[Simultaneous determination of seven artemisinin-related compounds in Artemisia annua by UPLC-QQQ-MS/MS].

A variety of compounds in Artemisia annua were simultaneously determined to evaluate the quality of A. annua from multiple perspectives. A method based on ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry(UPLC-QQQ-MS/MS) was established for the simultaneous determination of seven compounds: amorpha-4,11-diene, artemisinic aldehyde, dihydroartemisinic acid, artemisinic acid, artemisinin B, artemisitene, and artemisinin, in A. annua. The content of the seven compounds in different tissues(roots, stems, leaves, and lateral branches) of A. annua were compared. The roots, stems, leaves, and lateral branches of four-month-old A. annua were collected and the content of seven artemisinin-related compounds in different tissues was determined. A multi-reaction monitoring(MRM) acquisition mode of UPLC-QQQ-MS/MS was used, with a positive ion mode of atmospheric pressure chemical ion source(APCI). Chromatographic separation was achieved on an Eclipse Plus RRHD C_(18) column(2.1 mm×50 mm, 1.8 µm). The gradient elution was performed with the mobile phase consisted of formic acid(0.1%)-ammonium formate(5 mmol·L~(-1))(A) and the methanol(B) gradient program of 0-8 min, 55%-100% B, 8-11 min, 100% B, and equilibrium for 3 min, the flow rate of 0.6 mL·min~(-1), the column temperature of 40 ℃, the injection volume of 5 µL, and the detection time of 8 min. Through methodological investigation, a method based on UPLC-QQQ-MS/MS was established for the simultaneous quantitative determination of seven representative compounds involved in the biosynthesis of artemisinin. The content of artemisinin in A. annua was higher than that of artemisinin B, and the content of artemisinin and dihydroartemisinic acid were high in all the tissues of A. annua. The content of the seven compounds varied considerably in different tissues, with the highest levels in the leaves and neither artemisinene nor artemisinic aldehyde was detected in the roots. In this study, a quantitative method based on UPLC-QQQ-MS/MS for the simultaneous determination of seven representative compounds involved in the biosynthesis of artemisinin was established, which was accurate, sensitive, and highly efficient, and can be used for determining the content of artemisinin-related compounds in A. annua, breeding new varieties, and controlling the quality of Chinese medicinal materials.

[Chemical constituents from Pulsatillae Radix].

Chemical constituents of ethanol extract of Pulsatillae Radix were investigated. The n-butanol fraction of ethanol extract of Pulsatillae Radix was isolated and purified by macroporous resin and silica gel column chromatography and semi-preparative high performance liquid chromatography. The triterpenoid glycosides were identified by multiple spectral methods. Six compounds were obtained from the n-butanol fraction of ethanol extract of Pulsatillae Radix and identified as 23-aldehyde-cussosaponin C(1), cussosaponin C(2), anemoside B4(3), akebia saponin D(4), pulchinenoside E3(5), and hederacoside C(6). Among them, compound 1 was a new compound.

Integration of the metabolome and transcriptome reveals indigo biosynthesis in Phaius flavus flowers under freezing treatment.

Background: Indigo-containing plant tissues change blue after a freezing treatment, which is accompanied by changes in indigo and its related compounds. Phaius flavus is one of the few monocot plants containing indigo. The change to blue after freezing was described to explore the biosynthesis of indigo in P. flavus. Methods: In this study, we surveyed the dynamic change of P. flavus flower metabolomics and transcriptomics. Results: The non-targeted metabolomics and targeted metabolomics results revealed a total of 98 different metabolites, the contents of indole, indican, indigo, and indirubin were significantly different after the change to blue from the freezing treatment. A transcriptome analysis screened ten different genes related to indigo upstream biosynthesis, including three anthranilate synthase genes, two phosphoribosyl-anthranilate isomerase genes, one indole-3-glycerolphosphate synthase gene, five tryptophan synthase genes. In addition, we further candidate 37 cytochrome P450 enzyme genes, one uridine diphosphate glucosyltransferase gene, and 24 ß-D-glucosidase genes were screened that may have participated in the downstream biosynthesis of indigo. This study explained the changes of indigo-related compounds at the metabolic level and gene expression level during the process of P. flavus under freezing and provided new insights for increasing the production of indigo-related compounds in P. flavus. In addition, transcriptome sequencing provides the basis for functional verification of the indigo biosynthesis key genes in P. flavus.

[Research progress on mechanism of phytohormones in regulating flavonoid metabolism].

Phytohormones play an important role at all stages of plant growth, influencing plant growth and development and regulating plant secondary metabolism, such as the synthesis of flavone, flavonol, anthocyanin, and other flavonoids. Flavonoids, a group of important secondary metabolites ubiquitous in plants, have antioxidative, anti-microbial, and anti-inflammatory activities and thus have a wide range of potential applications in Chinese medicine and food nutrition. With the development of biotechnology, phytohormones' regulation on flavonoids has become a research focus in recent years. This study reviewed the research progress on the mechanism of common phytohormones, such as abscisic acid, gibberellin, methyl jasmonate, and salicylic acid, in regulating flavonoid metabolism, and discussed the molecular mechanism of the synthesis and accumulation of flavonoids, aiming at clarifying the key role of phytohormones in modulating flavonoid metabolism. The result is of guiding significance for improving the content of flavonoids in plants through rational use of phytohormones and of reference value for exploring the mechanism of hormones in regulating flavonoid metabolism.

Paeoniflorin exerts antidepressant-like effects through enhancing neuronal FGF-2 by microglial inactivation.

Ethnopharmacological relevance Paeonia lactiflora is a famous Traditional Chinese medicine widely used for immunological regulation. Paeoniflorin, the main component of Paeonia lactiflora, exerts neuroprotective and antidepressant-like effects in rodents. AIM OF THE STUDY: Fibroblast growth factor 2 (FGF-2) is essentially required in the central nervous system as it acts as both a neurotrophic factor and an anti-inflammatory factor participating in the regulation of proliferation, differentiation and apoptosis of neurons in the brain. However, it is unclear whether paeoniflorin could exert antidepressant effects via regulating FGF-2. MATERIALS AND METHODS: In the present study, the effects of paeoniflorin were evaluated in depressive mice induced by the endotoxin lipopolysaccharide (LPS) injection. RESULTS: The results showed that paeoniflorin markedly increased sucrose preference and reduced immobility time in LPS mice, indicating antidepressant effects. Consistent with the results from molecular docking showing paeoniflorin antagonizes TLR4, NF-κB and NLRP3, the biochemical analysis also indicated paeoniflorin inhibited TLR4/NF-κB/NLRP3 signaling, decreased proinflammatory cytokine levels and microglial activation in the hippocampus of LPS induced mice. In addition, the levels of neuronal FGF-2 and the density of dendritic spine were improved by paeoniflorin. More importantly, the FGFR1 inhibitor SU5402 prevented the antidepressant effects of paeoniflorin and blocked the neuroinflammatory and neurogenic regulatory effects of paeoniflorin, indicating that FGF-2/FGFR1 activation was required for the effects of paeoniflorin. CONCLUSION: Taken together, the results demonstrate that paeoniflorin exhibits neuroprotective and antidepressant effects in mice, which may be mediated by activating neuronal FGF-2/FGFR1 signaling via the inhibition of microglial activation in the hippocampus.

Boosting photocatalytic degradation of antibiotic wastewater by synergy effect of heterojunction and phosphorus doping.

Phosphorus-doped g-C3N4/ZnIn2S4 (PCN/ZIS) heterojunction photocatalysts were constructed by solvothermal method. The physical and chemical properties were investigated with X-ray diffraction (XRD), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), UV-vis diffuse reflectance spectroscopy (DRS), etc. The degradation of antibiotic wastewater was used to investigate the photocatalytic activities of composites under visible-light irradiation. The 10% PCN/ZIS had the best photocatalytic degradation performance for tetracycline with a photodegradation rate of 0.0874 min-1, which is respectively about 2.9 and 52.0 times than that of pure ZIS and PCN. Meanwhile, it was concluded that the holes and ⋅O2- (superoxide radicals) play dominant roles in the photocatalytic reactions through radicals trapping experiments, while ⋅OH (hydroxyl radicals) has a negative effect. In addition, 10%PCN/ZIS, with excellent stability and recyclability, also exhibited high photocatalytic activity for terramycin, chlortetracycline and ofloxacin. Overall, with the enhanced photocatalytic performance, PCN/ZIS could be potentially applied for photocatalytic degradation of antibiotic wastewater.

[Advances in biosynthesis of indigo in plants].

Natural indigo, as one of the oldest dyes, is also a pivotal dye utilized in cotton fabrics today. A diversity of plants rich in indigo compounds belong to traditional Chinese herbal medicines. Indigo compounds have a variety of biological and pharmacological activities, including anticonvulsant, antibacterial, antifungal, antiviral and anticancer activities. A substantial progress in indigo biosynthesis has been made lately. This paper summarizes the value of indigo from the aspects of cultural history, biosynthetic pathways and the medicinal activities of its related derivatives involved in the pathways. In addition, the latest research advancements in indigo biosynthetic pathways is demonstrated in this paper, which would lay the theoretical foundation for the exploration and utilization of natural indigo.

[Molecular genetics of medicinal plants, past achievement, and new era].

Unraveling the genetic basis of medicinal plant metabolism and developmental traits is a long-standing goal for pharmacologists and plant biologists. This paper discusses the definition of molecular genetics of medicinal plants, which is an integrative discipline with medicinal plants as the research object. This discipline focuses on the heredity and variation of medicinal plants, and elucidates the relationship between the key traits of medicinal plants(active compounds, yield, resistance, etc.) and genotype, studies the structure and function, heredity and variation of medicinal plant genes mainly at molecular level, so as to reveal the molecular mechanisms of transmission, expression and regulation of genetic information of medicinal plants. Specifically, we emphasize on three major aspects of this discipline.(1)Individual and population genetics of medicinal plants, this part mainly highlights the genetic mechanism of the domestication, the individual genomics at the species level, and the formation of genetic diversity of medicinal plants.(2)Elucidation of biosynthetic pathways of active compounds and their evolutionary significance. This part summarizes the biosynthesis, diversity and molecular evolution of active compounds in medicinal plants.(3) Molecular mechanisms that shaping the key agronomic traits by internal and external factors. This part focuses on the accumulation and distribution of active compounds within plants and the regulation of metabolic network by environmental factors. Finally, we prospect the future direction of molecular genetics of medicinal plants based on the rapid development of multi-omics technology, as well as the application of molecular genetics in the future strategies to achieve conservation and breeding of medicinal plants and efficient biosynthesis of active compounds.

N,N'-Diacetyl-p-phenylenediamine restores microglial phagocytosis and improves cognitive defects in Alzheimer's disease transgenic mice.

As a central feature of neuroinflammation, microglial dysfunction has been increasingly considered a causative factor of neurodegeneration implicating an intertwined pathology with amyloidogenic proteins. Herein, we report the smallest synthetic molecule (N,N'-diacetyl-p-phenylenediamine [DAPPD]), simply composed of a benzene ring with 2 acetamide groups at the para position, known to date as a chemical reagent that is able to promote the phagocytic aptitude of microglia and subsequently ameliorate cognitive defects. Based on our mechanistic investigations in vitro and in vivo, 1) the capability of DAPPD to restore microglial phagocytosis is responsible for diminishing the accumulation of amyloid-ß (Aß) species and significantly improving cognitive function in the brains of 2 types of Alzheimer's disease (AD) transgenic mice, and 2) the rectification of microglial function by DAPPD is a result of its ability to suppress the expression of NLRP3 inflammasome-associated proteins through its impact on the NF-κB pathway. Overall, our in vitro and in vivo investigations on efficacies and molecular-level mechanisms demonstrate the ability of DAPPD to regulate microglial function, suppress neuroinflammation, foster cerebral Aß clearance, and attenuate cognitive deficits in AD transgenic mouse models. Discovery of such antineuroinflammatory compounds signifies the potential in discovering effective therapeutic molecules against AD-associated neurodegeneration.

Polyoxygenated cyathane diterpenoids from the mushroom Cyathus africanus, and their neurotrophic and anti-neuroinflammatory activities.

In a previous study, we reported ten new polyoxygenated cyathane diterpenoids, neocyathins A-J, and their anti-neuroinflammatory effects from the liquid culture of the medicinal Basidiomycete Cyathus africanus. In the present study, eight new highly polyoxygenated cyathane diterpenoids, named neocyathins K-R (1-8), were isolated from the solid culture of C. africanus cultivated on cooked rice, together with three known congeners (9-11). The structures and the absolute configurations of the new compounds were elucidated through comprehensive NMR and HRESIMS spectroscopic data, electronic circular dichroism (ECD) data, and chemical conversion. Compounds 1 and 2 represent the first reported naturally occurring compounds with 4,9-seco-cyathane carbon skeleton incorporating an unprecedented medium-sized 9/7 fused ring system, while the 3,4-seco-cyathane derivative (3) was isolated from Cyathus species for the first time. All compounds were evaluated for their neurotrophic and anti-neuroinflammatory activity. All the isolates at 1-25 µM displayed differential nerve growth factor (NGF)-induced neurite outgrowth-promoting activity in PC-12 cells, while one of the compounds, allocyathin B2 (11), inhibited NO production in lipopolysaccharide (LPS)-stimulated microglia BV-2 cells. In addition, molecular docking studies showed that compound 11 generated interactions with the inducible nitric oxide synthase (iNOS) protein.

Cytotoxic indole alkaloids from Nauclea orientalis.

A new indole alkaloid, nauclorienine (1), along with seven known alkaloids (2-8), were isolated from the stems and leaves of Nauclea orientalis. Among them, nauclorienine (1) was a new indole alkaloid holding a rare corynanthe-type skeleton, and the known compounds (2-8) were isolated from N. orientalis for the first time. Their structures were elucidated on the basis of extensive spectroscopic data analyses. All isolated compounds were evaluated for their cytotoxicities against five human cancer cell lines: HL-60, SMMC-7721, A-549, MCF-7 and SW480 in vitro. Alkaloids 1-4 exhibited significant inhibitory effects against various human cancer cell lines with IC50 values comparable to those of cisplatin.

A randomized, double-blind, placebo- controlled study on the anti-haemostatic effects of Curcuma longa, Angelica sinensis and Panax ginseng.

PURPOSE: Herbs with "blood-activating" properties by traditional medicine theory often raise concerns for their possible anti-platelet or anticoagulation effects based on reports from in vitro studies. Such herbs have been implicated for bleeding manifestations based on only anecdotal reports. In particular, the combination of such herbs with anti-platelet agents is often empirically advised against despite lack of good clinical evidence. Here we studied 3 commonly used herbal preparations Curcuma longa, Angelica sinensis and Panax ginseng on their respective anti-platelet and anticoagulation effect, alone and in combination with aspirin. STUDY DESIGN: This is a randomized, double-blind, placebo-controlled trial involving 25 healthy volunteers for each herbal preparation. METHODS: Each subject underwent 3 phases comprising of herbal product alone, aspirin alone and aspirin with herbal product, where each phase lasted for 3 weeks with 2 weeks of washout between phases. PT/APTT, platelet function by light transmission aggregometry and thrombin generation assay by calibrated automated thrombogram were measured at baseline and after each phase. Information on adverse reaction including bleeding manifestations was collected after each phase. RESULTS: On the whole there was no clinically relevant impact on platelet and coagulation function. With the exception of 5 of 24 subjects in the Curcuma longa group, 2 of 24 subjects in the Angelica sinensis group and 1 of 23 subjects in the Panax ginseng group who had an inhibition in arachidonic-acid induced platelet aggregation, there was no effect of these 3 herbals products on platelet aggregation by other agonists. Combination of these herbal products with aspirin respectively did not further aggravate platelet inhibition caused by aspirin. None of the herbs impaired PT/APTT or thrombin generation. There was no significant bleeding manifestation. CONCLUSIONS: This study on healthy volunteers provides good evidence on the lack of bleeding risks of Curcuma longa, Angelica sinensis and Panax ginseng either used alone or in combination with aspirin.

Study on effect of Traditional Chinese Medicine Jianpi Chushi decoction and ointment on chronic eczema.

OBJECTIVE: To study the effects of Traditional Chinese Medicine Jianpi Chushi decoction and ointment on chronic eczema. METHODS: DNCB acetone solution was used to sensitize the skin of back and ears of 36 rats in order to establish chronic eczema model. A total of 36 rats were divided into four groups of 9 randomly including oral medicine group, external inunctum group, combination therapy group, and model control group respectively. Besides, the blank group of 4 healthy rats were set. The oral medicine group was given Traditional Chinese Medicine Jianpi Chushi decoction [(Poria cocos, Chinese yam, Cortex dictamni, Zaocys dhumnade, Rhizoma atractylodis, Pericarpium citri reticulatae, Scutellaria baicalensis, Radix Sophorae Flavescentis, Raw Radix Paeoniae Alba, Licorice roots (Northwest Origin)] by gastric infusion (1.6 g/mL·5 mL/d); the external inunctum group was given Qingpeng ointment on the skin, the combination therapy group was given Jianpi Chushi decoction by gastric infusion and Qingpeng ointment combination therapy. The model control group was given normal saline (NS) of the same volume by gastric infusion and vaseline on skin. Continuous administration 15 d and stopped for 3 d. The thickness difference and weight difference of left and right ear of every group were measured and the degree of ear swelling were evaluated. The CD4+ and CD8+ content and the IL-2, IL-4 level of serum were detected, and the inflammatory cells counts of back skin were recorded. RESULTS: After treatment, the degree of ear swelling of oral medicine group, external inunctum group and combination therapy group significant decreased compared with model control group (P < 0.05). The CD4+, CD8+ cell content and IL-2 level of oral medicine group, external inunctum group, combination therapy group and model control group significant decreased compared with blank group, and IL-2 level and the inflammatory cells count increased. After 15 d of treatment, the CD4+, CD8+ cell content and IL-2 level of serum of oral medicine group, external inunctum group and combination therapy group raised and the IL-4 level and the inflammatory cells count had significant decreased compared with model control group, and the effect of combination therapy group was more obvious (P < 0.05). CONCLUSIONS: Qral Jianpi Chushi decoction could treat chronic eczema effectively, and oral Chinese medicine combined with ointment could enhance and speed up the efficacy.

A Practical Guide to Ordering and Interpreting Coagulation Tests for Patients on Direct Oral Anticoagulants in Singapore.

INTRODUCTION: Direct oral anticoagulants (DOACs) are establishing themselves as principle choices for the treatment of a variety of thrombotic disorders. DOACs are also known to affect common coagulation tests which are routinely performed for patients in clinical practice. An understanding of their varied effects is crucial for the appropriate ordering of coagulation tests and their interpretation. MATERIALS AND METHODS: Laboratories in public and private healthcare institutions and commercial sectors were surveyed on coagulation tests offered and their methods. A Medline and bibliography search, including a search on search engines, was performed for publications reporting the effects of dabigatran, apixaban and rivaroxaban on these coagulation tests. These papers were reviewed and summarised for consensus recommendations. RESULTS: Prothrombin time (PT) and activated partial thromboplastin time (aPTT) are variably affected by the DOACs and dependent of the coagulation assays used. Clinicians must know which laboratory has performed these tests to logically interpret test results. A normal PT or aPTT does not exclude the presence of residual DOACs effect. The thrombin time is sensitive to dabigatran but not apixaban or rivaroxaban. Specialised coagulation tests such as thrombophilia tests are also variably affected by the DOACs. All laboratories in Singapore however, employ similar test methods permitting a common set of recommendations for specialised coagulation testing. CONCLUSION: Knowledge of the effects of DOACs on coagulation testing is essential to determine the appropriateness of performing such tests and interpreting them coherently. Practical recommendations which are tests and location-specific are set out in this paper.

Transcriptional analysis of micronutrient zinc-associated response for enhanced carbohydrate utilization and earlier solventogenesis in Clostridium acetobutylicum.

The micronutrient zinc plays vital roles in ABE fermentation by Clostridium acetobutylicum. In order to elucidate the zinc-associated response for enhanced glucose utilization and earlier solventogenesis, transcriptional analysis was performed on cells grown in glucose medium at the exponential growth phase of 16 h without/with supplementary zinc. Correspondingly, the gene glcG (CAC0570) encoding a glucose-specific PTS was significantly upregulated accompanied with the other two genes CAC1353 and CAC1354 for glucose transport in the presence of zinc. Additionally, genes involved in the metabolisms of six other carbohydrates (maltose, cellobiose, fructose, mannose, xylose and arabinose) were differentially expressed, indicating that the regulatory effect of micronutrient zinc is carbohydrate-specific with respects to the improved/inhibited carbohydrate utilization. More importantly, multiple genes responsible for glycolysis (glcK and pykA), acidogenesis (thlA, crt, etfA, etfB and bcd) and solventogenesis (ctfB and bdhA) of C. acetobutylicum prominently responded to the supplementary zinc at differential expression levels. Comparative analysis of intracellular metabolites revealed that the branch node intermediates such as acetyl-CoA, acetoacetyl-CoA, butyl-CoA, and reducing power NADH remained relatively lower whereas more ATP was generated due to enhanced glycolysis pathway and earlier initiation of solventogenesis, suggesting that the micronutrient zinc-associated response for the selected intracellular metabolisms is significantly pleiotropic.

Growth but not photosynthesis response of a host plant to infection by a holoparasitic plant depends on nitrogen supply.

Parasitic plants can adversely influence the growth of their hosts by removing resources and by affecting photosynthesis. Such negative effects depend on resource availability. However, at varied resource levels, to what extent the negative effects on growth are attributed to the effects on photosynthesis has not been well elucidated. Here, we examined the influence of nitrogen supply on the growth and photosynthesis responses of the host plant Mikania micrantha to infection by the holoparasite Cuscuta campestris by focusing on the interaction of nitrogen and infection. Mikania micrantha plants fertilized at 0.2, 1 and 5 mM nitrate were grown with and without C. campestris infection. We observed that the infection significantly reduced M. micrantha growth at each nitrate fertilization and more severely at low than at high nitrate. Such alleviation at high nitrate was largely attributed to a stronger influence of infection on root biomass at low than at high nitrate fertilization. However, although C. campestris altered allometry and inhibited host photosynthesis, the magnitude of the effects was independent of nitrate fertilizations. The infection reduced light saturation point, net photosynthesis at saturating irradiances, apparent quantum yield, CO2 saturated rate of photosynthesis, carboxylation efficiency, the maximum carboxylation rate of Rubisco, and maximum light-saturated rate of electron transport, and increased light compensation point in host leaves similarly across nitrate levels, corresponding to a similar magnitude of negative effects of the parasite on host leaf soluble protein and Rubisco concentrations, photosynthetic nitrogen use efficiency and stomatal conductance across nitrate concentrations. Thus, the more severe inhibition in host growth at low than at high nitrate supplies cannot be attributed to a greater parasite-induced reduction in host photosynthesis, but the result of a higher proportion of host resources transferred to the parasite at low than at high nitrate levels.

[Classification of Cimicifuga species based on 1H-NMR fingerprint combined with pattern recognition technique].

The metabolomic analysis of three Cimicifuga species was performed using H-NMR spectroscopy and pattern recognition (PR) techniques. A broad range of metabolites could be detected by 'H-NMR spectroscopy without any chromatographic separation. The analysis using principal component analysis (PCA) and discriminant partial least square (DPLS) of the 1H-NMR spectrum showed a clear discrimination between C. foetida and the other two species. The major metabolites responsible for the discrimination were triterpenoid saponins and saccharides. These results indicated that the combination of 1H-NMR and PR provides a useful tool for chemotaxonomic analysis and authentification of Cimicifuga species, and could used for the quality control of plant materials.

Classification of Cimicifuga species based on 1H-NMR fingerprint combined with pattern recognition technique / 中国中药杂志

The metabolomic analysis of three Cimicifuga species was performed using H-NMR spectroscopy and pattern recognition (PR) techniques. A broad range of metabolites could be detected by 'H-NMR spectroscopy without any chromatographic separation. The analysis using principal component analysis (PCA) and discriminant partial least square (DPLS) of the 1H-NMR spectrum showed a clear discrimination between C. foetida and the other two species. The major metabolites responsible for the discrimination were triterpenoid saponins and saccharides. These results indicated that the combination of 1H-NMR and PR provides a useful tool for chemotaxonomic analysis and authentification of Cimicifuga species, and could used for the quality control of plant materials.