What exactly does the PfK13 C580Y mutation in Plasmodium falciparum influence?

BACKGROUND: The emergence and spread of artemisinin resistance threaten global malaria control and elimination goals, and encourage research on the mechanisms of drug resistance in malaria parasites. Mutations in Plasmodium falciparum Kelch 13 (PfK13) protein are associated with artemisinin resistance, but the unique or common mechanism which results in this resistance is unclear. METHODS: We analyzed the effects of the PfK13 mutation on the transcriptome and proteome of P. falciparum at different developmental stages. Additionally, the number of merozoites, hemozoin amount, and growth of P. falciparum 3D7C580Y and P. falciparum 3D7WT were compared. The impact of iron supplementation on the number of merozoites of P. falciparum 3D7C580Y was also examined. RESULTS: We found that the PfK13 mutation did not significantly change glycolysis, TCA, pentose phosphate pathway, or oxidative phosphorylation, but did reduce the expression of reproduction- and DNA synthesis-related genes. The reduced number of merozoites, decreased level of hemozoin, and slowed growth of P. falciparum 3D7C580Y were consistent with these changes. Furthermore, adding iron supply could increase the number of the merozoites of P. falciparum 3D7C580Y. CONCLUSIONS: These results revealed that the PfK13 mutation reduced hemoglobin ingestion, leading to artemisinin resistance, likely by decreasing the parasites' requirement for haem and iron. This study helps elucidate the mechanism of artemisinin resistance due to PfK13 mutations.

Chemical profile of Roselle extract and its inhibitory activities on three digestive enzymes in vitro and in vivo.

Roselle is rich in an extensive diversity of beneficial substances, including phenolic acids, amino acids, anthocyanins, vitamins, and flavonoids. Herein, the chemical constituents in Roselle extract (RE) were identified by UPLC-DAD-QTOF-MS. Besides, its inhibitory effects on three digestive enzymes, i.e. α-amylase, α-glucosidase, and pancreatic lipase, were investigated in both in vitro and in vivo. Thirty-three constituents including hibiscus acid, 18 phenolic acids, 2 anthocyanins and 12 flavonoids were identified. The anthocyanins content in RE was 21.44 ± 0.68 %, while the contents of chlorogenic acids, rutin and quercetin were 17.76 ± 2.28 %, 0.31 ± 0.01 % and 0.32 ± 0.01 %, respectively. RE inhibited pancreatic lipase in a non-competitive way with an IC50 value of 0.84 mg/mL. Besides, it demonstrated a mixed-type inhibition on both α-glucosidase and α-amylase with IC50 values of 0.59 mg/mL and 1.93 mg/mL, respectively. Fluorescence quenching assays confirmed the binding of RE to the enzyme proteins. Furthermore, rats pre-treated with RE at doses of 50 and 100 mg/kg body weight (bwt) exhibited significant reductions in fat absorption and improvements in fat excretion through feces. Additionally, the in vivo study revealed that RE was effective in suppressing the increase of blood glucose after starch consumption, while its effects on maltose and sucrose consumption were relatively weak.

In vitro and in vivo Inhibitory Activity of C-glycoside Flavonoid Extracts from Mung Bean Coat on Pancreatic Lipase and α-glucosidase.

Mung bean is a kind of legume commonly eaten by human. In the present study, a HPLC method for analyzing of two C-glycoside flavonoids, isovitexin and vitexin, in Mung bean was developed. Results showed that the flavonoids are mainly existed in Mung bean coat (MBC), while kernel contains very trace. The extraction of C-glycoside flavonoids from MBC was optimized. MBC extracts with isovitexin and vitexin contents of 29.0 ± 0.28% and 35.8 ± 0.19% were obtained with yield of 1.6 ± 0.21%. MBC extracts exhibited inhibitory activities on pancreatic lipase and α-glucosidase with IC50 values of 0.147 mg/ml and 0.226 mg/ml, respectively. The inhibitory kinetics revealed that MBC extracts showed mixed-type inhibition on these enzymes. Fluorescence quenching titration confirmed the binding of MBC extracts with the enzyme proteins. In vivo study revealed that pre-administration with MBC extracts significantly reduced the triglyceride absorption. Furthermore, it also improved postprandial hyperglycemia in rats through the inhibition of α-glucosidase.

Extraction, identification, and starch-digestion inhibition of phenolics from Euryale ferox seed coat.

BACKGROUND: Euryale ferox is an important cash crop and valuable tonic in traditional medicine. The seeds of E. ferox are rich in starch, which is hard to digest, and the digestion speed is significantly slower than that of rice starch. The goal of this study was to evaluate the effects of E. ferox seed-coat phenolics (EFCPs) on the digestion of E. ferox seed starch. RESULTS: EFCPs were extracted and identified by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. We optimized the extraction parameters, and the final extraction yield was about 1.49%. We identified seven phenolics from the E. ferox seed-coat extracts: gallic acid, digalloylhexoside, catechin, procyanidin B2, epicatechin, ellagic acid, and epicatechin gallate. Quantitative analysis results showed that the E. ferox seed phenolics mainly distributed in the seed coat and the gallic acid, digalloylhexoside, and epicatechin gallate were three main phenolic compounds. The phenolics displayed strong inhibitory activities on α-glucosidase and α-amylase with an IC50 of 3.25 µg mL-1 and 1.36 mg mL-1 respectively. Furthermore, these phenolics could interact with starch by hydrogen bonds, which might make its starch more difficult to digest. CONCLUSION: Our investigation suggests that the EFCPs can strongly inhibit the digestion of E. ferox seed starch by inhibiting the α-amylase and α-glucosidase activities and interacting with starch by hydrogen bonds; therefore, E. ferox seeds have a promising application prospect in foods for hypoglycemia. © 2023 Society of Chemical Industry.

Randomised, controlled crossover trial of intermittent and continuous transcutaneous electrical stimulation of the genioglossus muscle for obstructive sleep apnoea.

PURPOSE: Continuous transcutaneous electrical stimulation (CTES) of the genioglossus muscle may benefit patients with obstructive sleep apnoea (OSA). However, the therapeutic value of intermittent transcutaneous electrical stimulation (ITES) for OSA is unclear. METHODS: This was a randomised, controlled, crossover study to compare the effects of ITES and CTES of the genioglossus muscle. Over three single-night sessions, participants were alternately subjected to three genioglossus stimulation modalities during sleep (sham, CTES and ITES). The apnoea-hypopnoea index (AHI) and oxygen desaturation index (ODI) were used for OSA diagnosis and to evaluate efficacy. A responder was defined as an individual with a ≥50% reduction in AHI together with <10 AHI events per hour and/or an ODI reduction of ≥25% between sham stimulation and electrical stimulation nights. RESULTS: Fifteen men with OSA completed the study. Compared with sham, the median AHI with ITES decreased by 13.3 events/hour (95% CI 3.1 to 23.5, p=0.030) and by 7.3 events/hour (95% CI -3.9 to 18.5, p=0.825) with CTES. The median ODI was reduced by 9.25 events/hour (95% CI 0.5 to 18.0) with ITES and 3.3 events/hour (95% CI -5.6 to 12.2) with CTES; however, there was no significant difference between groups. Furthermore, ITES outperformed CTES with respect to longest apnoea duration (median (95% CI), 9.5 (0.0 to 19.0), p=0.011)) and the highest sleep efficiency (12.2 (2.7 to 21.7), p=0.009). Of the 15 participants, 8 responded to ITES and 3 responded to CTES (p=0.058), of whom all eight cases and two out of three cases had ODIs <5 events/hour, respectively. All participants tolerated ITES well. CONCLUSIONS: ITES improved upper airway obstruction in patients with OSA, suggesting that further prospective validation of the intermittent approach is warranted. TRIAL REGISTRATION NUMBER: ChiCTR2100050138.

Gougunao tea polysaccharides ameliorate high-fat diet-induced hyperlipidemia and modulate gut microbiota.

Many natural polysaccharides have been proven to have ameliorative effects on high-fat diet-induced hyperlipidemia with fewer side effects. However, similar data on Gougunao tea polysaccharides remain obscure. In this study, we aimed to investigate the role of Gougunao tea polysaccharides (GTP40) in the alleviation of hyperlipidemia and regulation of gut microbiota in C57BL/6J mice induced by a high-fat diet. The results indicated that GTP40 intervention inhibited the abnormal growth of body weight and the excessive accumulation of lipid droplets in the livers and ameliorated the biochemical parameters of serum/liver related to lipid metabolism in hyperlipidemia mice. The elevated levels of antioxidant enzyme and anti-inflammation cytokine in serum, as well as the up-regulating anti-inflammation gene in the liver, reflected that GTP40 might mitigate the oxidative and inflammatory stress induced by a high-fat diet. In addition, GTP40 could modulate the composition, abundance, and diversity of gut microbiota in hyperlipidemia mice. Besides, Spearman's correlation analysis implied that GTP40 intervention could enrich beneficial bacteria (e.g., Akkermansia, Bacteroides, Roseburia, and Alistipes), and decrease harmful bacteria (e.g., Blautia, Faecalibaculum, Streptococcus, and norank_f_Desulfovibrionaceae), which were correlated with the lipid metabolic parameters associated with hyperlipidemia. Moreover, it also indicated that there was a significant correlation between gut microbiota and SCFAs. Thus, GTP40 may be a novel strategy against fat accumulation, oxidative stress, and inflammation, as well as restoring the normal microbial balance of the gut in hyperlipidemia mice.

Therapeutic Value of Traditional Chinese Massage plus Moxibustion for Degenerative Knee Osteoarthritis.

Objective: To evaluate the therapeutic value of traditional Chinese massage plus moxibustion for degenerative knee osteoarthritis (DKOA). Methods: From January 2019 to October 2021, 152 patients with DKOA were enrolled. All patients were randomly divided into the treatment group and the control group. The control group was treated with oral glucosamine hydrochloride capsules. The treatment group was treated with traditional Chinese massage and moxibustion on the basis of the control group. The duration of treatment in the two groups was 8 weeks. The effectiveness, visual analog scale (VAS) score, the hospital for special surgery (HSS) score, World Health Organization Quality of Life Scale (WHOQOL-BREF) score, serum high-sensitivity C-reactive protein (CRP), and interleukin-6 (IL-6) levels were compared between the two groups before and after treatment. Results: There were no significant differences in age, sex, and the duration of disease between the two groups (P > 0.05). The overall response rate in the treatment group was significantly higher than that of the control group after treatment (92.1% vs 78.9%; P=0.038); the VAS score of both two groups were significantly decreased, and the VAS score in the treatment group was significantly lower than that of the control group (3.5 ± 1.4 vs 4.8 ± 1.1; P < 0.001); the HSS score and WHOQOL-BREF score significantly improved in both groups, and the HSS score and WHOQOL-BREF score improved more in the treatment group than those of the control group (P < 0.05). The high-sensitivity CRP level significantly decreased in both groups, and the high-sensitivity CRP level in treatment group is lower than that of the control group (2.79 ± 1.65 vs 4.37 ± 1.54; P < 0.001); the IL-6 level was significantly decreased in the treatment group than in the control group (7.22 ± 3.41 vs 4.59 ± 2.98; P < 0.001). Conclusion: Traditional Chinese massage plus moxibustion is worthy of clinical application, which has a significant clinical effect on DKOA, reducing pain and improving knee joint function and quality of life.

Regulating the Imbalance of Gut Microbiota by Smilax china L. Polyphenols to Alleviate Dextran Sulfate Sodium-induced Inflammatory Bowel Diseases.

Smilax china L. is used not only as a kind of traditional Chinese herbal medicinal ingredients with various pharmacological properties, but also as food in certain parts of China. However, it is by far still unclear whether Smilax china L. polyphenols (SCP), as important bioactive constituents in Smilax china L., have effects on inflammatory bowel diseases (IBD). This study investigated the impact of SCP on the dextran sulfate sodium (DSS)-induced IBD and gut microbiota in mice. SCP treatments ameliorated typical symptoms of IBD as what was reflected through suppressing body weight loss, colonic shortening, intestinal barrier damage, and increasing intestinal disease activity index. SCP treatments simultaneously decreased the release of proinflammatory cytokines and oxidative stress, as well as promoted the release of anti-inflammatory factors. Furthermore, SCP ameliorated the ecological imbalance of gut microbiota and regulated the key bacteria associated with IBD (including Akkermansiaceae, Ruminococcaceae, Acidaminococcaceae, Muribaculaceae, and Anaeroplasmataceae). In general, SCP may improve DSS-induced IBD in mice by regulating inflammatory factors, inhibiting oxidative stress, reducing intestinal tissue damage, and regulating the ecological imbalance of intestinal microbiota. Thus, SCP might serve as a potential therapeutic agent against the inflammation-driven diseases.

Dihydromyricetin Improves Cognitive Impairments in d-Galactose-Induced Aging Mice through Regulating Oxidative Stress and Inhibition of Acetylcholinesterase.

SCOPE: Alzheimer's disease (AD) is a neurodegenerative disease with phenomena of cognitive impairments. Oxidative stress and cholinergic system dysfunction are two widely studied pathogenesis of AD. Dihydromyricetin (DMY) is a natural dihydroflavonol with many bioactivities. In this study, it is aimed to investigate the effects of DMY on cognitive impairment in d-galactose (d-gal) induced aging mice. METHODS AND RESULTS: Mice are intraperitoneally injected with d-gal for 16 weeks, and DMY is supplemented in drinking water. The results show that DMY significantly improves d-gal-induced cognitive impairments in novel object recognition and Y-maze studies. H&E and TUNEL staining show that DMY could improve histopathological changes and cell apoptosis in mice brain. DMY effectively induces the activities of catalase, superoxide dismutase and glutathione peroxidase, and reduces malondialdehyde level in mice brain and liver. Furthermore, DMY reduces cholinergic injury by inhibiting the activity of Acetylcholinesterase (AChE) in mice brain. In vitro studies show that DMY is a non-competitive inhibitor of AChE with IC50 value of 161.2 µg mL-1 . CONCLUSION: DMY alleviates the cognitive impairments in d-gal-induced aging mice partly through regulating oxidative stress and inhibition of acetylcholinesterase.

In vivo and in vitro comparison of three astilbin encapsulated zein nanoparticles with different outer shells.

Three shell materials, lecithin (ZNP-L), chitosan (ZNP-CH) and sodium caseinate (ZNP-SC), were used to prepare core-shell zein nanoparticles. Astilbin was encapsulated as a model flavonoid to compare the influence of the shell materials on zein nanoparticles both in vitro and in vivo. The particle size was moderately increased by lecithin and sodium caseinate, but notably increased by chitosan. All the shell materials provided good redispersibility for the nanoparticles and significantly improved the colloidal stability. Chitosan and sodium caseinate significantly delayed and decreased the feces excretion of astilbin in rats, while lecithin exhibited a very weak effect. The results may be attributed to the difference in mucoadhesive properties between the shell materials. As a consequence, the bioavailability values of astilbin in rats were 18.2, 9.3 and 1.89 times increased through ZNP-CH, ZNP-SC and ZNP-L compared with that of free astilbin, respectively.

Pancreatic lipase inhibitory activity of Bambusa multiplex cv. Fernleaf leaf extract in vitro and in vivo.

Bambusa multiplex cv Fernleaf (B. multiplex) is a species of bamboo. In the present study, B. multiplex leaf extract was prepared through the resin absorption/desorption procedure and analyzed by HPLC. C-Glycosyl flavonoids are the main constituents of B. multiplex extract, and the content of isoorientin and vitexin was 51.8 and 23.1 mg g-1, respectively. Besides, the extract exhibited inhibitory activities on pancreatic lipase and α-glucosidase with IC50 values of 0.91 and 1.16 mg mL-1, respectively. The extract could bind to pancreatic lipase and showed mixed-type inhibition. An in vivo study showed that pre-administration of B. multiplex extract significantly reduced the fat absorption in rats and increased fat excretion through feces. The change in the C-glycosyl flavonoid content in feces was the same as that in the triglyceride content. The inhibitory activity of B. multiplex leaf extract on pancreatic lipase was confirmed both in vitro and in vivo.

Physicochemical properties of dihydromyricetin and the effects of ascorbic acid on its stability and bioavailability.

BACKGROUND: Dihydromyricetin (DMY) is a natural dihydroflavonol with many bioactive effects. However, the physicochemical properties of DMY related to its bioavailability, especially its stability, are unclear. RESULTS: The effects of pH, temperature, metal ions and ascorbic acid (AA) on the stability of DMY were studied using high-performance liquid chromatography (HPLC). The bioavailability of DMY in the presence and absence of AA was compared. Dihydromyricetin was unstable in weak alkaline solutions, and the degradation was significantly accelerated in the presence of Cu2+ and Fe3+ . The degradation process followed the first-order kinetic model. The degradation rate constant (k) increased with increasing pH and temperature. The remaining DMY was only 49% of its initial concnentration after 4 h in simulated intestinal fluid (SIF) at 37 °C. However, by supplementing with AA, the degradation of DMY was rarely occured within 6 h. The solubility of DMY at pH 3-5 was about 750 µg mL-1 , slightly increasing to 853 µg mL-1 at pH 6. Pharmacokinetic studies showed that the bioavailability of DMY increased from 0.122% to 0.341% by supplementing with AA (10% of DMY). CONCLUSION: The degradation of DMY is one reason for its poor bioavailability. The presence of AA could significantly improve the stability of DMY, and further improve its bioavailability in rats. © 2020 Society of Chemical Industry.

A Comparison of Solubility, Stability, and Bioavailability between Astilbin and Neoastilbin Isolated from Smilax glabra Rhizoma.

Astilbin and neoastilbin are two flavonoid stereoisomers. In the present study, their solubility, stability, and bioavailability were compared in a rat. The results revealed that the water solubility of astilbin and neoastilbin was 132.72 µg/mL and 217.16 µg/mL, respectively. The oil-water distribution coefficient (log P) of astilbin and neoastilbin in simulated gastric fluid (SGF) was 1.57 and 1.39, and in simulated intestinal fluid (SIF) was 1.09 and 0.98, respectively. In SIF, about 78.6% astilbin remained after 4 h of incubation at 37 °C, while this value was 88.3% for neoastilbin. Most of the degraded astilbin and neoastilbin were isomerized into their cis-trans-isomer, namely neoisoastilbin and isoastilbin, respectively, and the decomposed parts were rare. For bioavailability comparison in a rat, an HPLC method for trace amounts of astilbin and neoastilbin determination in plasma was developed, and the pretreatment of plasma was optimized. A pharmacokinetic study showed that the absolute bioavailability of astilbin and neoastilbin in a rat showed no significant difference with values of 0.30% and 0.28%, respectively.

Antibacterial mechanism of the synergistic combination between streptomycin and alcohol extracts from the Chimonanthus salicifolius S. Y. Hu. leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: Chimonanthus salicifolius S. Y. Hu. Is a unique traditional medicinal plant in ancient China, and it can eliminate turbid pathogens with aromatics, clear heat, detoxify, prevent colds and influenza, Xinhua Compendium of Materia Medica records that. AIM OF THE STUDY: In previous study, we investigated the regulation of ethanol extracts (EEs) from C. salicifolius S. Y. Hu. leaves on three common antibiotics (chloramphenicol, streptomycin, imipenem) by the checkerboard method. The combination exhibited the best synergy among all combinations, which were composed of streptomycin and 50% EE (SE) from the C. salicifolius S. Y. Hu. leaves. The aim of this study was to investigate the antibacterial mechanism of the SE against Escherichia coli (E. coli, G-) and Staphylococcus aureus (S. aureus, G+). MATERIALS AND METHODS: The antibacterial mechanism of the SE was explored by the time-kill test, the phosphorus metabolism, cell membrane integrity assays, the SDS-PAGE, the SEM and TEM observation. RESULTS: The time-kill test illustrated that the SE was bacteriostatic with a time-dependent relationship, not sterilization. The phosphorus metabolism indicated that the SE lowered phosphorus consumption. The cell membrane integrity assays demonstrated that the cell membrane was damaged, with the nucleic acid flowing out. The SDS-PAGE analysis found that the SE inhibited the synthesis of the total protein. The SEM and TEM results revealed that the surface and internal ultrastructure of bacteria were damaged. The surface of the bacteria was shriveled and deformed, and the internal structure of the cells was also mutilated. CONCLUSIONS: The SE damaged the cell membrane, with the cytoplasm flowing out, disturbed the synthesis of total protein and phosphorus metabolism, and ultimately killed the bacteria.

Identification and quantification of main flavonoids in the leaves of Bambusa multiplex cv. Fernleaf.

The chemical profile of Bambusa multiplex cv. Fernleaf (B. multiplex) leaves was analysed by UPLC-DAD-Q-TOF-MS. Twelve compounds were identified and C-glycosyl flavonoids, including vitexin, isovitexin, isoorientin and its derivatives, are the main constitutes of the plant. Besides, a HPLC method for isoorientin quantification was developed. The RSD of retention time and peak area were 0.05% and 2.04% for six times analysis of isoorientin with concentration of 20 µg/mL. The recovery of isoorientin in real sample was 99.2%. The general trend of isoorientin content in B. multiplex leaves was that it steady increased from Jan. to May, and then quickly decreased. The maximum was found on May with value of 4.7 mg/g. The lowest level of isoorientin was found during Aug. to Nov. with value of about 1.66 mg/g. In different seasons, isoorientin is always the most dominant flavonoid which was accounted for about 50% of total flavonoids in the sample.

Metabolism, Excretion, and Tissue Distribution of Astilbin-Zein Nanoparticles in Rats.

The excretion, tissue distribution, and metabolic profile of astilbin in rat were studied by HPLC and UPLC-QTOF-MS. Astilbin underwent isomerization in the small intestine, and its four isomers were found in feces. Besides, taxifolin, the aglycone of astilbin, and its further metabolites by gut microbes through hydrogenation, dehydration, and ring-fission were found. The total feces excretion of astilbin was about 14.4% of administration. The forming of zein-caseinate nanoparticles can significantly delay and reduce the feces excretion of astilbin. Astilbin and its isomers were absorbed in their intact form. The main metabolites found in plasma and tissues were the methylated products. Astilbin was rapidly distributed in various tissues including brain and maintained relatively high concentration in heart. Compared with other tissues, significantly higher concentration and longer duration of astilbin were found in the gastrointestinal tract. Astilbin and its isomers were excreted in their intact and methylated form in urine.

Bioavailability Enhancement of Astilbin in Rats through Zein-Caseinate Nanoparticles.

Astilbin-encapsulated zein-caseinate nanoparticles were fabricated through the antisolvent method. The encapsulation and loading efficiency of astilbin in the nanoparticles were determined by high-performance liquid chromatography. The nanoparticles were characterized by particle size, ζ potential, redispersibility, scanning electron microscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy, and differential scanning calorimetry (DSC). Under the optimal formulation of astilbin, zein, and sodium caseinate with a mass ratio of 1:1:2, the size and ζ potential of the nanoparticles were 152.9 nm and -40.43 mV, respectively, while the encapsulation and loading efficiency of astilbin were 80.1 and 21.8%, respectively. The nanoparticles had good redispersibility in water without a particle size change after freeze drying. The nanoparticles showed a spherical shape with a smooth surface. XRD and DSC analyses showed that astilbin existed in amorphous form in the nanoparticles. The interactions between astilbin and the protein were found, and astilbin was encapsulated in nanoparticles rather than adsorbed. The diffusion of astilbin from nanoparticles was significantly faster than that of astilbin suspensions in both simulated gastric and intestinal fluids. Astilbin was relatively stable in simulated intestinal fluids, and the encapsulation in the nanoparticles showed a slight stability improvement effect. A pharmacokinetic study showed that the absolute bioavailability of astilbin was improved from 0.32 to 4.40% in rats through nanoparticle fabrication.

Isomerization of astilbin and its application for preparation of the four stereoisomers from Rhizoma Smilacis Glabrae.

Astilbin is the most predominant flavonoid in Rhizoma Smilacis Glabrae (RSG) with many bioactivities. The interconversion of the astilbin and its three stereoisomers was found with incubation of RSG extract at different temperatures, and the equilibrium ratios were calculated. Under certain conditions, neoastilbin would replace astilbin and become the predominant flavonoid in RSG extract. The effects of ascorbic acid, sucrose, sodium benzoate, ß-cyclodextrin (ß-CD) and common metal ions on the isomerization and decomposition of astilbin were studied. Ascorbic acid showed the best protective effect on the decomposition of astilbin and its isomers, which may be attributed to its reducing and radical scavenging ability. Besides, ascorbic acid also accelerated the isomerization of astilbin. ß-CD suppressed both isomerization and decomposition of astilbin through complexation between them. Most metal ions had inhibition effects on the isomerization of astilbin. Al3+ could almost completely inhibit the isomerization. The presence of Fe3+ caused the rapid decomposition of astilbin, and Cu2+ also showed weak effects. Based on the isomerization study, a novel and simple method for preparative separation of astilbin and neoastilbin from RSG sample was developed. Astilbin and neoastilbin with purity of 93% and yield of 0.86% and 0.48% were obtained, respectively, which represent 46.8% of total flavonoids in RSG sample. By controlling the isomerization conditions, astilbin and neoastilbin could be used as the initial reactants to produce neoisoastilbin and isoastilbin, respectively.

Production of chlorogenic acid and its derivatives in hairy root cultures of Stevia rebaudiana.

Chlorogenic acid and its derivatives (CADs) are valuable bioactive plant secondary metabolites with many health benefits. In the present study, Stevia rebaudiana hairy root cultures were established, and the culture conditions for the production of CADs were optimized. The hairy roots were induced by coculture of S. rebaudiana leaves and Agrobacterium rhizogenes (C58C1) after infection, which were further verified by PCR detection of rolB and rolC genes. HPLC-MS and HPLC analysis showed that chlorogenic acid (3-caffeoylquinic acid, 3-CQA), 3,5-dicaffeoylquinic acid (3,5-CQA), and 4,5-dicaffeoylquinic acid (4,5-CQA) were the major CADs in the hairy roots. Eight single roots with rapid growth rate were selected. Among them, T3 had the highest yield of CADs. B5 medium supplemented with 40 g/L sucrose was more suitable for the production of CADs than others. Under optimal culture conditions, the total content of these three compounds reached 105.58 mg/g and total yield was 234.40 mg/100 mL.

A fluorescence turn-on detection of copper(II) based on the template-dependent click ligation of oligonucleotides.

In this work, a fluorescence turn-on method for copper(II) detection is reported. A molecular beacon (MB) was designed as a template. Cu(2+) was reduced to Cu(+) in the presence of a reductant (ascorbic acid). Two short single-stranded oligonucleotides one was labeled with a 5'-alkyne and the other with 3'-azide group, proceeded a template-dependent chemical ligation through the Cu(I)-catalyzed azide-alkyne cycloaddition. The newly generated click-ligated long chain oligonucleotide, which was complementary to the MB, opened the MB hairpin structure and resulted in a turn on fluorescence. The increase in fluorescence intensity is directly proportional to the amount of Cu(2+) added to the assay solution. The present assay is quite sensitive and allows the detection of 2 nM Cu(2+). The described assay also exhibits high selectivity over other metal ions.