Cloning and Functional Characterization of Two Germacrene A Oxidases Isolated from Xanthium sibiricum.

Sesquiterpene lactones (STLs) from the cocklebur Xanthium sibiricum exhibit significant anti-tumor activity. Although germacrene A oxidase (GAO), which catalyzes the production of Germacrene A acid (GAA) from germacrene A, an important precursor of germacrene-type STLs, has been reported, the remaining GAOs corresponding to various STLs' biosynthesis pathways remain unidentified. In this study, 68,199 unigenes were studied in a de novo transcriptome assembly of X. sibiricum fruits. By comparison with previously published GAO sequences, two candidate X. sibiricum GAO gene sequences, XsGAO1 (1467 bp) and XsGAO2 (1527 bp), were identified, cloned, and predicted to encode 488 and 508 amino acids, respectively. Their protein structure, motifs, sequence similarity, and phylogenetic position were similar to those of other GAO proteins. They were most strongly expressed in fruits, according to a quantitative real-time polymerase chain reaction (qRT-PCR), and both XsGAO proteins were localized in the mitochondria of tobacco leaf epidermal cells. The two XsGAO genes were cloned into the expression vector for eukaryotic expression in Saccharomyces cerevisiae, and the enzyme reaction products were detected by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) methods. The results indicated that both XsGAO1 and XsGAO2 catalyzed the two-step conversion of germacrene A (GA) to GAA, meaning they are unlike classical GAO enzymes, which catalyze a three-step conversion of GA to GAA. This cloning and functional study of two GAO genes from X. sibiricum provides a useful basis for further elucidation of the STL biosynthesis pathway in X. sibiricum.

[Changes of transport sugar content in different organs of Rehmannia glutinosa].

Raffinose series oligosaccharides are the transport and storage sugars of many plants, Rehmannia glutinosa is one of the commonly used Chinese herbal medicines, medicinal parts ist he roots. Root and tuber of R. glutinosa contains stachyose, raffinose and other oligosaccharides, but the study about the process of growth and development of other organs in the non-structural changes in sugar content is rare.In this study, leaves, stems and roots of R. glutinosa were used as materials to analyze the diurnal variation and the changes of sugar content of sucrose, raffinose and stachyose in different organs of R. glutinosa. The results showed that the content of sucrose in R. glutinosa leaves gradually increased from seedling stage.However, the content of stachyose did not change much at the early stage of growth, and the stachyose rapidly increased at the later stage of growth. The raffinose content gradually decreased throughout the growing season, young leaves of R. glutinosa have higher ability to sucrose synthesis than mature leaves, while mature leaf has higher raffinose and stachyose synthesis ability than young leaves. Sucrose and stachyose content in stem gradually increased, while there was little change in raffinose content. The content of raffinose and stachyose in root increased rapidly from the beginning of fast growing period, while the content of sucrose did not change much. The content of sucrose in leaves of R. glutinosa did not change much at day and night, while the daily changes of raffinose and stachyose contents were very obvious. The contents of raffinose and stachyose in daytime were higher than those at night. The content of raffinose in root and stem was not changed much, but the change of stachyose in root, stem and leaf was very obvious, especially in stem and leaf. In summary, the leaf is the main synthetic organ of raffinose, leaves, stems and roots are stachyose synthesis organ. Sucrose, raffinose and stachyose are the major transport forms of carbohydrates in R. glutinosa.

[A simple method for quantification of tapentadol in dog plasma by liquid chromatography-tandem mass spectrometry and evaluation of the effects of conjugated metabolites on tapentadol].

Tapentadol is a novel drug of opioid pain reliever, which is extensively metabolized primarily through conjugation. Tapentadol glucuronide and tapentadol sulfate are major drug-related metabolites in circulation. The objectives of this study were to develop a simple and rapid method to determine tapentadol and evaluate the effects of conjugated metabolites on tapentadol quantification using liquid chromatography with tandem mass spectrometry in dog plasma. The analyte and tramadol（IS） were extracted from plasma by protein precipitation with methanol, and chromatographied on a XDB C(18)（50 mm × 4.6 mm, 1.8 µm） column using a mobile phase of methanol and 5 mmol·L(-1) ammonium acetate（0.01% ammonia）. Mass spectrometric detection was performed using the m/z 222 → 121 transition for tapentadol and the m/z 264 → 58 transition for the internal standard tramadol, the m/z 398 → m/z 121 transition for glucuronides conjugate and the m/z 302 → m/z 222 transition for sulfate conjugate. Conjugated metabolites could undergo in-source conversion to generate an ion that interfered the quantification of tapentadol. Chromatographic separation was achieved to elimination interferences due to in-source conversion of the conjugated metabolites. The standard curves were demonstrated to be linear in the range of 0.100 to 20.0 ng·m L(-1) for tapentadol. The intra- and inter-day precisions were within 5.1%, and accuracy ranged from -3.2% to 0. This method was successfully applied to the pharmacokinetics of tapentadol hydrochloride sustained release tablets in Beagle dogs.

[Identification of six species of medicinal Diospyros plants based on leaf macro- and micro-morphology].

To establish a method for the identification of five species and one variety of medicinal plants from Diospyros, their leaf veins, epidermis, anatomic and powder characters were observed and compared with macro-morphological and microscopic methods. The results indicated the differences of secondary and tertiary veins among those Diospyros species. The single cell non-glandular hair and glandular hair exist in most species' epidermis while stone cells were only found in the leaf powders of two species. Through the study, the main differences of leaf macro- and micro-morphology of these species were obtained and practical keys were also established, which can provide scientific base not only for identification of these species during their vegetative stages, but also for accuracy authentication of the source of Kaki Folium.

[Morphological and cytohistological observations of seed germination and protocorm development of Bletilla striata].

In order to investigate the mechanism of growth for Bletilla striata, which could be applied for rapid propagation, morphological and cytohistological of seed germination and protocorm development in vitro culture were observed using paraffin section techniques. In this study, we have found that the development of B. striata goes through four stages： embryo, protocorm, rhizome and pseudobulb. The end away from embryo suspensor is able to differentiate green buds after the seed of B. striata swelling, growing point. At the same time, the other end of embryo grows many white villous roots, with the green bud differentiating into cotyledon, the embryo breaking through seed coat and being protocorm. The shoot apical meristem of protocorm consists of tunica, corpus and leaf primordium, whose developmental flowing tunica-corpus theory. After more vascular bundle appeared from the leaf primordium, B. striata grows into the stage of rhizome. While in the stage of rhizome, the root primordium of tissue culture seedlings are differentia initially that derived from rhizome vascular bundle, belonging to internal origin. Subsequently, the pseudobulb forms by the inner meristem growing into mature parenchymatous tissue and the rhizome enlargement gradually.

Ilexgenin A inhibits endoplasmic reticulum stress and ameliorates endothelial dysfunction via suppression of TXNIP/NLRP3 inflammasome activation in an AMPK dependent manner.

Ilexgenin A is a natural triterpenoid with beneficial effects on lipid disorders. This study aimed to investigate the effects of ilexgenin A on endothelial homeostasis and its mechanisms. Palmitate (PA) stimulation induced endoplasmic reticulum stress (ER stress) and subsequent thioredoxin-interacting protein (TXNIP)/NLRP3 inflammasome activation in endothelial cells, leading to endothelial dysfunction. Ilexgenin A enhanced LKB1-dependent AMPK activity and improved ER stress by suppression of ROS-associated TXNIP induction. However, these effects were blocked by knockdown of AMPKα, indicating AMPK is essential for its action in suppression of ER stress. Meanwhile, ilexgenin A inhibited NLRP3 inflammasome activation by down-regulation of NLRP3 and cleaved caspase-1 induction, and thereby reduced IL-1ß secretion. It also inhibited inflammation and apoptosis exposed to PA insult. Consistent with these results in endothelial cells, ilexgenin A attenuated ER stress and restored the loss of eNOS activity in vascular endothelium, and thereby improved endothelium-dependent vasodilation in rat aorta. A further analysis in high-fat fed mice showed that oral administration of ilexgenin A blocked ER stress/NLRP3 activation with reduced ROS generation and increased NO production in vascular endothelium, well confirming the beneficial effect of ilexgenin A on endothelial homeostasis in vivo. Taken together, these results show ER stress-associated TXNIP/NLRP3 inflammasome activation was responsible for endothelial dysfunction and ilexgenin A ameliorated endothelial dysfunction by suppressing ER-stress and TXNIP/NLRP3 inflammasome activation with a regulation of AMPK. This finding suggests that the application of ilexgenin A is useful in the management of cardiovascular diseases in obesity.

[Herbal textural research on species of Xanthii Fructus].

Xanthii Fructus is a traditional medicine for the treatment of nasal diseases in clinic, mainly come from the burs of Xanthium sibiricum with a worldwide distribution. By sorting and studying literature of Chinese medicine and comparing different figures recorded with the morphological description of several species from Xanthium (Asteraceae) in the Flora of China, combining the biological investigation in resource survey, the article pointed out that the burs or the whole herbs of X. mongolicum, as well as X. sibiricum, has been used by the traditional Chinese medicine in ancient time. It provides a reference for further studies in the future.

Dynamic changes of flavonoids contents in the different parts of rhizome of Belamcanda chinensis during the thermal drying process.

The dried rhizome of Belamcanda. chinensis (L.) DC. is an important traditional Chinese medicine. Previous chemical and pharmacological investigations indicated that flavonoids may be responsible for the bioactivity of the herb. In this paper, the effects on the contents of twelve flavonoids in the three subunit parts of the rhizome of B. chinensis during the thermal drying process under treatment temperatures ranging from 40 °C to 120 °C at 10 °C intervals were investigated. The results showed that the content of most of the individual flavonoids except that of tectorigenin in the fresh eldest parts of the rhizome that originate directly from the seedling was higher than those of the other junior parts. The change trends of flavonoids contents were similar for three subunit parts of the rhizome during the drying process under the same treatment temperature. Most of the individual flavonoid contents in the rhizome increased in the early stages of the drying processes and decreased as the process was prolonged. The durations required to reaching the points of the maximal amounts of flavonoids revealed a significant negative correlation with the temperature. The variation of the content of mangiferin, iristectorigenin A, irigenin, irilone and dichotomitin was positively correlated with irisflorentin that is the chemical marker used for the quality control of this herb. Taking into account of the production effectiveness and flavonoid yields, the appropriate drying temperature for this herb was suggested to be 100 °C.

Post-harvest induced production of salvianolic acids and significant promotion of antioxidant properties in roots of Salvia miltiorrhiza (Danshen).

Danshen, the dried roots of Salvia miltiorrhiza, is an extremely valued Traditional Chinese Medicine. Previously, we have demonstrated that salvianolic acid B (SaB), the important bioactive ingredient in this herb, was a post-harvest product. Here, we further reported that all salvianolic acids (SAs) in the roots were post-harvest products of the drying process. In addition, the results of various radical scavenging activity assays, including lipid peroxidation (1), DPPH (2), hydroxyl (3) and superoxide (4), were significantly increased along with the accumulation of total salvianolic acids in the process. The contents of chemical targets and antioxidant activities both reached the highest value under thermal treatment at 130 °C for 80 min. In this dehydration period, contents of SaB, and sum of nine SAs increased from 0.01% to 5.51%, and 0.20% to 6.61%; and IC50 of antioxidant activity decreased from 4.85 to 2.69 (1); 7.75 to 0.43 (2); 2.57 to 1.13 (3) and 17.25 to 1.10 mg/mL. These results further supported the hypothesis that the newly harvested plant roots were still physiologically active and the secondary metabolites might be produced due to dehydration stress after harvest. Our findings supplied an important and useful theoretical basis for promoting the quality of Danshen and other medicinal plant materials.

[Pharmacodynamic differences between hangmaidong and chuanmaidong based on metabonomics].

To evaluate the differences of Ophiopogonjaponicus from different cultivations, the metabolomics based method was conducted to compare the effects of Hangmaidong and Chuanmaidong (Chinese name) on plasma endogenous metabolites of normal rats. Data were collected by ultra performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-Q-TOF/MS), and were analyzed by multivariate statistical method, such as Principal Component Analysis and Orthogonal Signal Correction Partial Least Square Discriminant Analysis. Results revealed that the plasma metabolites profiling of low and middle dose group of Chuanmaidong were similar to the control group, but different from the high dose group obviously. Meanwhile the high, middle and low dose groups of Hangmaidong were different from control group notably, and the difference is dose dependent. Lysophosphatidylcholines, the possible endogenous metabolites which contribute to the classification most significantly, are closely related to cardiovascular system diseases. Compared with the group of Chuanmaidong, Hangmaidong has greater impact on the plasma metabolic profiling of normal rats. Hangmaidong and Chuanmaidong showed significant differences pharmacodynamically.

[Proposal for standardized authors' name citing in original plant Latin name listed in the Chinese Pharmacopoeia].

In 2010, Chinese Pharmacopoeia Committee officially enacted Chinese Pharmacopoeia (2010 edition). The Volume 1 of the pharmacopoeia is comprised of the medicinal materials and the decoction pieces, the essential oils and extracts of medicinal plants, prescription preparations and single preparation, etc., which not only provides Latin names of Chinese medicinal materials, also provided Latin names of the original medicinal plants to effectively control the quality of Chinese medicinal materials. In order to raise awareness of correctly citation and maintain the authority and standardization of Chinese Pharmacopoeia, this paper briefly describes abbreviations rules of authors' name of plant scientific name according to the 'International Code of Botanical Nomenclature, ICBN'. Through comparing with the rules of ICBN, 'Flora of China' (Chinese edition and English edition), and authority international plant catalogue databases, the authors made statistic and analysis of the non-standard cited authors' names phenomena of the original plant scientific names recorded in the Chinese Pharmacopoeia (2010 edition), and the revision suggestions are proposed.

[Study on chemical constituents from leaf of Bombax ceiba (II)].

OBJECTIVE: To study the chemical constituents from the leaf of Bombax ceiba. METHODS: The compounds were isolated and purified with silica gel and Sephadex LH-20 column chromatography. Their structures were determined on the basis of physicochemical properties and spectroscopic analysis. RESULTS: Eleven compounds were isolated and identified as taraxeryl acetate (1), squalene (2), taraxerone (3), beta-sitosterol palmitate (4), taraxerol (5), 4-methyl stigmast-7-en-3-ol (6), 1H-indole-3-carboxylic acid (7), 6-O-palmitoylsitosteryl-D-glucoside (8), 12beta-hydroxyl-pregnane-4, 16-diene-3, 20-dione (9), loliolide (10) and 5-(hydroxymethyl) furfural (11). CONCLUSION: All the compounds are isolated from this genus for the first time.

[Chemical constituents from rhizomes of Iris germanica].

Twenty-one compounds were isolated from the rhizomes of Iris germanica by various chromatographic techniques such as silica gel, ODS and Sephadex LH-20 chromatography. Their structures were established on basis of physical properties, MS and NMR spectroscopic data Their structures were identified as ombuin (1), 5, 3, 3'-trihydroxy-7, 4'-dimethoxyflavanone (2), naringenin (3), cirsiliol-4'-glucoside (4), 3beta, 4'-dihydroxy-7,3'-dimethoxyflavonone-5-O-beta-D-glucopyranoside (5), genistein (6), irilin D (7), muningin (8), 5, 7, 4'-trihydroxy-6, 3', 5'-trimethoxyisoflavone (9), tectorigenin (10), irigenin (11), tectoridin (12), iridin (13), mangiferin (14), irisxanthone (15), pyroglutamic acid (16), 2, 4', 6-trihydroxy-4-methoxybenzophenone-2-O-beta-D-glucoside (17), apocynin (18), androsin (19), beta-sitosterol (20), and daucosterol (21). Among them, compounds 1-9, 16, 17 were obtained from this plant for the first time, compounds 8 and 9 were separated from Iris species for the first time, compounds 1, 4, and 17 were obtained from the family for the first time.

[Fermentation of Bacillus subtilis ge25 strain and preliminary study on its antagonistic substances].

Panax ginseng is one of the most important traditional Chinese herbal medicine, soil borne diseases influenced the yield and quality severely. In our previous work, endophytic Bacillus subtilis ge25 strain was isolated from ginseng root, and which showed significant antagonistic activity against several most destructive ginseng phytopathogens. In the present work, crude protein and lipopeptid extracts were prepared from LB and Landy supernate by salting out, acid precipitation methods respectively. The antagonistic activity of crude extracts and stability to temperature and protease digestion were examined by ginseng phytopathogen Alternaria panax. Results showed that, the antagonistic activity of crude protein extracts from LB culture was complete and partially lost when treated by high temperature and proteinase K. However, crude lipopeptid from Landy culture showed significant stabile antagonistic activity to them. Acid-hydrolyzation and TLC-bioautography analysis showed, that the crude lipopeptide contained at least one cyclic lipopeptide. In consideration of the stability and perfect antagonistic activity of ge25, further researches will promote the biocontrol of ginseng diseases in the field.

[AS-PCR amplification in identification of Sedum sarmentosum with its relative species].

OBJECTIVE: To develop a scientific and effective method for identification of Sedum sarmentosum and its relative materials easily confused. METHODS: DNA templates were extracted from plants, and DNA fragments of cpDNA trnL (tRNA-Leu) gene and trnL-trnF intergenic spacer were amplified and sequenced subsequently. trnL-trnF sequences of all samples were aligned. RESULTS: The allele specific PCR method was studied with the designed allele-specific for distinguishing different samples. The result indicated that 300 bp and 500 bp DNA fragments were amplified from Sedum sarmentosum, where no any fragment was amplified from its relative species under the same reaction condition. CONCLUSION: The primers designed in this study are specific for Sedum sarmentosum from its relative species.

[Chemical constituents of Gentiana rhodantha].

OBJECTIVE: To determine the chemical constituents of Gentiana rhodantha. METHOD: To isolate the constituents, column chromatography over silica gel, MCI, Sephadex LH-20 and C18 reverse-phased silica gel were used. Spectroscopic methods were used to elucidate the structures of the isolated compounds. RESULT: Sixteen compounds were isolated and elucidated as ten phonemic compounds, namely 1,3,7,8-tetrahydroxylxanthone (1), rhodanthenone D (2), 1,3,6,7-tetrahydroxylxanthone (3), 1,3,7-trihydroxy-4,8-dimethoxyxanthone (4), quercetin (5), isoorientin (6), mangiferin (7), norswertianolin (8), gallic acid ethyl ester (9) and salicylic acid (10), and six triterpenes including alpha-amyrin (11), erythrodiol 3-O-palmitate (12), ursolic aldehyde (13), uvaol 3-O-acetyl (14), ursolic acid (15) and 2alpha-hydroxyursolic acid (16). CONCLUSION: Compounds 4-6, 8, 10-12, 15 and 16 were isolated from this plant for the first time. Compounds 1 and 3 were obtained firstly from the genus Gentiana and compounds 9, 13-14 were firstly from the family Gentianaceae.

[Chemical constitunents of seeds of Oroxylum indicum].

OBJECTIVE: To study the chemical constituents in the seeds of Oroxylum indicum. METHOD: Twenty compounds were isolated and purified by silica gel, and Sephadex LH-20 column chromatography, and their structures were determined by spectroscopic analysis including NMR and MS. RESULT: Twenty compounds were isolated and identified as oroxin A (1), oroxin B (2), chrysin (3), baicalein (4), quercetin (5), apigenin (6), kaempferol (7), quercetin-3-O-ara-binopyranoside (8), lupeol C9), lup-20 (29)-ene-2alpha,3beta-diol (10), pinosylvin (11), dihydropinosylvin (12), cholest-5-ene-3, 7-diol (13), rengyol (14), isorengyol (15), zarzissine (16), (E) -pinosylvin-3-O-beta-D-glucopyranoside (17), adenosine (18), sitosterol (19) and daucosterol (20). CONCLUSION: Compounds 11-13 and 15-18 were obtained from the genus Oroxylum for the first time, and except compound 18, the remaining 6 compounds were obtained from the family Bignoniaceae for the first time.

A new eudesmane sesquiterpene glycosides from Liriope muscari.

A new eudesmane sesquiterpene glycoside, ophiopogonoside B (1), along with five known compounds, ophiopogonoside A (2), ruscogenin-1-O-[ß-D-glucopyranosyl (1 → 2)]-[ß-D-xylopyranosyl (1 → 3)]-ß-D-fucopyranoside (3), palmitic acid (4), palmitic acid glyceride (5), and ß-sitosterol-D-glucopyranoside (6),was isolated from the tuberous roots of Liriope muscari (Decn.) Bailey (Liliaceae). Their structures were confirmed by 1D and 2D NMR spectroscopy. Among them, compounds 1, 2, 4, and 5 were the first reported from the genus Liriope. Ophiopogonoside B (1) showed moderate inhibitory activity to glycogen phosphorylase a.

[Determination of the content of the main components of flavonoids compounds in raw malt, torrefied malt and ustulate malt].

OBJECTIVE: To determine the content of Catechin, Myricetin, Quercetin and Kaempferol in barley grain, raw malt, torrefied malt and ustulate malt based on different barley cultivars. METHODS: HPLC method was used. Analysis was performed on Agilent ZORBAXSB-C18 (150 mm x 4. 6 mm, 3.5 microm) column with acetonitrile-0.1% acetic acid as mobile phase. The detection wavelength was 280 nm, flow rate was 0.8 mL/min, and the column temperature was 30 degrees C. RESULTS: Catechin was the main component of barley seeds and its processed products. Slight reduction of catechin was observed in processed and sprouting seeds. Sprouting significantly increased the content of myricetin. Both barley seeds and the processed products were lack of quercetin. The amounts of kaempferol in seed were higher than that in barley grain, but similar to that in ustulate malt. CONCLUSION: The content of flavonoids in raw malt and torrefied malt are significantly affected by sprouting and processing, and significance differences are presented among different varieties.

New features on the fragmentation patterns of homoisoflavonoids in Ophiopogon japonicus by high-performance liquid chromatography/diode-array detection/electrospray ionization with multi-stage tandem mass spectrometry.

Homoisoflavonoids, a special class of flavonoids, are mainly distributed in the Liliaceae family and have various biological activities. Previously, very little research has been reported on the gas-phase fragmentation patterns of homoisoflavonoids by electrospray ionization mass spectrometry. In this paper, we report the use of high-performance liquid chromatography with a diode-array detector (HPLC-DAD) and electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)) to study the fragmentation behavior of 11 homoisoflavonoid standards and to analyze homoisoflavonoids in Ophiopogon japonicus. In total, 28 homoisoflavonoids (including seven novel constituents) were characterized. The deprotonated [M--H](-) molecules of the homoisoflavonoids containing a saturated C2--C3 bond afforded the A or B product ion (base peak) according to whether the B-ring was substituted with a hydroxyl group. For the homoisoflavonoids containing a C-2-C-3 double bond, the product ions (A or C ion) were created from the precursor [M-H](-) ion as the base peak when the B-ring was substituted with a hydroxyl group. The homoisoflavonoids carrying a formyl group in the A-ring readily eliminated one molecule of CO to form the product ion [M + H-CO](-) (base peak) irrespective whether the C-2-C-3 bond was saturated or not. This product ion afforded the [M-H-CO-B-ring--CH(2) + H](-) ion by cleavage of the C3-C9 bond. This latter product ion always appeared in tandem mass (MS/MS) spectra of type I homoisoflavonoids. The common features of flavonoids observed during the gas-phase fragmentation mechanisms were the loss of the following groups: 15 Da (CH(3)), 18 Da (H(2)O), 28 Da (CO), 44 Da (CO(2)) and 46 Da (CH(2)O(2)). A retro-Diels-Alder (RDA)-like cleavage was also observed for the homoisoflavonoids. The different gas-phase fragmentation routes were characterized for the deprotonated molecules obtained from the various homoisoflavonoids and collision-induced dissociation (CID) fragmentation differences were noted for the different locations of the various substituents. In conclusion, we can say that this study allowed us to structurally elucidate and identify homoisoflavonoids distributed in related plants and their complex prescriptions.