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The rapidly growing field of cannabinoid research is gaining recognition for its impact in neuropsychopharmacology and mood regulation. However, prenyltransferase (NphB) (a key enzyme in cannabinoid precursor synthesis) still needs significant improvement in order to be usable in large-scale industrial applications due to low activity and limited product range. By rational design and high-throughput screening, NphB’s catalytic efficiency and product diversity have been markedly enhanced, enabling direct production of a range of cannabinoids, without the need for traditional enzymatic conversions, thus broadening the production scope of cannabinoids, including cannabigerol (CBG), cannabigerolic acid (CBGA), cannabigerovarin (CBGV), and cannabigerovarinic acid (CBGVA). Notably, the W3 mutant achieved a 10.6-fold increase in CBG yield and exhibited a 10.3- and 20.8-fold enhancement in catalytic efficiency for CBGA and CBGV production, respectively. The W4 mutant also displayed an 9.3-fold increase in CBGVA activity. Molecular dynamics simulations revealed that strategic reconfiguration of the active site’s hydrogen bonding network, disulfide bond formation, and enhanced hydrophobic interactions are pivotal for the improved synthetic efficiency of these NphB mutants. Our findings advance the understanding of enzyme optimization for cannabinoid synthesis and lay a foundation for the industrial-scale production of these valuable compounds.

To meet the demands for high-temperature performance and lightweight materials in aerospace engineering, the Au-Ni solder is often utilized for joining dissimilar materials, such as Ti3Al-based alloys and Ni-based high-temperature alloys. However, the interaction between Ti and Ni can lead to the formation of brittle phases, like Ti2Ni, TiNi, and TiNi3, which diminish the mechanical properties of the joint and increase the risk of crack formation during the welding process. Cu doping has been shown to enhance the mechanical properties and high-temperature stability of the Au-Ni brazed joint’s central area. Due to the difficulty in accurately controlling the solid solution content of Cu in the Au-Ni alloy, along with the high cost of Au, traditional experimental trial-and-error methods are insufficient for the development of Au-based solders. In this study, first principles calculations based on density functional theory were employed to analyze the effect of Cu content on the stability of the Au-2.0Ni-xCu (x = 0, 0.25, 0.5, 0.75, 1.0, 1.25 wt%) alloy phase structure. The thermal properties of the alloy were determined using Gibbs software fitting. The results indicate that the Au-2.0Ni-0.25Cu alloy exhibits the highest plastic toughness (B/G = 5.601, ν = 0.416, Cauchy pressure = 73.676 GPa) and a hardness of 1.17 GPa, which is 80% higher than that of Au-2.0Ni. This alloy balances excellent strength and plastic toughness, meeting the mechanical performance requirements of brazed joints. The constant pressure specific heat capacity (Cp) of the Au-2.0Ni-xCu alloy is higher than that of Au-2.0Ni and increases with Cu content. At 1000 K, the Cp of the Au-2.0Ni-0.25Cu alloy is 35.606 J·mol−1·K−1, which is 5.88% higher than that of Au-2.0Ni. The higher Cp contributes to enhanced high-temperature stability. Moreover, the linear expansion coefficient (CTE) of the Au-2.0Ni-0.25Cu alloy at 1000 K is 8.76 × 10−5·K−1, only 0.68% higher than Au-2.0Ni. The lower CTE helps to reduce the risk of solder damage caused by thermal stress. Therefore, the Au-2.0Ni-0.25Cu alloy is more suitable for brazing applications in high-temperature environments due to its excellent mechanical properties and thermal stability. This study provides a theoretical basis for the performance optimization and engineering application of the Au-2.0Ni-xCu alloy as a gold-based solder.

Developing clinically meaningful nanomedicines for cancer therapy requires the drugs to be effective, safe, simple, cheap, and easy to store. In the present work, we report that a simple cationic Fe(III)-rich salt of [FeIIICl(TMPPH2)][FeIIICl4]2 (Fe-TMPP) exhibits a superior anticancer performance on a broad spectrum of cancer cell lines, including breast, colorectal cancer, liver, pancreatic, prostate, and gastric cancers, with half maximal inhibitory concentration (IC50) values in the range of 0.098–3.97 μM (0.066–2.68 μg mL−1), comparable to the best-reported medicines. Fe-TMPP can form stand-alone nanoparticles in water without the need for extra surface modification or organic-solvent-assisted antisolvent precipitation. Critically, Fe-TMPP is TME-responsive (TME = tumor microenvironment), and can only elicit its function in the TME with overexpressed H2O2, converting H2O2 to the cytotoxic •OH to oxidize the phospholipid of the cancer cell membrane, causing ferroptosis, a programmed cell death process of cancer cells.

Mycobacterium tuberculosis (Mtb) is one of the major causes of human death. In its battle with humans, Mtb has fully adapted to its host and developed ways to evade the immune system. At the same time, the human immune system has developed ways to respond to Mtb. The immune system responds to viral and bacterial infections through a variety of mechanisms, one of which is alternative splicing. In this study, we summarized the overall changes in alternative splicing of the transcriptome after macrophages were infected with Mtb. We found that after infection with Mtb, cells undergo changes, including (1) directly reducing the expression of splicing factors, which affects the regulation of gene expression, (2) altering the original function of proteins through splicing, which can involve gene truncation or changes in protein domains, and (3) expressing unique isoforms that may contribute to the identification and development of tuberculosis biomarkers. Moreover, alternative splicing regulation of immune-related genes, such as IL-4, IL-7, IL-7R, and IL-12R, may be an important factor affecting the activation or dormancy state of Mtb. These will help to fully understand the immune response to Mtb infection, which is crucial for the development of tuberculosis biomarkers and new drug targets.

Oil-immersed transformers play a pivotal role owing to their environmentally friendly characteristics, compact footprint, and cost-effectiveness. Ensuring the online monitoring of oil-immersed transformers is a fundamental measure to ensure the secure and stable operation of modern power systems. In this paper, metal particle cluster-doped SnS is firstly used in the adsorption and sensing of decomposition components (CO, C2H2) under fault conditions in oil-immersed transformers. The study comprehensively analyzed band structure, differential charge density, density of states, and molecular orbital theory to unveil the adsorption and sensing mechanisms of target gases. The findings suggest that the modification of metal particle clusters can enhance the surface electronic properties of single-layer SnS. In the regions of metal particle clusters and the gas–surface reaction area, electronic activity is significantly heightened, primarily attributed to the contribution of d-orbital electrons of the metal cluster structures. The modified SnS exhibits adsorption capacity in the following order: Ru3-SnS > Mo3-SnS > Au3-SnS. Additionally, the modified material demonstrates increased competitiveness for C2H2, with adsorption types falling under physical chemistry adsorption. Different metal elements exert diverse effects on the electronic distribution of the entire system, providing a theoretical foundation for the preparation of corresponding sensors. The findings in this work offer numerical insights for the further preparation and development of SnS nanosensors, concurrently shedding light on the online monitoring of faults in oil-immersed transformers.

Bacterial infections pose a significant risk to human health. Magnolol, derived from Magnolia officinalis, exhibits potent antibacterial properties. Synthetic biology offers a promising approach to manufacture such natural compounds. However, the plant-based biosynthesis of magnolol remains obscure, and the lack of identification of critical genes hampers its synthetic production. In this study, we have proposed a one-step conversion of magnolol from chavicol using laccase. After leveraging 20 transcriptomes from diverse parts of M. officinalis, transcripts were assembled, enriching genome annotation. Upon integrating this dataset with current genomic information, we could identify 30 laccase enzymes. From two potential gene clusters associated with magnolol production, highly expressed genes were subjected to functional analysis. In vitro experiments confirmed MoLAC14 as a pivotal enzyme in magnolol synthesis. Improvements in the thermal stability of MoLAC14 were achieved through selective mutations, where E345P, G377P, H347F, E346C, and E346F notably enhanced stability. By conducting alanine scanning, the essential residues in MoLAC14 were identified, and the L532A mutation further boosted magnolol production to an unprecedented level of 148.83 mg/L. Our findings not only elucidated the key enzymes for chavicol to magnolol conversion, but also laid the groundwork for synthetic biology-driven magnolol production, thereby providing valuable insights into M. officinalis biology and comparative plant science.

Panax quinquefolius (PQ) has been widely used in traditional Chinese medicine and functional food. Ginsenosides are the important functional components of PQ. The ginsenosides’ diversity is deeply affected by the processing conditions. The ginsenosides in the steamed PQ have been not well-characterized yet because of the complexity of their structure. In the study, the comprehensive investigation of ginsenosides was performed on the steamed PQ with different steaming times and temperatures by UPLC-Q-TOF-MS. Based on the molecular weight, retention time and characterized fragment ions, 175 ginsenosides were unambiguously identified or tentatively characterized, including 45 protopanaxatriol type, 49 protopanaxadiol type, 19 octillol type, 6 oleanolic acid type ginsenosides, and 56 other ginsenosides. Ten new ginsenosides and three new aglycones were discovered in the steamed PQ samples through searching the database of CAS SciFindern. Principal component analysis showed the significant influence on the chemical components of PQ through different processing conditions. The steaming temperature was found to promote the transformation of ginsenosides more than the steaming time. The protoginsenosides were found to transform into the rare ginsenosides by elimination reactions. The malonyl ginsenosides were degraded into acetyl ginsenosides, and then degraded into neutral ginsenosides. The sugar chain experienced degradation, with position changes and configuration inversions. Furthermore, 20 (S/R)-ginsenoside Rh1, Rh2, Rg2, and Rh12 were found to transform from the S-configuration to the R-configuration significantly. This study could present a comprehensive ginsenosides profile of PQ with different steaming conditions, and provide technical support for the development and utilization of PQ.

Trilobatin (TBL) is a key sweet compound from the traditional Chinese sweet tea plant (Rubus suavissimus S. Lee). Because of its intense sweetness, superior taste profile, and minimal caloric value, it serves as an exemplary natural dihydrochalcone sweetener. It also has various health benefits, including anti-inflammatory and glucose-lowering effects. It is primarily produced through botanical extraction, which impedes its scalability and cost-effectiveness. In a novel biotechnological approach, phloretin is used as a precursor that is transformed into TBL by the glycosyltransferase enzyme ph-4′-OGT. However, this enzyme’s low catalytic efficiency and by-product formation limit the large-scale synthesis of TBL. In our study, the enzyme Mdph-4′-OGT was used to screen 17 sequences across species for TBL synthesis, of which seven exhibited catalytic activity. Notably, PT577 exhibited an unparalleled 97.3% conversion yield within 3 h. We then optimized the reaction conditions of PT577, attaining a peak TBL bioproduction of 163.3 mg/L. By employing virtual screening, we identified 25 mutation sites for PT577, thereby creating mutant strains that reduced by-products by up to 50%. This research enhances the enzymatic precision for TBL biosynthesis and offers a robust foundation for its industrial-scale production, with broader implications for the engineering and in silico analysis of glycosyltransferases.

The removal of antibiotics from wastewater to prevent their environmental accumulation is significant for human health and ecosystems. Herein, iron (Fe)-atom-doped anatase TiO2 nanofibers (Fe-TNs) were manufactured for the photocatalytic Fenton-like decomposition of tylosin (TYL) under LED illumination. Compared with the pristine TiO2 nanofibers (TNs), the optimized Fe-TNs exhibited improved visible-light-driven photocatalytic Fenton-like activity with a TYL degradation efficiency of 98.5% within 4 h. The effective TYL degradation could be attributed to the expanded optical light absorption and accelerated separation and migration of photogenerated electrons and holes after the introduction of Fe. The photogenerated electrons were highly conducive to the generation of active SO4•− radicals as they facilitated Fe(III)/Fe(II) cycles, and to oxidizing TYL. Moreover, the holes could be involved in TYL degradation. Thus, a significant enhancement in TYL degradation could be achieved. This research verifies the use of iron-doped anatase nanofibers as an effective method to synthesize novel photocatalytic Fenton-like catalysts through surface engineering for wastewater remediation.

Amaranth plants contain abundant betalains and flavonoids. Anthocyanins are important flavonoids; however, they cannot coexist in the same plant with betalains. Blue light influences metabolite synthesis and hypocotyl elongation; accordingly, analyses of its effects on betalain and flavonoid biosynthesis in Amaranthus tricolor may provide insight into the distribution of these plant pigments. We analyzed the betalain and flavonoid content and transcriptome profiles in amaranth hypocotyls under blue light and dark conditions. Furthermore, we analyzed the expression patterns of key genes related to betalains and flavonoids. Amaranth hypocotyls were shorter and redder and showed higher betalain and flavonoid content under blue light than in dark conditions. Key genes involved in the synthesis of betalains and flavonoids were upregulated under blue light. The gene encoding DELLA was also upregulated. These results suggest that blue light favors the synthesis of both betalains and flavonoids via the suppression of bioactive gibberellin and the promotion of DELLA protein accumulation, which also suppresses hypocotyl elongation. The metabolite profiles differed between plants under blue light and dark conditions. These findings improve our understanding of the environmental cues and molecular mechanisms underlying pigment variation in Amaranthus.

Fabric-reinforced thin film composite (TFC) membranes exhibit outstanding mechanical durability over free-standing membranes for commercial applications. In this study, polyethylene glycol (PEG) was incorporated to modify the polysulfone (PSU) supported fabric-reinforced TFC membrane for forward osmosis (FO). The effects of PEG content and molecular weight on the structure, material property and FO performance of the membrane were investigated comprehensively, and the corresponding mechanisms were revealed. The membrane prepared by using 400 g/mol PEG exhibited better FO performances than those of membranes with 1000 and 2000 g/mol PEG, and 20 wt.% was demonstrated to be the optimal PEG content in the casting solution. The permselectivity of the membrane was further improved by reducing the PSU concentration. The optimal TFC-FO membrane had a water flux (Jw) of 25.0 LMH using deionized (DI) water feed and 1 M NaCl draw solution, and the specific reverse salt flux (Js/Jw) was as low as 0.12 g/L. The degree of internal concentration polarization (ICP) was significantly mitigated. The membrane behaved superior to the commercially available fabric-reinforced membranes. This work provides a simple and low-cost approach in the development TFC-FO membrane and shows great potential in the large-scale production for practical applications.

With the switchability between transparent and light-scattering states, polymer-dispersed liquid crystals (PDLC) are widely used as smart windows, flexible display devices, projectors, and other devices. In outdoor applications, in addition to excellent electro-optical properties, there is also a high demand for film stability. In this work, a PDLC film with high mechanical strength and structural stability is prepared that can maintain stability at 80 °C for 2000 h. By choosing liquid crystals with a wide temperature range, adopting acrylate polymer monomers containing hydroxyl groups, and adjusting the polymer content, the PDLC film can work well from −20 °C to 80 °C. On this basis, the effects of the introduction of rigid monomers on the mechanical properties and electro-optical properties of PDLC films are investigated.

With the wide application of nuclear energy, the problem of radioactive pollution has attracted worldwide attention, and the research on the treatment of radioactive wastewater is imminent. How to treat radioactive wastewater deeply and efficiently has become the most critical issue in the development of nuclear energy technology. The radioactive wastewater produced after using nuclear technology has the characteristics of many kinds, high concentration, and large quantity. Therefore, it is of great significance to study the treatment technology of radioactive wastewater in reprocessing plants. The process flow and waste liquid types of the post-treatment plant are reviewed. The commonly used evaporation concentration, adsorption, precipitation, ion exchange, biotechnology, membrane separation, and photocatalysis are summarized. The basic principles and technological characteristics of them are introduced. The advantages and disadvantages of different single and combined processes are compared, and the development trend of future processing technology is prospected.

Neuroblastoma has obvious heterogeneity. It is one of the few undifferentiated malignant tumors that can spontaneously degenerate into completely benign tumors. However, for its high-risk type, even with various intensive treatment options, the prognosis is still unsatisfactory. At the same time, a large number of research data show that the abnormal amplification and high-level expression of the MYCN gene are positively correlated with the malignant progression, poor prognosis, and mortality of neuroblastoma. In this context, this article explores the role of the N-Myc, MYCN gene expression product on its target genes related to the cell cycle and reveals its regulatory network in promoting tumor proliferation and malignant progression. We hope it can provide ideas and direction for the research and development of drugs targeting N-Myc and its downstream target genes.

Genetic improvement of milk fatty acid content traits in dairy cattle is of great significance. However, chromatography-based methods to measure milk fatty acid content have several disadvantages. Thus, quick and accurate predictions of various milk fatty acid contents based on the mid-infrared spectrum (MIRS) from dairy herd improvement (DHI) data are essential and meaningful to expand the amount of phenotypic data available. In this study, 24 kinds of milk fatty acid concentrations were measured from the milk samples of 336 Holstein cows in Shandong Province, China, using the gas chromatography (GC) technique, which simultaneously produced MIRS values for the prediction of fatty acids. After quantification by the GC technique, milk fatty acid contents expressed as g/100 g of milk (milk-basis) and g/100 g of fat (fat-basis) were processed by five spectral pre-processing algorithms: first-order derivative (DER1), second-order derivative (DER2), multiple scattering correction (MSC), standard normal transform (SNV), and Savitzky–Golsy convolution smoothing (SG), and four regression models: random forest regression (RFR), partial least square regression (PLSR), least absolute shrinkage and selection operator regression (LassoR), and ridge regression (RidgeR). Two ranges of wavebands (4000~400 cm−1 and 3017~2823 cm−1/1805~1734 cm−1) were also used in the above analysis. The prediction accuracy was evaluated using a 10-fold cross validation procedure, with the ratio of the training set and the test set as 3:1, where the determination coefficient (R2) and residual predictive deviation (RPD) were used for evaluations. The results showed that 17 out of 31 milk fatty acids were accurately predicted using MIRS, with RPD values higher than 2 and R2 values higher than 0.75. In addition, 16 out of 31 fatty acids were accurately predicted by RFR, indicating that the ensemble learning model potentially resulted in a higher prediction accuracy. Meanwhile, DER1, DER2 and SG pre-processing algorithms led to high prediction accuracy for most fatty acids. In summary, these results imply that the application of MIRS to predict the fatty acid contents of milk is feasible.

Esophageal squamous cell carcinoma is a severe malignancy for its high mortality and poor prognosis. Mainstay chemotherapies cause serious side effects for their ways of inducing cell death. Oridonin is the main bioactive constituent from natural plants that has anticancer ability and weak side effects. The proteomics method is efficient to understand the anticancer mechanism. However, proteins identified by proteomics aimed at understanding oridonin’s anticancer mechanism is seldom overlapped by different groups. This study used proteomics based on two-dimensional electrophoresis sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2-DE SDS-PAGE) integrated with mass spectrometry and Gene Set Enrichment Analysis (GSEA) to understand the anticancer mechanism of oridonin on esophageal squamous cell carcinoma (ESCC). The results showed that oridonin induced ESCC cell death via apoptosis by decreasing the protein expression of LASP1 and PDLIM1.

Tuberculosis remains an important cause of morbidity and mortality throughout the world. Notably, an important number of multi drug resistant cases is an increasing concern. This problem points to an urgent need for novel compounds with antimycobacterial properties and to improve existing therapies. Whole-cell-based screening for compounds with activity against Mycobacterium tuberculosis complex strains in the presence of linezolid was performed in this study. A set of 15 bioactive compounds with antimycobacterial activity in vitro were identified with a minimal inhibitory concentration of less than 2 µg/mL. Among them, compound 1 is a small molecule with a chemical structure consisting of an adamantane moiety and a hydrazide–hydrazone moiety. Whole genome sequencing of spontaneous mutants resistant to the compounds suggested compound 1 to be a new inhibitor of MmpL3. This compound binds to the same pocket as other already published MmpL3 inhibitors, without disturbing the proton motive force of M. bovis BCG and M. smegmatis. Compound 1 showed a strong activity against a panel ofclinical strains of M. tuberculosis in vitro. This compound showed no toxicity against mammalian cells and protected Galleria mellonella larvae against M. bovis BCG infection. These results suggest that compound 1 is a promising anti-TB agent with the potential to improve TB treatment in combination with standard TB therapies.

Cotton is one of the Uyghur medical materials in China and is rich in flavonoids. Flavonoids have important pharmacological effects. The yield of flavonoids in traditional extraction methods is low, which affects the development of flavonoids. Therefore, it is urgent to optimize the extraction techniques. The yield of flavonoids in cotton flowers was effectively improved by response surface methodology, and the highest yield of flavonoids reached 5.66%, and the optimal extraction process conditions were obtained. The DPPH free radical scavenging rate, hydroxyl free radical scavenging rate, superoxide anion free radical scavenging rate, and reducing ability were tested to reflect the antioxidant capacity of flavonoids. The flavonoids had an excellent antioxidant effect. Cell experiments suggested that the flavonoids had the effect of protecting glutamate-induced damage to HT-22 cells. The results of this study provide a theoretical basis for the extraction of cotton flowers flavonoids and the comprehensive evaluation of antioxidant products, as well as the extraction of other plant flavonoids.

A series of novel pleuromutilin derivatives containing nitrogen groups on the side chain of C14 were synthesized under mild conditions. Most of the synthesized derivatives displayed potent antibacterial activities. Compound 9 was found to be the most active antibacterial derivative against MRSA (MIC = 0.06 μg/mL). Furthermore, the result of time-kill curves showed that compound 9 had a certain inhibitory effect against MRSA in vitro. Moreover, according to a surface plasmon resonance (SPR) study, compound 9 (KD = 1.77 × 10−8 M) showed stronger affinity to the 50S ribosome than tiamulin (KD = 2.50 × 10−8 M). The antibacterial activity of compound 9 was further evaluated in an MRSA-infected murine thigh model. Compared to the negative control group, tiamulin reduced MRSA load (~0.7 log10 CFU/mL), and compound 9 performed a treatment effect (~1.3 log10 CFU/mL). In addition, compound 9 was evaluated in CYP450 inhibition assay and showed only moderate in vitro CYP3A4 inhibition (IC50 = 2.92 μg/mL).

The well-known toxic medicine Gelsemium elegans is widely and historically used to treat bone fracture and skin ulcers by the folk people of China. Two new monoterpenoid indole alkaloids, gelselegandines D and E, together with the known analogue gelegamine A were isolated from G. elegans. Their structures were elucidated by means of spectroscopic techniques and quantum chemical calculations. All isolated compounds were tested for the effects on RANKL-induced osteoclast formation. Interestingly, gelselegandine E and gelegamine A, respectively, showed significant promoting and inhibitory activities on osteoclastogenesis, while gelselegandine D had no activity under the same concentration. This work suggested the different configurations for the carbons near the C-19/20 oxygen rings of the isolated compounds may be the key active groups on osteoclast formation and provided the evidence for the rationality as the traditional treatment for bone-related diseases of G. elegans.

Epigenetic regulation plays a critical role in controlling fungal secondary metabolism. Here, we report the pleiotropic effects of the epigenetic regulator HdaA (histone deacetylase) on secondary metabolite production and the associated biosynthetic gene clusters (BGCs) expression in the plant endophytic fungus Penicillium chrysogenum Fes1701. Deletion of the hdaA gene in strain Fes1701 induced a significant change of the secondary metabolite profile with the emergence of the bioactive indole alkaloid meleagrin. Simultaneously, more meleagrin/roquefortine-related compounds and less chrysogine were synthesized in the ΔhdaA strain. Transcriptional analysis of relevant gene clusters in ΔhdaA and wild strains indicated that disruption of hdaA had different effects on the expression levels of two BGCs: the meleagrin/roquefortine BGC was upregulated, while the chrysogine BGC was downregulated. Interestingly, transcriptional analysis demonstrated that different functional genes in the same BGC had different responses to the disruption of hdaA. Thereinto, the roqO gene, which encodes a key catalyzing enzyme in meleagrin biosynthesis, showed the highest upregulation in the ΔhdaA strain (84.8-fold). To our knowledge, this is the first report of the upregulation of HdaA inactivation on meleagrin/roquefortine alkaloid production in the endophytic fungus P. chrysogenum. Our results suggest that genetic manipulation based on the epigenetic regulator HdaA is an important strategy for regulating the productions of secondary metabolites and expanding bioactive natural product resources in endophytic fungi.

The aim of this paper was to seek an efficient method to preparative separation of alkaloid compounds from Aconitum coreanum (Guanbaifu), a well-known traditional Chinese medicinal plant for heart disease. Seven alkaloid compounds were successfully purified by pH-zone-refining counter-current chromatography with two-phase solvent system of petroleum ether–ethyl acetate–methanol–water (5:5:1:9, v/v/v/v), 10 mM triethylamine in upper phase and 10 mM hydrochloric acid in lower phase. From 3.5 g of crude extract, 356 mg of Guanfu base I, 578 mg of Guanfu base A, 74 mg of atisine, 94 mg of Guanfu base F, 423 mg of Guanfu base G, 67 mg of Guanfu base R and 154 mg of Guanfu base P were obtained with the purity of 96.40%, 97.2%, 97.5%, 98.1%, 98.9%, 98.3% and 98.4%. Their chemical structures were identified by TOF-MS and 1H-NMR. This study indicated that pH-zone-refining counter-current chromatography was an efficient method for separating the kind of alkaloids with low absorbance values.

Two high-speed countercurrent chromatography (HSCCC) modes were compared by separation of major alkaloids from crude extract of Picrasma quassioides. The conventional HSCCC separation was performed with a two-phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (5:5:4.5:5.5, v/v/v/v) with 200 mg loading. pH-Zone-refining CCC was performed with two-phase solvent system composed of petroleum ether–ethyl acetate–n-butanol–water (3:2:7:9, v/v/v/v) where triethylamine (10 mM) was added to the upper organic stationary phase and hydrochloric acid (5 mM) was added to the lower aqueous phase with 2 g loading. From 2 g of crude extract, 87 mg of 5-methoxycanthin-6-one (a), 38 mg of 1-methoxy-β-carboline (b), 134 mg of 1-ethyl-4,8-dimethoxy-β-carboline (c), 74 mg of 1-ethoxycarbonyl-β-carboline (d), 56 mg of 1-vinyl-4,8-dimethoxy-β-carboline (e) and 26 mg of 1-vinyl-4-dimethoxy-β-carboline (f) were obtained with purities of over 97.0%. The results indicated that pH-zone-refining CCC is an excellent separations tool at the multigram level.

To explore the transformation mechanisms of free gastrodin and combined gastrodin before and after steaming of Gastrodia elata (G. elata), a fresh G. elata sample was processed by the traditional steaming method prescribed by Chinese Pharmacopoeia (2015 version), and HPLC-ESI-TOF/MS method was used to identify the chemical composition in steamed and fresh G. elata. Finally, 25 components were identified in G. elata based on the characteristic fragments of the compounds and the changes of the 25 components of fresh and steamed G. elata were compared by the relative content. Hydrolysis experiments and enzymatic hydrolysis experiments of 10 monomer compounds simulating the G. elata steaming process were carried out for the first time. As a result, hydrolysis experiments proved that free gastrodin or p-hydroxybenzyl alcohol could be obtained by breaking ester bond or ether bond during the steaming process of G. elata. Enzymatic experiments showed that steaming played an important role in the protection of gastrodin, confirming the hypothesis that steaming can promote the conversion of chemical constituents of G. elata—inhibiting enzymatic degradation. This experiment clarified the scientific mechanism of the traditional steaming method of G. elata and provided reference for how to apply G. elata decoction to some extent.

Seven diterpene lactones, andrographolide (1), isoandrographolide (2), neo-andrographolide (3), 14-deoxy-11,12-didehydroandrographolide (4), 14-deoxyandrographiside (5), 14-deoxy-11,12-didehydroandrographiside (6), 3,14-dideoxyandrographolide (10), and three flavones, andrographidine C (7), andrographidine A (8), 5-hydroxy-7,8-dimethoxyflavanone (9) have been successfully and efficiently isolated from A. paniculata using an off-line two dimensional (2D) high-speed counter-current chromatography (HSCCC) method for the first time. For the first dimension HSCCC separation, petroleum ether-ethyl acetate-methanol-water 3:7:5:5 (v/v) was employed to isolate 14.4 mg of compound 1, 3.1 mg of compound 2, 7.8 mg of compound 3, and 18.0 mg of compound 4 from 200 mg of the A. paniculata extract. For the second dimension HSCCC separation, petroleum ether-ethyl acetate-methanol-water 2:8:1:9 (v/v) and 5:5:6:4 (v/v) were employed to isolate the collected fractions ranged from 55 to 79 min and the flow out fraction, respectively, which led to 5.1 mg of compound 5, 4.4 mg of compound 6, 2.4 mg of compound 7, 3.3 mg of compound 8, 4.0 mg of compound 9, 7.0 mg of compound 10. The structures of these diterpene lactones and flavones were elucidated by extensive spectroscopic methods.

Gastrodia elata Blume (G. elata) is a prominent traditional herb and its dry tuber is officially listed in the Chinese Pharmacopoeia. To ensure the quality of dried G. elata, the establishment of a nondestructive and convenient method to monitor the drying process is necessary. In this study, a nondestructive low-field nuclear magnetic resonance (LF-NMR) and magnetic resonance imaging (MRI) method was introduced to monitor the drying process of G. elata. Three water states (bound, immobilized, and free) in G. elata samples were investigated through multiexponential fitting and inversion of the NMR data. The variation and distribution of the three water states during drying were monitored by LF-NMR, and the spatial distribution of water and internal structural changes were analyzed by MRI. Linear analysis of the moisture content, L* (lightness), b* (yellowness), and NMR parameters showed good correlations among them. Furthermore, partial least squares regression (PLSR) model analysis, which takes into account all NMR parameters, also showed good correlations among these parameters. All results showed that LF-NMR was feasible and convenient for monitoring moisture content. Therefore, LF-NMR and MRI could be used to monitor the moisture content nondestructively in the drying process of Chinese traditional herbs.

pH-Zone refining counter-current chromatography was successfully applied to the preparative isolation and purification of six alkaloids from the ethanol extracts of Uncaria macrophylla Wall. Because of the low content of alkaloids (about 0.2%, w/w) in U. macrophylla Wall, the target compounds were enriched by pH-zone refining counter-current chromatography using a two-phase solvent system composed of petroleum ether–ethyl acetate–isopropanol–water (2:6:3:9, v/v), adding 10 mM triethylamine in organic stationary phase and 5 mM hydrochloric acid in aqueous mobile phase. Then pH-zone refining counter-current chromatography using the other two-phase solvent system was used for final purification. Six target compounds were finally isolated and purified by following two-phase solvent system composed of methyl tert-butyl ether (MTBE)–acetonitrile–water (4:0.5:5, v/v), adding triethylamine (TEA) (10 mM) to the organic phase and HCl (5 mM) to aqueous mobile phase. The separation of 2.8 g enriched total alkaloids yielded 36 mg hirsutine, 48 mg hirsuteine, 82 mg uncarine C, 73 mg uncarine E, 163 mg rhynchophylline, and 149 mg corynoxeine, all with purities above 96% as verified by HPLC Their structures were identified by electrospray ionization-mass spectrometry (ESI-MS) and 1H-NMR spectroscopy.

A method combining hydrophilic interaction chromatography (HILIC) and electrospray ionization mass spectrometry (ESI-MS) was developed for the characterization and determination of natural Cordyceps. Separation was achieved on a Waters Xbridge Amide column with gradient elution. Identification of 15 target nucleosides and nucleobases was based on retention time, UV spectra and mass measurements of the protonated molecules ([M+H]+) and main fragment ions (ESI-TOF/MS). Eight non-target compounds were tentatively identified by ESI-TOF/MS. The 15 target compounds were quantified by HILIC-ESI-MS/MS using time-programmed selective ion monitoring or multiple reaction monitoring in positive-ion mode under optimized mass conditions. This technique showed good linearity, repeatability and recovery. This approach was also successfully implemented in the analysis of nucleosides and nucleobases in 12 batches of natural Cordyceps samples that were collected from different regions in China. The developed HILIC-ESI-MS method exhibited clear advantages in identifying and determining highly polar bioactive components in Cordyceps, as well as their quality control.

pH-Zone-refining counter-current chromatography (pH-zone-refining CCC) was successfully applied for the large-scale separation of alkaloids from Corydalis bungeana. The crude extract was separated by a two-phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (5:5:2:8, v/v) where triethylamine (10 mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as a displacer. As a result, 285 mg of protopine, 86 mg of corynoloxine, 430 mg of coryno1ine, and 115 mg of acetylcorynoline were obtained from 3.0 g of crude extract in a one-step separation. The purities of these compounds were 99.1%, 98.3%, 99.0% and 98.5%, respectively, as determined by HPLC. The chemical structures of these isolated compounds were confirmed by ESI-MS, 1H-NMR and 13C-NMR.

We previously reported 18F-labeled pyrazolo[1,5-a]pyrimidine derivatives: 7-(2-[18F]fluoroethylamino)-5-methylpyrazolo[1,5-a]pyrimidine-3-carbonitrile ([18F]1) and N-(2-(3-cyano-5-methylpyrazolo[1,5-a]pyrimidin-7-ylamino)ethyl)-2-[18F]fluoro-4-nitro- benzamide ([18F]2). Preliminary biodistribution experiments of both compounds showed s slow clearance rate from excretory tissues which warranted further investigation for tumor imaging with PET. Here we modified [18F]1 and [18F]2 by introducing polar groups such as ester, hydroxyl and carboxyl and developed three additional 18F-18 labeled pyrazolo[1,5-a] pyrimidine derivatives: (3-Cyano-7-(2-[18F]fluoroethylamino)pyrazolo[1,5-a]-pyrimidin-5- yl)methyl acetate ([18F]3), 7-(2-[18F]fluoroethylamino)-5-(hydroxymethyl)pyrazolo[1,5-a]- pyrimidine-3-carbonitrile ([18F]4) and (S)-6-(3-cyano-5-methylpyrazolo[1,5-a]pyrimidin-7-ylamino)-2-(2-[18F]fluoro-4-nitrobenzamido)hexanoic acid ([18F]5). The radiolabeled probes were synthesized by nucleophilic substitution of the corresponding tosylate and nitro precursors with 18F-fluoride. In Vitro studies showed higher uptake of [18F]3 and [18F]4 than that of [18F]5 by S180 tumor cells. In Vivo biodistribution studies in mice bearing S180 tumors showed that the uptake of both [18F]3 and [18F]4 in tumors displayed an increasing trend while the uptake of [18F]5 in tumor decreased through the course of the 120 min study. This significant difference in tumor uptake was also found between [18F]1 and [18F]2. Thus, we compared the biological behavior of the five tracers and reported the tumor uptake kinetic differences between 2-[18F]fluoroethylamino- and 2-[18F]fluoro-4-nitro- benzamidopyrazolo[1,5-a] pyrimidine derivatives.

Treatment of substituted benzyl alcohols with tosyl chloride resulted in the formation of the corresponding chlorides, not the usual tosylates. A series of experiments demonstrated that it was possible to predict whether chlorination or tosylation would occur for substituted benzyl alcohols and pyridine methanols. Treatment of electron withdrawing group-substituted benzyl alcohols with tosyl chloride gave the corresponding chlorides in moderate yields under mild conditions, which provided a simple way to directly prepare chlorides from alcohols.

Three phenylethanoid glycosides, echinacoside (1), salidroside (3), and acteoside (6), and three secoiridoid glycosides, isonuezhenide (2), nuezhenoside G13 (4), and specnuezhenide (5), have been extracted and separated by a combined method of ultrahigh pressure extraction (UPE) and high-speed counter-current chromatography (HSCCC) from Ligustri Lucidi Fructus. For the UPE, the optimal extraction was developed with conditions including solvent of 90% ethanol, sample to solvent ratio of 1:20 g/mL, pressure of 200 MPa, and time of 2 min, which rendered the yields of compounds 4 and 5 were 15.0 and 78.0 mg/g, respectively. For the HSCCC separation, the strategy of changing flow rates between 1.0 and 2.0 mL/min allowed the acquisition for 2.7 mg of compound 1, 4.5 mg of compound 2, 6.8 mg of compound 3, 5.9 mg of compound 4, 11.2 mg of compound 5, and 2.2 mg of compound 6 in one separation run under the solvent system of ethyl acetate:n-butanol:water (2:1:3, v/v) from 200 mg of the UPE extract. The structures of these phenylethanoid and secoiridoid glycosides were elucidated by extensive spectroscopic methods.

The compound 5-((2-aminoethylamino)methyl)-7-(4-bromoanilino)-3-cyano-pyrazolo[1,5-a]pyrimidine (ABCPP) was synthesized and conjugated with N-mercapto-acetylglycine (MAG), N-mercaptoacetylphenylalanine (MAF) and N-mercaptoacetylvaline (MAA), respectively. These three compounds were labeled successfully with [99mTcN]2+ intermediate in high radiochemical purities. Biodistribution in tumor-bearing mice demonstrated that the three new complexes showed tumor accumulation, high tumor-to-muscle (T/M) ratios and fast clearance from blood and muscle. Among them, the 99mTcN-MAG-ABCPP showed the most favorable characteristics, with tumor/blood and tumor/ muscle ratios reaching 1.51 and 2.97 at 30 min post-injection, 1.84 and 2.49 at 60 min post-injection, suggesting it could be further studied as potential tumor imaging agent for single photon emission computed tomography (SPECT).

One new monoterpene glycoside (1), one new phenyl glycoside (2), one new caffeoyl derivative (3), were isolated from Scindapsus officinalis (Roxb.) Schott., along with four known compounds (4–7). Structures of the isolated compounds were elucidated by extensive analysis of spectroscopic data, especially 2D NMR data and comparison with literatures. All isolates were evaluated for anti-inflammatory activity against nitric oxide (NO) production in vitro. Compounds 3 and 7 exhibited moderate inhibitory effects on NO production with IC50 values of 12.2 ± 0.8 and 18.9 ± 0.3 μM, respectively.

High-speed counter-current chromatography was used to separate and purify galloyl, caffeoyl, and hexahydroxydiphenoyl esters of glucoses from the aerial parts of the parasitic plant Balanophora simaoensis for the first time using n-hexane-ethyl acetate-methanol-water (1:2:1:2, v/v) as the optimum solvent system. Accordingly, 1-O-(E)-caffeoyl-3-O-galloyl-β-d-glucopyranose (I, 12.5 mg), 1-O-(E)-caffeoyl-3-O-galloyl-4,6-(S)-hexahydroxydiphenoyl-β-d-glucopyranose (II, 27.2 mg), and 1-O-(E)-caffeoyl-4,6-(S)-hexahydroxydiphenoyl-β-d-glucopyranose (III, 52.8 mg) with 98.0%, 98.5%, and 98.7% purities, respectively, were purified from 210 mg crude extract of B. simaoensis in a one-step separation. The structures of the glucose esters were identified by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance spectra (NMR). Their antioxidant activities were evaluated by measuring their inhibition activity on liver microsomal lipid peroxidation induced by the Fe2+-Cys system in vitro. Compounds I–III showed significant antioxidant activities with IC50 values ranging from 2.51 to 6.68 μm, respectively.

Chemical composition of secondary metabolites is of great importance for quality control of agricultural products. Black sesame seeds are significantly more expensive than white sesame seeds, because it is thought that black sesame seeds are more beneficial to human health than white sesame seeds. However, the differences in nutrient composition between black sesame seeds and white sesame seeds are still unknown. The current study examined the levels of different metabolites in black and white sesame seeds via the use of a novel metabolomics strategy. Using widely targeted metabolomics data, we obtained the structure and content of 557 metabolites, out of which 217 metabolites were identified, and discovered 30 metabolic pathways activated by the secondary metabolites in both black and white sesame seeds. Our results demonstrated that the main pathways that were differentially activated included: phenylpropanoid biosynthesis, tyrosine metabolism, and riboflavin metabolism. More importantly, the biomarkers that were significantly different between black seeds and white sesame seeds are highly related to the functions recorded in traditional Chinese medicine. The results of this study may serve as a new theoretical reference for breeding experts to promote the genetic improvement of sesame seeds, and therefore the cultivation of higher quality sesame varieties.

Wheat aphids are major pests during the seed filling stage of wheat. Plant lectins are toxic to sap-sucking pests such as wheat aphids. In this study, Pinellia pedatisecta agglutinin (ppa), a gene encoding mannose binding lectin, was cloned, and it shared 92.69% nucleotide similarity and 94% amino acid similarity with Pinellia ternata agglutinin (pta). The ppa gene, driven by the constitutive and phloem-specific ribulose bisphosphate carboxylase small subunit gene (rbcs) promoter in pBAC-rbcs-ppa expression vector, was transferred into the wheat cultivar Baofeng104 (BF104) by particle bombardment transformation. Fifty-four T0 transgenic plants were generated. The inheritance and expression of the ppa gene were confirmed by PCR and RT-PCR analysis respectively, and seven homozygous transgenic lines were obtained. An aphid bioassay on detached leaf segments revealed that seven ppa transgenic wheat lines had lower aphid growth rates and higher inhibition rates than BF104. Furthermore, two-year aphid bioassays in isolated fields showed that aphid numbers per tiller of transgenic lines were significantly decreased, compared with wild type BF104. Therefore, ppa could be a strong biotechnological candidate to produce aphid-resistant wheat.

Burdock roots are healthy dietary supplements and a kind of famous traditional Chinese medicine, which contains large amounts of caffeoylquinic acid derivatives. However, little research has been reported on the preparative separation of these compounds from burdock roots. In the present study, a combinative method of HSCCC and semi-preparative HPLC was developed for the semi-preparative separation of caffeoylquinic acid derivatives from the burdock roots. The ethyl acetate extract of burdock roots was first fractionated by MCI macroporous resin chromatography and give three fractions (Fr. 1–3) from the elution of 40% methanol. Then, these three fractions (120 mg) were separately subjected to HSCCC for purification with the solvent system composed of petroleum ether-ethyl acetate-methanol-water at different volume ratios, and the mixtures were further purified by semi-preparative HPLC. As a result, a total of eight known caffeoylquinic acid derivatives including 3-O-caffeoylquinic acid (32.7 mg, 95.7%), 1,5-O- dicaffeoylquinic acid (4.3 mg, 97.2%), 3-O-caffeoylquinic acid methyl ester (12.1 mg, 93.2%), 1,3-O-dicaffeoylquinic acid (42.9 mg, 91.1%), 1,5-O-dicaffeoyl-3-O-(4-maloyl)-quinic acid (4.3 mg, 84.5%), 4,5-O-dicaffeoylquinic acid (5.3 mg, 95.5%), 1,5-O-dicaffeoyl-3-O-succinylquinic acid (8.7 mg, 93.4%), and 1,5-O-dicaffeoyl-4-O-succinylquinic acid (1.7 mg, 91.8%), and two new compounds were obtained. The new compounds were 1,4-O-dicaffeoyl-3-succinyl methyl ester quinic acid (14.6 mg, 96.1%) and 1,5-O-dicaffeoyl-3-O-succinyl methyl ester quinic acid (3.1 mg, 92.6%), respectively. The research indicated that the combination of HSCCC and semi-preparative HPLC is a highly efficient approach for preparative separation of the instability and bioactive caffeoylquinic acid derivatives from natural products.

An offline two-dimensional recycling high-speed countercurrent chromatography (2D R-HSCCC) strategy with extrusion mode was developed for isolating polyphenols from the rhizome of Smilax glabra. Firstly, the ethyl acetate extract was divided into two fractions, Fr.1 and Fr.2, by silica gel column chromatography. Then, HSCCC was applied to separate polyphenols from the two fractions using a solvent system consisting of petroleum ether–ethyl acetate–methanol–water (1:3:0.5:5, v/v). Fifty milligrams of Fr.1 was separated by conventional HSCCC, yielding 5-O-caffeoylshikimic acid (1, 15.8 mg) and taxifolin (2, 4.8 mg). Offline 2D R-HSCCC with extrusion mode was used to separate Fr.2, and astilbin (4, 37.3 mg), neoisoastilbin (5, 8.8 mg), engeletin (7, 7.9 mg), and a mixture of two polyphenols were obtained from 100 mg of Fr.2. The mixture of two polyphenols was further separated by pre-HPLC, yielding neoastilbin (3, 15.2 mg) and isoastilbin (6, 9.9 mg). The purities of these seven compounds were all over 96.0%. Their structures were identified by MS and NMR. The results demonstrated that the strategy based on offline 2D R-HSCCC with extrusion mode was a powerful tool to separate the main compounds from the rhizome of Smilax glabra and valued for the preparative separation compounds with broad K-values and similar structures.

Diaphragma juglandis fructus contains various bioactive constituents. Fourteen compounds were isolated from Diaphragma juglandis fructus by preparative high performance liquid chromatography (pre-HPLC) and high-speed counter-current chromatography (HSCCC). Their structures were identified by nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS). Compounds (+)-dehydrovomifoliol (12), (6R,9R)-9-hydroxymegastigman-4-en-3-one (13) and (6R,9S)-9-hydroxymegastigman-4-en-3-one (14) are found from Juglans regia L. for the first time. Compounds dihydrophaseic acid (2), blumenol B (3) and (4S)-4-hydroxy-1-tetralone (11) are isolated from Diaphragma juglandis fructus for the first time. The anti-inflammatory effects of isolated compounds were evaluated by an in vitro model of lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Compounds gallic acid (1), ethyl gallate (9) and (+)-dehydrovomifoliol (12) exhibited inhibitory activity on the nitric oxide production of RAW 264.7 at a concentration of 25 μM. The result indicated that the combination HSCCC with pre-HPLC is an effective way for compound separation and purification. And Diaphragma juglandis fructus constituents have the potential for the treatment of inflammatory-related diseases.

Ten compounds, including three lignan glycosides and seven lignans, were purified from Justicia procumbens L. in 8 h using an efficient strategy based on high-speed counter-current chromatography (HSCCC). The two-phase solvent system composed of petroleum–ethyl acetate–methanol–H2O (1:0.7:1:0.7, v/v) was firstly employed to separate the crude extract (320 mg), from which 19.3 mg of justicidin B (f), 10.8 mg of justicidin A (g), 13.9 mg of 6′-hydroxyjusticidin C (h), 7.7 mg of justicidin E (i), 6.3 mg of lignan J1 (j) were obtained with 91.3 mg of enriched mixture of compounds a–e. The enriched mixture (91.3 mg) was further separated using the solvent system consisting of petroleum–ethyl acetate–methanol–H2O (3:3.8:3:3.8, v/v), yielding 12.1 mg of procumbenoside E (a); 7.6 mg of diphyllin-1-O-β-d-apiofuranoside (b); 7.4 mg of diphyllin (c); 8.3 mg of 6′-hydroxy justicidin B (d); and 7.9 mg of diphyllin acetyl apioside (e). The purities of the 10 components were all above 94%, and their structures were identified by NMR and ESI-MS spectra. The results demonstrated that the strategy based on HSCCC for the separation of lignans and their glycosides was efficient and rapid.

High-speed counter-current chromatography was used to separate and purify ecdysteroids for the first time from the stems of Diploclisia glaucescens using a two-phase solvent system composed of ethyl acetate–n-butanol–ethanol–water (3:0.2:0.8:3, v/v). Three ecdysteroids were obtained from 260 mg of ethyl acetate extract of the residue obtained after evaporation of the crude ethanolicextractof D. glaucescens in one-step separation, which were identified as paristerone (I, 30.5 mg), ecdysterone (II, 7.2 mg), and capitasterone (III, 8.1 mg) by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR). Their anti-inflammatory activities were evaluated by measuring the inhibitory ratios of β-glucuronidase release in rat polymorphonuclear leukocytes (PMNs) induced by platelet-activating factor. Compounds I–III showed significant anti-inflammatory activities with IC50-values ranging from 1.51 to 11.68 μM, respectively.

In this work, flavonoid fraction from the leaves of Crataegus pinnatifida was separated into its seven main constituents using a combination of HSCCC coupled with pre-HPLC. In the first step, the total flavonoid extract was subjected to HSCCC with a two-solvent system of chloroform/methanol/water/n-butanol (4:3:2:1.5, v/v), yielding four pure compounds, namely (–)-epicatechin (1), quercetin-3-O-(2,6-di-α-l-rhamnopyranosyl)-β-d-galactopyranoside (2), 4′′-O-glucosylvitexin (3) and 2′′-O-rhamnosylvitexin (4) as well as a mixture of three further flavonoids. An extrusion mode was used to rapidly separate quercetin-3-O-(2,6-di-α-l-rhamnopyranosyl)-β-d-galactopyranoside with a big KD-value. In the second step, the mixture that resulted from HSCCC was separated by pre-HPLC, resulting in three pure compounds including: vitexin (5), hyperoside (6) and isoquercitrin (7). The purities of the isolated compounds were established to be over 98%, as determined by HPLC. The structures of these seven flavonoids were elucidated by ESI-MS and NMR spectroscopic analyses.

In this work, the n-butanol extract from leaves of Lonicera japonica Thunb. (L. japonica) was reacted with DPPH and subjected to a HPLC analysis for the guided screening antioxidants (DPPH-HPLC experiments). Then, nine antioxidants, including flavonoid glycosides and caffeoylquinic acid derivatives, were isolated and purified from leaves of L. japonica using high speed counter-current chromatography (HSCCC) and prep-HPLC. The n-butanol extract was firstly isolated by HSCCC using methyl tert-butyl ether/n-butanol/acetonitrile/water (0.5% acetic acid) (2:2:1:5, v/v), yielding five fractions F1, F2 (rhoifolin), F3 (luteoloside), F4 and F5 (collected from the column after the separation). The sub-fractions F1, F4 and F5 were successfully separated by prep-HPLC. Finally, nine compounds, including chlorogenic acid (1), lonicerin (2), rutin (3), rhoifolin (4), luteoloside (5), 3,4-Odicaffeoylquinic acid (6), hyperoside (7), 3,5-O-dicaffeoylquinic acid (8), and 4,5-O-dicaffeoylquinic acid (9) were obtained, respectively, with the purities over 94% as determined by HPLC. The structures were identified by electrospray ionization mass spectrometry (ESI-MS), 1H- and 13C-NMR. Antioxidant activities were tested, and the isolated compounds showed strong antioxidant activities.

High-speed counter-current chromatography (HSCCC) has been successfully used for the separation of eight compounds from Chimonanthus praecox flowers. Firstly, the crude extract of Chimonanthus praecox flowers was dissolved in a two-phase solvent system composed of petroleum ether–ethyl acetate–methanol–H2O (5:5:3:7, v/v) and divided into two parts: the upper phase (part I) and the lower phase (part II). Then, HSCCC was applied to separate the phenolic acids from part I and part II, respectively. Considering the broad polarity range of target compounds in part I, a stepwise elution mode was established. Two optimal solvent systems of petroleum ether–ethyl acetate–methanol–H2O–formic acid (FA) (5:5:3:7:0.02, 5:5:4.3:5.7:0.02, v/v) were employed in this separation. Five phenylpropanoids and two flavonoids were successfully separated from 280 mg of part I, including 8.7 mg of 3,4-dihydroxy benzoic acid (a, 95.3% purity), 10.9 mg of protocatechualdehyde (b, 96.8% purity), 11.3 mg of p-coumaric acid (c, 98.9% purity), 12.2 mg of p-hydroxybenzaldehyde (d, 95.9% purity), 24.7 mg of quercetin (e, 97.3% purity), 33.8 mg of kaempferol (f, 96.8% purity), and 24.6 mg of 4-hydroxylcinnamic aldehyde (g, 98.0% purity). From 300 mg of part II, 65.7 mg of rutin (h, 98.2% purity), 7.5 mg of 3,4-dihydroxy benzoic acid (a, 77.4% purity), and 4.7 mg of protocatechualdehyde (b, 81.6% purity) were obtained using the solvent system EtOAc–n-butanol (n-BuOH)–FA–H2O (4:1:0.5:5, v/v). The structures of the eight pure compounds were confirmed by electrospray ionization-mass spectrometry (ESI-MS), 1H-NMR and 13C-NMR. To the best of our knowledge, compounds a–d and f were the first separated and reported from the Chimonanthus praecox flower extract.