnetin (7-O-methylquercetin), and rhamnazin (7, 3'-di-O-methylquercetin). No flavonoids or anthocyanins were detected in other tissues, and procyanidins were undetectable. The dimeric piperidine alkaloids azimine, azcarpine, and carpaine were found in all tissues of A. tetracantha.     

Effects of ergot alkaloids on food preference and satiety in rabbits, as assessed with gene-knockout endophytes in perennial ryegrass (Lolium perenne)

Neotyphodium species are fungal endophytes best known for their protection of grass hosts and production of bioactive metabolites including ergot alkaloids. Perennial ryegrass-Neotyphodium sp. Lp1 symbiota that have altered ergot alkaloid profiles (resulting from knockouts in two different endophyte genes) were fed, along with controls, to rabbits to test the effects of ergot alkaloids on food preference and satiety. Interestingly, rabbits dramatically preferred plants that were endophyte-infected but free of ergot alkaloids over endophyte-free plants (P = 0.01). Accumulation of ergot alkaloids of the clavine class counteracted the added appeal of endophyte-infected plants. In satiety tests, consumption of ergovaline (the ultimate ergot pathway product in wild-type endophyte), but not of several other ergot alkaloids, during an initial meal had a negative effect on subsequent rabbit chow consumption (P < 0.05). The data indicate that clavines were sufficient to reduce the appeal of endophyte-infected grasses, whereas only ergovaline reduced appetite.     

Crotalaria medicaginea associated with horse deaths in northern Australia: new pyrrolizidine alkaloids

Crotalaria medicaginea has been implicated in horse poisoning in grazing regions of central-west Queensland, which resulted in the deaths of more than 35 horses from hepatotoxicosis in 2010. Liver pathology was suggestive of pyrrolizidine alkaloidosis, and we report here the isolation of two previously uncharacterized pyrrolizidine alkaloids from C. medicaginea plant specimens collected from pastures where the horses died. The first alkaloid was shown by mass spectometric and NMR analyses to be 1β,2β-epoxy-7β-hydroxy-1α-methoxymethyl-8α-pyrrolizidine, which, like other alkaloids previously isolated from C. medicaginea, lacks the requisite functionality for hepatotoxcity. The second alkaloid isolated in this investigation was a new macrocyclic diester of otonecine, which we have named cromedine. The (1)H and (13)C NMR spectra of cromedine were fully assigned by 2D NMR techniques and allowed the constitution of the macrocyclic diester to be assigned unambiguously. C. medicaginea specimens implicated in this investigation do not belong to any of the three recognized Australian varieties (C. medicaginea var. neglecta, C. medicaginea var. medicaginea, and C. medicaginea var. linearis) and appear to be a local variant or form, referred to here as C. medicaginea (chemotype cromedine).     

Cancer-preventive peptide lunasin from Solanum nigrum L. inhibits acetylation of core histones H3 and H4 and phosphorylation of retinoblastoma protein (Rb)

Lunasin, a unique 43 amino acid, 4.8 kDa cancer-chemopreventive peptide initially reported in soybean and now found in barley and wheat, has been shown to be cancer-chemopreventive in mammalian cells and in a skin cancer mouse model against oncogenes and chemical carcinogens. To identify bioactive components in traditional herbal medicines and in search for new sources of lunasin, we report here the properties of lunasin from Solanum nigrum L. (SNL), a plant indigenous to northeast Asia. Lunasin was screened in the crude extracts of five varieties of the medicinal plants of Solanaceae origin and seven other major herbal plants. An in vitro digestion stability assay for measuring bioavailability was carried out on SNL crude protein and autoclaved SNL using pepsin and pancreatin. A nonradioactive histone acetyltransferase (HAT) assay and HAT activity colorimetric assay were used to measure the inhibition of core histone acetylation. The inhibitory effect of lunasin on the phosphorylation of retinoblastoma protein (Rb) was determined by immunoblotting against phospho-Rb. Lunasin isolated from autoclaved SNL inhibited core histone H3 and H4 acetylation, the activities of the HATs, and the phosphorylation of the Rb protein. Lunasin in the crude protein and in the autoclaved crude protein was very stable to pepsin and pancreatin in vitro digestion, while the synthetic pure lunasin was digested at 2 min after the reaction. We conclude that lunasin is a bioactive and bioavailable component in SNL and that consumption of SNL may play an important role in cancer prevention.     

Identification of Chinese medicinal fungus Cordyceps sinensis by PCR-single-stranded conformation polymorphism and phylogenetic relationship

Fungi belonging to the Cordyceps species have long been used as food and herbal medicines in Asia and are especially popular as commercially available powdered supplements. Despite this acceptance and use, little is known of the phylogenetic relationships of the genus. Presently, the neighbor-joining method based on the ITS1, 5.8S rRNA, and ITS2 regions was used to construct a phylogenetic tree of 17 Cordyceps isolates. Five major groups were evident. Cordyceps sinensis was less closely related to 15 Cordyceps species but shared a closer relationship with Cordyceps agriota. PCR-single-stranded conformational polymorphism was applied to differentiate seven Cordyceps isolates: five were different from those used to construct the phylogenetic tree, based on differences in the internal spacer 2 (ITS2). The length of ITS2, amplified by primers 5.8SR and ITS4, vary between 334 and 400 bp. This segment could be used for intraspecies classification or detection of mutations and represents potential novel means of identification of this fungal genus in herbal medicines and in quality control applications in the fermentation industry.     

Assessment of the floral origin of honey by SDS-page immunoblot techniques

We report on the development of a novel alternative method for the assessment of floral origin in honey samples based on the study of honey proteins using immunoblot assays. The main goal of our work was to evaluate the use of honey proteins as chemical markers of the floral origin of honey. Considering that honeybee proteins should be common to all types of honey, we decided to verify the usefulness of pollen proteins as floral origin markers in honey. We used polyclonal anti-pollen antibodies raised in rabbits by repeated immunization of Sunflower (Elianthus annuus) and Eucalyptus (Eucalyptus sp.) pollen extracts. The IgG fraction was purified by immunoaffinity. These antibodies were verified with nitrocellulose blotted pollen and unifloral honey protein extracts. The antibodies anti-Sunflower pollen, bound to the 36 and 33 kDa proteins of Sunflower unifloral honey and to honey containing Sunflower pollen; and the antibodies anti-Eucalyptus sp. pollen bound to the 38 kDa proteins of Eucalyptus sp. unifloral honey in immunoblot assays. Satisfactory results were obtained in differentiating between the types of pollen analyzed and between Sunflower honey and Eucalyptus honey with less cross reactivity with other types of honey from different origin and also with good sensitivity in the detection. This immunoblot method opens an interesting field for the development of new antibodies from different plants, which could serve as an alternative or complementary method to the usual melissopalynological analysis to assess honey floral origin.     

Development of a DNA microarray for authentication of ginseng drugs based on 18S rRNA gene sequence

Ginseng drugs, derived from underground parts of Panax species (Araliaceae), are the most important group of herbal medicines in the Orient. Previously, the nucleotide sequences of the nuclear 18S rRNA gene of 13 Panax taxa were determined, as were the specific polymorphic nucleotides for identification of each species. On the basis of the nucleotide difference, a DNA microarray (PNX array) was developed for the identification of various Panax plants and drugs. Thirty-five kinds of specific oligonucleotide were designed and synthesized as probes spotting on a decorated glass slide, which included 33 probes corresponding to the species-specific nucleotide substitutions and 2 probes as positive and negative controls. The species-specific probes were of 23-26 bp in length, in which the substitution nucleotide was located at the central part. Triplicate probes were spotted to warrant accuracy by correcting variation of fluorescent intensity. Partial 18S rRNA gene sequences amplified from Panax plants and drugs as well as their derived health foods were fluorescently labeled as targets to hybridize to the PNX array. After hybridization under optimal condition, specific fluorescent patterns were detected for each Panax species, and the analyzed results could be indicated as barcode patterns for quick distinction. The developed PNX array provided an objective and reliable method for the authentication of Panax plants and drugs as well as their derived health foods.     

河西走廊东部不同类型植物物候对气候变化的响应

基于甘肃省武威市气象局1980-2004年对多年生植物旱柳和中国槐物候期、1981-2008年对一年生植物春小麦和玉米发育期的观测资料,运用线性倾向估计法、膨化相关、SPSS统计软件进行逐步回归和方差分析,建立了关键物候期的气候预测模型。结果表明：（1）在分析期内,多年生木本植物相同物候期最晚与最早出现日期相差达20～38d,一年生草本植物达11～30d,反映出物候期对气象条件年际变化非常敏感。（2）分析期内多年生植物春季多数物候期提前（线性倾向率-2.008～-3.246d/10a,P〈0.05）,秋季物候期推迟（线性倾向率6.631～7.108d/10a,P〈0.01）。一年生植物的春、夏季物候期均有提前的趋势（线性倾向率-1.494～-4.122d/10a,P〈0.05）,秋季物候期推后不显著。（3）从各物候间隔期变化看,多年生植物绿叶期延长速率为1.046～7.738d/10a,营养生长期延长速率为0.877～8.454d/10a,干物质积累期延长速率为4.392～7.738d/10a,秋季生长期延长速率为0.477～3.015 d/10a。一年生植物春季营养生长期缩短速率为2.17～3.41d/10a,秋季生殖生长期延长速率为3.322d/10a。（4）对多年生植物而言,平均气温、平均最低气温、平均最高气温和20cm、80cm、160cm地温是影响春、夏、秋季物候期的主要气象因子,温度越高春季物候期越提前,夏、秋季物候期越推迟。对一年生植物而言,平均气温、平均最低气温、平均最高气温和20cm、80cm、160cm地温也是主要影响因子,气候变暖,春、夏、秋季物候期均提前。     

Initial study of honey adulteration by sugar solutions using midinfrared (MIR) spectroscopy and chemometrics

Fourier transform infrared (FTIR) spectroscopy and attenuated total reflection (ATR) sampling have been used to detect adulteration of honey samples. The sample set comprised 320 spectra of authentic (n = 99) and adulterated (n = 221) honeys. Adulterants used were solutions containing both d-fructose and d-glucose prepared in the following respective weight ratios: 0.7:1.0, 1.2:1.0 (typical of honey composition), and 2.3:1.0. Each adulterant solution was added to individual honeys at levels of 7, 14, and 21% w/w. Spectral data were compressed and analyzed using k-nearest neighbors (kNN) and partial least squares (PLS) regression techniques. A number of data pretreatments were explored. Best classification models were achieved with PLS regression on first derivative spectra giving an overall correct classification rate of 93%, with 99% of samples adulterated at levels of 14% w/w or greater correctly identified. This method shows promise as a rapid screening technique for detection of this type of honey adulteration.     

Development of enzyme-linked immunosorbent assays for toxic larkspur (Delphinium spp.) alkaloids

Larkspur (Delphinium spp.) poisons thousands of cattle on western rangelands each year. Because poisoning does not cause specific lesions, and poisoned animals are rarely found before they die, definitively identifying poisoned animals is difficult. Additionally, toxin concentrations in larkspur plants vary with environment, plant, and location. Rapid, sensitive, and specific diagnostic techniques are needed to identify poisoned animals and to determine when and what plants are likely to poison livestock. In this study, three competitive inhibition enzyme-linked immunosorbent assays (CI-ELISA) for toxic larkspur alkaloids were developed. One assay is class-specific toward the N-(methylsuccinimido)anthranoyllycoctonine (MSAL) alkaloids, and two assays are specific for individual alkaloids. The assay with the lowest limit of detection had an I(50) of 191 pg with a limit of detection of 30.5 pg for methyllycaconitine. Spike and recovery studies using bovine blood and brain tissue ranged from 52 to 89%. These findings suggest that with additional development these techniques are likely to be excellent tools for diagnosing poisoned animals and identifying highly toxic plants.