Development and validation of a UHPLC-ESI-QTOF mass spectrometry method to analyze opines, plant biomarkers of crown gall or hairy root diseases.

Opines are low-molecular-weight metabolites specifically biosynthesized by agrobacteria-transformed plant cells when plants are struck by crown gall and hairy root diseases, which cause uncontrolled tissue overgrowth. Transferred DNA is sustainably incorporated into the genomes of the transformed plant cells, so that opines constitute a persistent biomarker of plant infection by pathogenic agrobacteria and can be targeted for crown gall/hairy root disease diagnosis. We developed a general, rapid, specific and sensitive analytical method for overall opine detection using ultra-high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-MS-QTOF), with easy preparation of samples. Based on MS, MS/MS and chromatography data, the detection selectivity of a wide range of standard opines was validated in pure solution and in different plant extracts. The method was successfully used to detect different structural types of opines, including opines for which standard compounds are unavailable, in tumors or hairy roots induced by pathogenic strains. As the method can detect a wide range of opines in a single run, it represents a powerful tool for plant gall analysis and crown gall/hairy root disease diagnosis. Using an appropriate dilution of plant extract and a matrix-based calibration curve, the quantification ability of the method was validated for three opines belonging to different families (nopaline, octopine, mannopine), which were accurately quantified in plant tissue extracts.

Content of phenolic compounds and mannitol in olive leaves extracts from six Spanish cultivars: Extraction with the Soxhlet method and pressurized liquids.

Olive leaves are considered a promising source of bioactives such as phenolic compounds and mannitol. The extraction of high added value products is an issue of great interest and importance from the point of view of their exploitation. However, the content of these compounds can differ between cultivars and extraction methods. In this work, six olive leaves cultivars, including three wild cultivars, and two extraction processes (an innovative and alternative technique, pressurized liquid extraction, and a conventional Soxhlet extraction) were evaluated and compared towards the selective recovery of bioactive compounds. The wild cultivars showed the highest content of phenolic and flavonoid compounds, being oleuropein the compound present in higher amount. Findings also revealed that the highest mannitol content in the extracts was observed with the commercial cultivars, specifically in Arbequina. It is thus possible to decide which cultivars to use in order to obtain the highest yield of each bioproduct.

1H-NMR-Based Metabolic Profiling of Cordyceps militaris to Correlate the Development Process and Anti-Cancer Effect.

The study of metabolomics in natural products using the diverse analytical instruments including GC-MS, LC-MS, and NMR is useful for the exploration of physiological and biological effects and the investigation of drug discovery and health functional foods. Cordyceps militaris has been very attractive to natural medicine as a traditional Chinese medicine, due to its various bioactive properties including anti-cancer and anti-oxidant effects. In this study, we analyzed the metabolite profile in 50% ethanol extracts of C. militaris fruit bodies from three development periods (growth period, matured period, and aging period) using 1H-NMR, and identified 44 metabolites, which are classified as 16 amino acids, 10 organic acids, 5 carbohydrates, 3 nucleotide derivatives, and 10 other compounds. Among the three development periods of the C. militaris fruit body, the aging period showed significantly higher levels of metabolites including cordycepin, mannitol (cordycepic acid), and ß-glucan. Interestingly, these bioactive metabolites are positively correlated with antitumor growth effect; the extract of the aging period showed significant inhibition of HepG2 hepatic cancer cell proliferation. These results showed that the aging period during the development of C. militaris fruit bodies was more highly enriched with bioactive metabolites that are associated with cancer cell growth inhibition.

NMR and IR analysis of natural substances isolated from Cordyceps medicinal mushrooms.

There exist about 750 species of Cordyceps at present. A high price of natural Cordyceps and its lack in nature caused that the attention has been focused to its cultivation in laboratory conditions. The demand for this “fungus-parasite” is still quite high nowadays, as shown by the amount of commercial nutritional supplements. Phytochemical diversity has ensured that Cordyceps is used as an immunomodulatory and an antioxidant; it has anti-cancer, anti-inflammatory, anti-diabetic, antibacterial, anti-HIV effects. In the present study we focused on NMR and IR analyses of natural substances isolated from two species of Cordyceps: Cordyceps sinensis MFTCCB025/0216, MFTCCB026/0216 and Paecilomyces hepiali MFTCCB023/0216. Two types of rice substrates (Oryza sativa Indica and Oryza sativa Japonica) were used for cultivation. A total of five methanol extracts obtained by a reflux method of the ground mushroom were analysed. To determine the quality and quantity of the major chemical compounds, 1D and 2D NMR analysis has been used with 1H, 13C, COSY, NOESY, HSQC, HMBC and DEPT spectra. IR spectroscopy was chosen as a complementary analysis to determine functional groups. Linoleic acid, oleic acid and mannitol were identified as major compounds of the methanol extracts. Tyrosine, alanine, urea and the others biologically interesting substances were found as minor components.

Study of artemisinin and sugar accumulation in Artemisia vulgaris and Artemisia dracunculus "hairy" root cultures.

We studied the effect of genetic transformation on biologically active compound (artemisinin and its co-products (ART) as well as sugars) accumulation in Artemisia vulgaris and Artemisia dracunculus "hairy" root cultures. Glucose, fructose, sucrose, and mannitol were accumulated in A. vulgaris and A. dracunculus "hairy" root lines. Genetic transformation has led in some cases to the sugar content increasing or appearing of nonrelevant for the control plant carbohydrates. Sucrose content was 1.6 times higher in A. vulgaris "hairy" root lines. Fructose content was found to be 3.4 times higher in A. dracunculus "hairy" root cultures than in the control roots. The accumulation of mannitol was a special feature of the leaves of A. vulgaris and A. dracunculus control roots. A. vulgaris "hairy" root lines differed also in ART accumulation level. The increase of ART content up to 1.02 mg/g DW in comparison with the nontransformed roots (up to 0.687 mg/g DW) was observed. Thus, Agrobacterium rhizogenes-mediated genetic transformation can be used for obtaining of A. vulgaris and A. dracunculus "hairy" root culture produced ART and sugars in a higher amount than mother plants.

Comparison of Major Bioactive Compounds of the Caterpillar Medicinal Mushroom, Cordyceps militaris (Ascomycetes), Fruiting Bodies Cultured on Wheat Substrate and Pupae.

In this study, the main bioactive compounds of the fruit bodies of Cordyceps militaris-such as adenosine, cordycepin, polysaccharides, mannitol, superoxide dismutase (SOD), and carotenoids-were cultivated on wheat and pupae, as well as sclerotium (the pupae portion) and sclerotium with fruiting bodies. The amounts of adenosine and polysaccharide in all the tested samples (except for the polysaccharides of sclerotium) are higher than the quality standards (adenosine ≥0.055% and polysaccharide ≥2.5%) determined by the Ministry of Health of the People's Republic of China. As the most important bioactive compound in C. militaris, cordycepin is the highest in the fruiting bodies on pupae than in other samples, whereas it is the lowest in the sclerotium. The amounts of cordycepin, carotenoids, and SOD were higher in the fruiting bodies on pupae than that in the fruiting bodies on wheat, whereas the amounts of adenosine, polysaccharides, and mannitol were higher in the fruiting bodies on wheat than in the fruiting bodies on pupae. There was no significant difference in the amounts of cordycepin, carotenoids, and SOD in the sclerotium with fruiting bodies and the fruiting bodies on wheat. The adenosine, polysaccharide, and mannitol contents in the sclerotium with fruiting bodies were significantly lower than those of the fruiting bodies on wheat. Overall, the results of this evaluation could not distinguish which is better: the fruiting bodies on pupae or those on wheat; each has its own merits. The fruiting bodies of C. militaris cultivated on both wheat and pupae are important candidates for medicinal and tonic use for the welfare of humankind.

Pressurized liquid extraction of Aglaonema sp. iminosugars: Chemical composition, bioactivity, cell viability and thermal stability.

Pressurized liquid extraction of Aglaonema sp. iminosugars has been optimized. A single cycle under optimal conditions (80mg, 100°C, 2min) was enough to extract ⩾96% of most iminosugars. Further incubation with Saccharomyces cerevisiae for 5h removed coextracted interfering low molecular weight carbohydrates from extracts of different Aglaonema cultivars. A complete characterization of these extracts was carried out by gas chromatography-mass spectrometry: three iminosugars were tentatively identified for the first time; α-homonojirimycin and 2,5-dideoxy-2,5-imino-d-mannitol were the major iminosugars determined. α-Glucosidase inhibition activity, cell viability and thermal stability of Aglaonema extracts were also evaluated. Extracts with IC50 for α-glucosidase activity in the 0.010-0.079mgmL(-1) range showed no decrease of Caco-2 cell viability at concentrations lower than 125µgmL(-1) and were stable at 50°C for 30days. These results highlight the potential of Aglaonema extracts as a source of bioactives to be used as functional ingredients.

Iodine contributes to osmotic acclimatisation in the kelp Laminaria digitata (Phaeophyceae).

Iodide (I⁻) retained by the brown macroalga Laminaria digitata at millimolar levels, possesses antioxidant activities, but the wider physiological significance of its accumulation remains poorly understood. In its natural habitat in the lower intertidal, L. digitata experiences salinity changes and osmotic homeostasis is achieved by regulating the organic osmolyte mannitol. However, I⁻ may also holds an osmotic function. Here, impacts of hypo- and hypersaline conditions on I⁻ release from, and accumulation by, L. digitata were assessed. Additionally, mannitol accumulation was determined at high salinities, and physiological responses to externally elevated iodine concentrations and salinities were characterised by chl a fluorometry. Net I⁻ release rates increased with decreasing salinity. I⁻ was accumulated at normal (35 S A) and high salinities (50 S A); this coincided with enhanced rETRmax and qP causing pronounced photoprotection capabilities via NPQ. At 50 S A elevated tissue iodine levels impeded the well-established response of mannitol accumulation and prevented photoinhibition. Contrarily, low tissue iodine levels limited photoprotection capabilities and resulted in photoinhibition at 50 S A, even though mannitol was accumulated. The results indicate a, so far, undescribed osmotic function of I⁻ in L. digitata and, thus, multifunctional principles of this halogen in kelps. The osmotic function of mannitol may have been substituted by that of I⁻ under hypersaline conditions, suggesting a complementary role of inorganic and organic solutes under salinity stress. This study also provides first evidence that iodine accumulation in L. digitata positively affects photo-physiology.

The methanolic extract of Cordyceps militaris (L.) Link fruiting body shows antioxidant, antibacterial, antifungal and antihuman tumor cell lines properties.

Being Cordyceps militaris (L.) Link recognized as a medicinal and edible mushroom, this work intends to reveal new interesting bioactive molecules that could be isolated from this species. Hydrophilic and lipophilic compounds were analyzed by chromatographic techniques coupled to different detectors. The methanolic extract of C. militaris was tested for its antioxidant, antibacterial, antifungal and anti-proliferative properties in different human tumor cell lines. Mannitol (2.01 g/100 g dw) and trehalose (24.71 g/100 g) were the free sugars found in C. militaris. Polyunsaturated fatty acids (68.87%) predominated over saturated fatty acids (23.40%) and δ-tocopherol was the only isoform of vitamin E detected (55.86 µg/100 g). The organic acids found in this mushroom were oxalic, citric and fumaric acids (0.33, 7.97 and 0.13 g/100 g, respectively). p-Hydroxybenzoic acid was the only phenolic acid quantified in this species (0.02 mg/100 g); although cinnamic acid was also found (0.11 mg/100 g). The methanolic extract of C. militaris proved to inhibit lipid peroxidation, have reducing power and scavenge free radicals. This extract also revealed strong antibacterial and antifungal properties. Finally, the C. militaris extract was able to inhibit the proliferation of MCF-7 (breast), NCI-H460 (non-small lung), HCT-15 (colon) and HeLa (cervical) human carcinoma cell lines.

Effect of electroporation on viability and bioconversion of isoflavones in mannitol-soymilk fermented by lactobacilli and bifidobacteria.

BACKGROUND: The aim of this study was to evaluate the effect of electroporation (2.5-7.5 kV cm⁻¹ for 3.0-4.0 ms) on the growth of lactobacilli and bifidobacteria, membrane properties and bioconversion of isoflavones in mannitol-soymilk. RESULTS: The viability of lactobacilli and bifidobacteria decreased immediately after electroporation. This was attributed to lipid peroxidation, which led to alterations in the membrane phospholipid bilayer, specifically at the polar head, interface and apolar tail regions. Such alterations also resulted in decreased membrane fluidity and increased membrane permeability upon electroporation (P < 0.05). However, the effect was reversible and treated cells showed better growth than the control upon fermentation for 24 h at 37 °C (P < 0.05). Additionally, electroporation increased the bioconversion of glucosides to bioactive aglycones in mannitol-soymilk, which was attributed to increased intracellular and extracellular ß-glucosidase activities of cells upon treatment (P < 0.05). CONCLUSION: Application of electroporation on lactobacilli and bifidobacteria could be beneficial for the development of fermented soymilk with enhanced bioactivity. Considering the enhanced bioactive aglycones, this soymilk could be useful for the prevention of hormone-dependent disorders.

[Study on quality of medicinal material of Thesium chinense].

OBJECTIVE: To study the quality of medicinal material of Thesium chinense. METHOD: The active ingredients of total flavonoids, kaempferol, mannitol, polysaccharides were selected as the evaluative indicators to comprehensively evaluate the quality of medicinal material of T. chinense. RESULT: The total flavone 3.38%, kaempferol 0.984 7 mg x g(-1), mannitol 6.12%, polysaccharides 14.08% and water soluble extract 30.28% were measured in the sample of T. chinense. There was a certain correlation among these selected active ingredients. The compositions of most biological activities component in T. chinense of different populations were similar, but the contents were different significantly. The content of activities component in T. chinense showed a general tendency of declining during growth period and with the increase time of storage. CONCLUSION: Flavone, kaempferol, mannitol, polysaccharides should be selected as the indicators to evaluate the quality of medicinal material of T. chinense.

Identification and quantitative determination of carbohydrates in ethanolic extracts of two conifers using 13C NMR spectroscopy.

We developed a method for the direct identification and quantification of carbohydrates in raw vegetable extracts using (13)C NMR spectroscopy without any preliminary step of precipitation or reduction of the components. This method has been validated (accuracy, precision and response linearity) using pure compounds and artificial mixtures before being applied to authentic ethanolic extracts of pine needles, pine wood and pine cones and fir twigs. We determined that carbohydrates represented from 15% to 35% of the crude extracts in which pinitol was the principal constituent accompanied by arabinitol, mannitol, glucose and fructose.

Quality control of Cordyceps sinensis, a valued traditional Chinese medicine.

Cordyceps sinensis, a well-known and valued traditional Chinese medicine, is also called DongChongXiaCao (winter worm summer grass) in Chinese. It is commonly used to replenish the kidney and soothe the lung for the treatment of fatigue, night sweating, hyposexualities, hyperglycemia, hyperlipidemia, asthemia after severe illness, respiratory disease, renal dysfunction and renal failure, arrhythmias and other heart disease, and liver disease. As the rarity and upstanding curative effects of natural Cordyceps, several mycelial strains have been isolated from natural Cordyceps and manufactured in large quantities by fermentation technology, and they are commonly sold as health food products in Asia. In addition, some substitutes such as Cordyceps militaris also have been used and adulterants also confused the market. Therefore, quality control of C. sinensis and its products is very important to ensure their safety and efficacy. Herein, markers and analytical methods for quality control of Cordyceps were reviewed and discussed.

Antioxidant and immunostimulating activities of the fruiting bodies of Paecilomyces japonica, a new type of Cordyceps sp.

Cordyceps is negative for its many biological activities and a tonic for restoring vital functions in traditional Chinese medicine. In an effort to evaluate the pharmacological effects, including the antiaging effect of the fruiting bodies of the cultivated Paecilomyces japonica fungus, a new type of Cordyceps sp. was investigated. This investigation was focused on ultimately revealing its biologically active principles, its effects on free-radical scavenging enzymes, lipid peroxidation, as well as its immunological functions. As a result, both water and methanol extracts were found to cause not only significant increases in rat liver cytosolic SOD, catalase, and GSEH-px activities, but also a significant decrease in MDA production in TBA reactant assay in rats. The extracts also showed immunostimulating activity as measured by carbon clearance, weight-loaded forced swimming performances, and immobilizing stress in mice. Using bioassay-guided systematic fractionation of the extracts, two pure compounds were isolated as active principles from low molecular-weight fraction, a protein-bound polysaccharide was isolated that showed a marked increase in the liver enzyme activities, as well as a significant inhibition of lipid peroxidation.

[A study on quality standard for herba cistanches].

OBJECTIVE: To establish the quality standard for Herba Cistanches. METHOD: A qualitative identification method by TLC was established for five kinds of active components(acteoside, echinacoside, cistanoside A, betaine and mannitol) and RP-HPLC was used to quantify the acteoside contents. RESULTS: Qualitative and quantitative analyses were carried out for fourteen kinds of Herba Cistanches and eleven kinds of Yinpian. This method is accurate, reliable and of good separability and reproducibility. CONCLUSION: This method can be applied as standard for the quality control of cistanche deserticola.

Comparison of clinical outcome between histidine-triptophan-ketoglutalate solution and cold blood cardioplegic solution in mitral valve replacement.

BACKGROUND: This study was conducted to compare the effect of histidine-triptophan-ketoglutalate solution (HTK) with that of cold blood cardioplegic solution (CBC) in mitral valve surgery. METHOD: Forty-six patients who underwent mitral valve replacement between January 1994 and December 1996 were enrolled in this study. Twenty patients received HTK (HTK group), while 27 patients had CBC (CBC group) as myocardial protection. HTK was given as a single high dose, whereas CBC was used in the usual multidose format. RESULT: The doses of inotropic agent at the end of extracorporeal circulation did not differ between the HTK group and the CBC group. Creatine kinase values (units) on day 1 and day 2 were 1140+/-412, 921+/-436 for the HTK group and 904+/-335, 816+/-420 for the CBC group, respectively (p = NS). Spontaneous defibrillation occurred in 26% of the CBC group and 90% of the HTK group (p < 0.05). Pacing was temporarily used in 20% of the HTK group and 44% of the CBC group after extracorporeal circulation (p < 0.05). CONCLUSIONS: These results suggest that HTK provided more adequate myocardial protection in mitral valve surgery.

Adulteration of soluble coffee with coffee husks and parchments.

Commercial soluble coffee can be adulterated with coffee husks or parchments. Xylose is a good tracer for this type of mispractice. The analysis of total xylose in a wide selection of green beans and the assessment of its fate during processing allowed the derivation of a maximum total xylose limit of 0.40%, above which a soluble coffee should be considered as adulterated. Out of the 700 commercial soluble coffees analyzed, 81 exhibited a total xylose level above this limit. Of the samples with total xylose level lower than the limit, 99% displayed concentrations in free mannitol and total glucose below 0.30 and 2.10%, respectively.

[Study of chemical pattern recognition as applied to quality assessment of the traditional Chinese medicine "wei ling xian"].

Chemical pattern recognition was applied to the quality assessment of the traditional Chinese medicine "wei ling xian". The dried roots and rhizomes of Clematis chinensis Osbeck, Clematis hexapetalo Pall, and Clematis manshurica Rupr, 21 samples of "wei ling xian" of six different species which were collected from different regions of China, were extracted with methanol. The extracts were analyzed by gas chromatography and the GC data of "wei Ling xian" samples were correlated with their anti-inflammatory activity in pharmacologic experiments. Positive anti-inflammatory activity is regarded as the quality assurance of medicinal "wei ling xian". According to the results, the samples were classified as two groups, one having and the other not having distinct anti-inflammatory activity at the 5% significance level. Some samples were used as the training set, while some as the test set. The SIMCA program was used for the extraction of features and classification, whereas the PCA program for displaying the data in a two-dimensional space. The quality of unknown samples of "Wei Ling Xian" can be assessed as a result of the classification or the two-dimensional display.

Pesquisa de adocantes nao caloricos sinteticos em adocante natural de Stevia rebaudiana (BERT.) Bertoni / Investigation of synthetic sweeteners in Stevia rebaudiana (BERT.) Bertoni natural sweetener

Foram analisadas 19 amostras de "Adocantes Naturais de Stevia", tendo por objetivo verificar-se a presenca de outros adocantes nao caloricos sinteticos que nao sao declarados no rotulo do produto. Constatou-se que em 18 amostras, alem de componentes do extrato de Stevia rebaudiana (BERT.) Bertoni, a sacarina estava presente e que so uma delas declarava tal fato no rotulo. Em tres dessas amostras foi encontrado tambem aspartame e em uma, ciclamato. Finalmente, em uma amostra nao foi encontrado nenhum componente do extrato de Stevia rebaudiana (BERT.) Bertoni, mas sim uma mistura de manitol e aspartame.