Preparation of magnetic dialdehyde starch-immobilized phospholipase A1 and acyl transfer in reflection.

In this paper, using a coprecipitation method to prepare Fe3O4 magnetic nanoparticles (Fe3O4 MNPS), magnetic dialdehyde starch nanoparticles with immobilized phospholipase A1 (MDSNIPLA) were successfully prepared by using green dialdehyde starch (DAS) instead of glutaraldehyde as the crosslinking agent. The Fe3O4 MNPS was characterized by infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), the Brunauer-Emmett-Teller (BET) surface area analysis method, thermogravimetric analysis (TGA), and transmission electron microscopy (TEM) et al. The results showed that the alkaline resistance and acid resistance of the enzyme were improved after the crosslinking of DAS. After repeated use (seven times), the relative activity of MDSNIPLA reached 56 %, and the magnetic dialdehyde starch nanoparticles (MDASN) had good carrier performance. MDSNIPLA was applied to enzymatic hydrolysis of phospholipids in the soybean oil degumming process. The results showed that the acyl transfer rate of sn-2-HPA was 14.01 %, and the content of free fatty acids was 1.144 g/100 g after 2 h reaction at 50 °C and pH 5.0 with appropriate boric acid. The immobilized enzyme has good thermal stability and storage stability, and its application of soybean oil improves the efficiency of the oil.

Mining Xanthine Oxidase Inhibitors from an Edible Seaweed Pterocladiella capillacea by Using In Vitro Bioassays, Affinity Ultrafiltration LC-MS/MS, Metabolomics Tools, and In Silico Prediction.

The prevalence of gout and the adverse effects of current synthetic anti-gout drugs call for new natural and effective xanthine oxidase (XOD) inhibitors to target this disease. Based on our previous finding that an edible seaweed Pterocladiella capillacea extract inhibits XOD, XOD-inhibitory and anti-inflammatory activities were used to evaluate the anti-gout potential of different P. capillacea extract fractions. Through affinity ultrafiltration coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS), feature-based molecular networking (FBMN), and database mining of multiple natural products, the extract's bioactive components were traced and annotated. Through molecular docking and ADMET analysis, the possibility and drug-likeness of the annotated XOD inhibitors were predicted. The results showed that fractions F4, F6, F4-2, and F4-3 exhibited strong XOD inhibition activity, among which F4-3 reached an inhibition ratio of 77.96% ± 4.91% to XOD at a concentration of 0.14 mg/mL. In addition, the P. capillacea extract and fractions also displayed anti-inflammatory activity. Affinity ultrafiltration LC-MS/MS analysis and molecular networking showed that out of the 20 annotated compounds, 8 compounds have been previously directly or indirectly reported from seaweeds, and 4 compounds have been reported to exhibit anti-gout activity. Molecular docking and ADMET showed that six seaweed-derived compounds can dock with the XOD activity pocket and follow the Lipinski drug-like rule. These results support the value of further investigating P. capillacea as part of the development of anti-gout drugs or related functional foods.

Site-Specific Covalent Immobilization of SMA-Stabilized ACE2 for SARS-CoV-2 Recognition and Drug Screening.

Membrane protein (MP)-based biomaterials have a wide range of applications in drug screening, antigen detection, and ligand-receptor interaction analysis. Traditional MP immobilization methods have the disadvantage of disordered protein immobilization orientation, leading to the shielded binding domain and unreliable binding pattern. Herein, we describe a site-specific covalent immobilization of MPs, which utilizes the styrene maleic acid (SMA) detergent-free extraction method of MPs as well as the covalent reaction between His-tag and divinyl sulfone (DVS). As an example, we covalently immobilized angiotensin-converting enzyme 2 (ACE2) on a cell membrane chromatography system (ACE2-His-SMALPs/CMC) in a site-specific manner and verified the specificity and stability of this system. This technique significantly improves the service life compared to the physisorption CMC column. The improved protein immobilization strategies of the ACE2-His-SMALPs/CMC system enable it to effectively recognize SARS-CoV-2 pseudoviral particles as well as detect viral particles in ambient air once combined with an aerosol collector; as a powerful ligand biosensor, the ACE2-His-SMALPs/CMC system was used to screen for compounds with anti-SARS-CoV-2 pseudovirus activity. In conclusion, the optimized MP immobilization strategy has been successfully applied to CMC technology, showing enhanced stability and sensitivity, which can provide an efficient and convenient membrane protein immobilization method for biomaterials.

Identification of lignans and quality assessment in Dendrobium officinale under different cultivation modes.

RATIONALE: Lignans have attracted much attention from researchers because of their wide distribution and industrial applications in plants, as well as the remarkable diversity of their biological activities. As the literature has mainly focused on the extraction and identification of monomeric compounds of lignans, most lignans in Dendrobium officinale, a traditional Chinese medicine with a long cultivation history and rich sources, have not been detected using quality control methods. The aim of this study was to identify the lignans in Dactilon officinale. METHODS: High-performance liquid chromatography (HPLC) coupled with diode array detection and HPLC multiple-stage tandem mass spectrometry was used to identify the chemical constituents of D. officinale. Simultaneously, the characteristic chromatograms of D. officinale were established. Additionally, a method was established to determine the content of syringaresinol-4,4'-di-O-ß-D-glucoside, syringaresinol-4-O-ß-D-glucoside and syringaresinol. RESULTS: Thirty-three lignans, including 17 tetrahydrofuran lignans, two dibenzylbutane lignans, three aryl tetrahydronaphthalene lignans and 11 8-O-4'-neolignans, were tentatively identified from the methanol extract of the stems of D. officinale. This is the first report of 8-O-4'-neolignans from D. officinale. In addition, a total of eight characteristic peaks were marked in characteristic chromatograms, which were identified as lyoniresinol-9'-O-ß-D-glucoside, syringaresinol-4,4'-di-O-ß-D-glucoside, 8-hydroxy-syringaresinol-4-O-ß-D-glucoside, 5,5'-dimethoxy-lariciresinol-4-O-ß-D-glucoside, syringaresinol-4-O-ß-D-glucoside, 4-hydroxy-3,3',5,5'-tetramethoxy-8,4'-oxyneoligna-7'-ene-9,9'-diol-9-O-ß-D-glucoside, 4-hydroxy-3,3',5,5'-tetramethoxy-8,4'-oxyneoligna-7'-ene-9,9'-diol-4-O-ß-D-glucoside and syringaresinol. Our results showed that no significant difference occurred in lignan composition among the 99 batches of D. officinale from different sources. However, the peak areas of the lignans of D. officinale planted under simulated wild culture were generally higher than those in greenhouses, and showed an upward trend with the increase in growth years. The average contents of syringaresinol-4,4'-di-O-ß-D-glucoside, syringaresinol-4-O-ß-D-glucoside and syringaresinol were 10.112-179.873, 51.227-222.294 and 6.368-120.341 µg/g, respectively. CONCLUSIONS: This study provided a basis for improving the quality control of D. officinale and could provide references for the identification of lignans in other Dendrobium species.

The Hidden Dangers of Plant-Based Diets Affecting Bone Health: A Cross-Sectional Study with U.S. National Health and Nutrition Examination Survey (NHANES) Data from 2005-2018.

The plant-based dietary pattern has been recommended for its potential health and environmental benefits, but its association with bone loss needs to be further explored. This study aimed to investigate the association between three plant-based diet indexes and bone loss in 16,085 adults, using data from the National Health and Nutrition Examination Survey. Three plant-based diet indexes (PDI, hPDI, and uPDI) were calculated from two NHANES 24-h dietary recall interviews, to characterize a plant-based diet. A multinomial logistic regression model was used to estimate the odds ratios (OR) and 95% confidence intervals (95% CI). Higher hPDI and PDI were associated with increased risk of bone loss (ORQ5 vs. Q1 = 1.50; 95% CI: 1.24-1.81 for hPDI; ORQ5 vs. Q1 = 1.22; 95% CI: 1.03-1.45 for PDI), while higher uPDI was associated with increased risk of osteoporosis (ORQ5 vs. Q1 = 1.48; 95% CI: 1.04-2.11). A harmful association between plant-based diet indexes (hPDI and PDI) and osteopenia was observed at the lumbar spine rather than the femoral neck. We conducted several sensitivity analyses to ensure the robustness of results, including subgroup analysis, exclusion of people taking anti-osteoporotic and estrogenic drugs, further adjustment for menopausal status, corticosteroid usage, and dietary supplements, and calculation of E-value. Our study demonstrates the deleterious effects of a plant-based diet on bone health and emphasizes the importance of a balanced diet.

Anti-fatigue properties of the ethanol extract of Moringa oleifera leaves in mice.

BACKGROUND: Moringa oleifera (M. oleifera) leaves are rich in nutrients and bioactive ingredients. This study was aimed at evaluating the anti-fatigue effect of the ethanol extract of M. oleifera leaves (MLEE) on mice and its primary mechanism of action using a weight-loaded forced swimming test. In the present study, MLEE was prepared by ultrasound-assisted extraction, and its anti-fatigue effect and antioxidant capacity were evaluated in mice. Mice were administrated MLEE (320 mg kg-1 body weight) for 15 days. RESULTS: MLEE supplementation significantly increased levels of glucose and non-esterified fatty acids (NEFA), while decreasing levels of lactate and blood urea nitrogen in serum (P < 0.05); the levels of glycogen in the liver and muscle were also increased, as was the activity of glycogen synthase and the level of NEFA in muscle (P < 0.05). According to a Western blot analysis, MLEE increased the expression of AMPKα1, JNK, AKT and STAT3 in the muscle of mice. CONCLUSION: Our findings indicate that MLEE has an anti-fatigue effect via the AMPK-linked route, which enables it to control energy metabolism and enhance antioxidant enzyme activity. © 2023 Society of Chemical Industry.

Proteomic analysis of rat colonic mucosa following acupuncture treatment for irritable bowel syndrome with diarrhea.

To investigate the molecular pathological mechanisms of irritable bowel syndrome with diarrhea (IBS-D) and elucidate the effects of acupuncture on IBS-D colonic mucosa protein abundance in rats, a label-free high-throughput liquid chromatography-tandem mass spectrometry (LC-MS)-based proteomics analysis was used to survey the global changes of colonic mucosa proteins between different groups. Sixteen Sprague-Dawley (SD) male rats were randomly divided into four groups: the control group (C); the IBS-D model group (M); the syndrome differentiation acupuncture group (SD) and the traditional acupuncture group (T). IBS-D model rats were obtained using the CAS (chronic acute combining stress model) method. Comparative bioinformatics analysis of the proteomic data was analyzed using MaxQuant software, Perseus software, online tools DAVID, VENNY and STRING. Functional enrichment and network analyses revealed a close relationship between IBS-D and several biological processes including energy metabolism, muscular excitation/contraction, and both traditional acupuncture and syndrome differentiation acupuncture can reverse the impairments of normal energy metabolism. Moreover, the syndrome differentiation acupuncture can regulate the protein cluster relating inflammation, wound repair and cell protection against oxidative stress which is associated with acupuncture analgesic effect. Differentially expressed proteins Atp5a1 and Bpnt1 were selected as representative proteins and subjected to western blotting. In conclusion, our study provides further insight into the pathological and molecular mechanisms of IBS-D and acupuncture treatments, and serves as an experimental basis for clinical applications.

[Structural Characteristics of Zooplankton and Phytoplankton Communities and Its Relationship with Environmental Factors in Different Regions of Nanhu Lake in Jiaxing City].

Nanhu Lake is a red tourist attraction in Jiaxing city and the birthplace of the "Red Boat Spirit."To identify the influence of environmental factors on the distribution of plankton communities after ecological restoration in different regions, the environmental factors and plankton community in areas A, B, C, D, and S of Nanhu Lake were investigated in January 2021 after the completion of the ecological restoration projects. The concentrations of total nitrogen(TN), dissolved total nitrogen (DTN), ammonia nitrogen (NH4+-N), nitrate nitrogen (NO3--N), total phosphorus (TP), and dissolved total phosphorus (DTP) in the ecological restoration areas were significantly lower, and the content of dissolved oxygen (DO) was significantly higher (P<0.05) than those in the non-restoration area. The main phytoplankton species in the study area belonged to Cyanophyta and Bacillariophyta, and the main zooplankton species were protozoans and rotifers. The phytoplankton biomass in the restored area was lower than that in the unrestored area, and the number of phytoplankton and zooplankton species increased. Clustering and principal coordinate analysis results showed significant differences in plankton communities among the restoration areas (P<0.05), and plankton structures in regions A and B were similar. Redundancy analysis (RDA) showed that the main environmental factors affecting the distribution of phytoplankton communities were DO, NO3--N, pH, and water temperature (WT). The main driving factors of zooplankton community distribution were DO, NO3--N, NH4+-N, and TP. The results clarified the phytoplankton community characteristics and environmental correlation in different regions of Nanhu Lake, which can provide data support and reference for water ecological restoration of the lake.

Disrupting Circadian Rhythm via the PER1-HK2 Axis Reverses Trastuzumab Resistance in Gastric Cancer.

Trastuzumab is the only approved targeted drug for first-line treatment of HER2-positive advanced gastric cancer, but the high rate of primary resistance and rapid emergence of secondary resistance limit its clinical benefits. We found that trastuzumab-resistant (TR) gastric cancer cells exhibited high glycolytic activity, which was controlled by hexokinase 2 (HK2)-dependent glycolysis with a circadian pattern [higher at zeitgeber time (ZT) 6, lower at ZT18]. Mechanistically, HK2 circadian oscillation was regulated by a transcriptional complex composed of PPARγ and the core clock gene PER1. In vivo and in vitro experiments demonstrated that silencing PER1 disrupted the circadian rhythm of PER1-HK2 and reversed trastuzumab resistance. Moreover, metformin, which inhibits glycolysis and PER1, combined with trastuzumab at ZT6, significantly improved trastuzumab efficacy in gastric cancer. Collectively, these data introduce the circadian clock into trastuzumab therapy and propose a potentially effective chronotherapy strategy to reverse trastuzumab resistance in gastric cancer. SIGNIFICANCE: In trastuzumab-resistant HER2-positive gastric cancer, glycolysis fluctuates with a circadian oscillation regulated by the BMAL1-CLOCK-PER1-HK2 axis, which can be disrupted with a metformin-based chronotherapy to overcome trastuzumab resistance.

Preparation of Highly Substituted Sulfated Alfalfa Polysaccharides and Evaluation of Their Biological Activity.

Alfalfa polysaccharides (AP) receive wide attention in the field of medicine, because of their anti-inflammatory property. However, AP has high molecular weight and poor water solubility, resulting in low biological activity. We wanted to obtain highly bioactive alfalfa polysaccharides for further research. Herein, we successfully synthesized highly substituted sulfated alfalfa polysaccharides (SAP) via the chlorosulfonic acid (CSA)-pyridine (Pyr) method, which was optimized using response surface methodology (RSM). Under the best reaction conditions, that is, the reaction temperature, time, and ratio of CSA to Pyr being 55 °C, 2.25 h, and 1.5:1, respectively, the maximum degree of substitution of SAP can reach up to 0.724. Fourier transform infrared spectroscopy also confirmed the existence of sulfonic acid groups on SAP. Despite the increased average molecular weight of SAP, its water solubility is improved, which is beneficial for its biological activity. Further in vitro results showed that SAP exhibited better antioxidant activity and antibacterial ability than AP. Besides, the former can efficiently enhance the viability of oxidatively stressed intestinal epithelial cells compared with the latter. Furthermore, SAP has the potential to inhibit obesity. It is concluded that sulfation modification could improve the antioxidant, antibacterial, bovine intestinal epithelial cells' proliferation-promoting, and the obesity inhibition abilities of AP. The improvement of AP biological activity may provide references for the utilization of plant extracts that have weaker biological activity.

Identification of C-glycosyl flavones and O-glycosyl flavones in five Dendrobium species by high-performance liquid chromatography coupled with electrospray ionization multi-stage tandem MS.

RATIONALE: Flavones are widely used in traditional Chinese medicine (TCM) and are the pharmacologically active ingredients of many medicinal plants, such as Dendrobium. With the increasing demand for medicinal Dendrobium, the identification of characteristic flavones that can serve as chemical markers for quality control is critical step for quality assurance and safety in the TCM industry. METHODS: High-performance liquid chromatography coupled with electrospray ionization multi-stage tandem mass spectrometry (HPLC/ESI-MSn ) was used to identify the chemical constituents in five types of Dendrobium: D. crystallinum, D. falconeri, D. strongylanthum, D. moniliforme, and D. gratiosissimum. RESULTS: A total of seventy-six C-glycosyl flavones and three O-glycosyl flavones were identified, of which fifteen C-glycosyl flavones were found in D. crystallinum, twenty four were found in D. falconeri, thirty were found in D. strongylanthum, seven were found in D. moniliforme (also called "Huangtongpi", from Anhui, China), fifteen were found in D. moniliforme (also called "Zitongpi", from Yunnan, China) and seventeen were found in D. gratiosissimum. Additionally, three flavone O-glycosides were all found in D. strongylanthum. CONCLUSIONS: The results of this study may be useful for the quality assessment and for the application of D. crystallinum, D. falconeri, D. strongylanthum, D. moniliforme, and D. gratiosissimum. This study provides comprehensive information for identification of flavones from other Chinese herbs.

[Material manufacturability classification in high shear wet granulation process of Chinese medicine].

The high shear wet granulation(HSWG) process of Chinese medicine has a complicated mechanism. There are many influencing factors that contribute to this process. In order to summarize the manufacturability of different kinds of materials in HSWG, this paper constructed a material library composed of 11 materials, including 4 Chinese medicine extracts and 7 pharmaceutical excipients. Each material was described by 22 physical parameters. Several binders were employed, and their density, viscosity and surface tension were characterized. Combining empirical constraints and the principle of randomization, 21 designed experiments and 8 verification experiments were arranged. The partial least squares(PLS) algorithm was used to establish a process model in prediction of the median granule size based on properties of raw materials and binders, and process parameters. The surface tension and density of binders, as well as the maximum pore saturation were identified as key variables. In the latent variable space of the HSWG process model, all materials could be divided into three categories, namely the Chinese medicine extracts, the diluents and the disintegrants. The granulation of Chinese medicine extracts required low viscosity and low amount of binder, and the resulted granule sizes were small. The diluent powders occupied a large physical space, and could be made into granules with different granule sizes by adjusting the properties of binders. The disintegrants tended to be made into large granules under the condition of aqueous binder. The combination use of material database and multivariate modeling method is conducive to innovate the knowledge discovery of the wet granulation process of Chinese medicine, and provides a basis for the formulation and process design based on material attributes.

Natural Bioactive Molecules as Potential Agents Against SARS-CoV-2.

In the past two decades, pandemics of several fatal coronaviruses have posed enormous challenges for public health, including SARS-CoV (2003), MERS-CoV (2012), and SARS-CoV-2 (2019). Among these, SARS-CoV-2 continues to ravage the world today and has lead to millions of deaths and incalculable economic damage. Till now, there is no clinically proven antiviral drug available for SARS-CoV-2. However, the bioactive molecules of natural origin, especially medicinal plants, have been proven to be potential resources in the treatment of SARS-CoV-2, acting at different stages of the viral life cycle and targeting different viral or host proteins, such as PLpro, 3CLpro, RdRp, helicase, spike, ACE2, and TMPRSS2. They provide a viable strategy to develop therapeutic agents. This review presents fundamental biological information on SARS-CoV-2, including the viral biological characteristics and invasion mechanisms. It also summarizes the reported natural bioactive molecules with anti-coronavirus properties, arranged by their different targets in the life cycle of viral infection of human cells, and discusses the prospects of these bioactive molecules for the treatment of COVID-19.

Association between dietary onion intake and subclinical hypothyroidism in adults: a population-based study from an iodine-replete area.

PURPOSE: The protective effect of onion against thyroid hypofunction has been reported in animal studies. However, in humans, the association between onion consumption and subclinical hypothyroidism (SCH) are unclear. The study sought to explore the association between habitual onion intake and SCH among adult population from an iodine-replete area. METHODS: A cross-sectional study (6515 men and 5290 women) was performed in Tianjin, China. Frequency of onion consumption was assessed using a valid self-administered food frequency questionnaire. Serum free triiodothyronine (FT3), free thyroxine (FT4), and thyroid-stimulating hormone (TSH) were determined by chemiluminescence immunoassay. SCH was diagnosed with TSH > 4.78 mIU/L. Multiple logistic regression was used to evaluate the association of onion intake with SCH. RESULTS: The prevalence of SCH was 2.56% in men and 7.18% in women, respectively. In women, the fully adjusted odds ratios [95% confidence interval (CI)] of having SCH across increasing frequency of onion intake were 1.00 (reference) for <1 time/week, 0.99 (0.73, 1.34) for 1-3 times/week, 0.74 (0.53, 1.03) for 4-6 times/week, and 0.67 (0.47, 0.97) for ≥7 times/week (P for trend <0.01). However, we observed no significant association between onion intake and SCH in men. Stratified analyses suggested a potential effect modification by age: the odds ratios (95% CI) across extreme quartiles was 0.37 (0.17, 0.80) in <40 women and 1.11 (0.51, 2.47) in >60 women. CONCLUSIONS: Frequent consumption of onion is inversely associated with SCH in adult women from an iodine-replete area. Further studies are needed to explore the casual relationship. TRIAL REGISTRATION WEBSITE: https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000031137.

Saltwater fish but not freshwater fish consumption is positively related to handgrip strength: The TCLSIH Cohort Study.

Fish contain many important nutrients and are primarily known for high n-3 polyunsaturated fatty acids (n-3 PUFA) content. Studies have shown that supplementation of fish oil-derived n-3 PUFA improves muscle mass and strength. Here, we hypothesized that fish consumption might improve muscle strength. To test this hypothesis, we performed this cross-sectional study (n = 29,084) in Tianjin, China. The frequency of fish consumption was assessed using a valid self-administered food frequency questionnaire. Handgrip strength (HGS) was used as the indicator of muscle strength, and was measured using a handheld digital dynamometer. Analysis of covariance was used to examine the relationship between fish consumption and HGS. In men, after adjusted potential confounding factors, the least square means (95% confidence intervals) of HGS across saltwater fish consumption categories were 41.5 (41.1, 43.7) kg for <1 time/week, 44.6 (43.2, 45.8) kg for 1 time/week, and 44.7 (43.3, 46.1) kg for ≥2 to 3 times/week (P for trend <0.001). In men, the least square means (95% confidence intervals) of HGS across the ascending quartiles of dietary n-3 PUFA intake were 43.6 (43.2, 44.4) kg, 43.7 (43.2, 44.6) kg, 44.4 (43.0, 45.8) kg, and 44.6 (43.1, 46.0) kg (P for trend <0.01). The results showed that saltwater fish consumption was positively related to HGS in men, but not in women, suggesting that saltwater fish contain nutrients that may be used to improve HGS.

Identification of C-glycosyl flavones and quality assessment in Dendrobium nobile.

RATIONALE: Flavones are significant indicators of quality in traditional Chinese medicines (TCMs) and thus play a significant role in the quality control of TCMs in the pharmaceutical industry. Most flavones in Dendrobium nobile Lindl, a TCM with a long cultivation history and rich sources, have not been identified. This study was aimed at identifying the flavones in D. nobile from various habitats. METHODS: High-performance liquid chromatography (HPLC) coupled with diode-array detection and HPLC multiple-stage tandem mass spectrometry was used to identify the chemical constituents of D. nobile from various habitats, and a method was established to determine the content of vicenin II, violanthin and isoviolanthin. Hierarchical cluster analysis, principal component analysis and orthogonal partial least-squares discriminant analysis were used to analyze the variations among 26 batches from different habitats. RESULTS: A total of 33 flavones were tentatively identified. Twenty-five flavones, previously undescribed in D. nobile, were acylated by p-coumaroyl, feruloyl, sinapoyl or 3-hydroxy-3-methylglutaryl. The D. nobile habitats were distinguished by significant differences in their flavone content. The C-glycosyl flavones were demonstrated to be characteristic compounds for evaluating D. nobile from various habitats. In particular, flavones acylated with 3-hydroxy-3-methylglutaryl were specific compounds that were only detected in samples from Yunnan. CONCLUSIONS: The results of this study could be used to improve the quality control of D. nobile and could provide references for the identification of acylated C-glycosyl flavones in other natural products.

HPLC coupled with electrospray ionization multistage MS/MS and TLC analysis of flavones-C-glycosides and bibenzyl of Dendrobium hercoglossum.

Dendrobium hercoglossum Rchb. f. (D. hercoglossum), as one of the origins of medicinal Dendrobium, has been widely used as a health food and nutrient source promoting fluid production. Due to a lack of quality control, it is often counterfeited or mixed with other Dendrobium. In this study, a high-performance liquid chromatography characteristic chromatogram method is established for the quality evaluation of D. hercoglossum. Based on the high-performance liquid chromatography characteristic chromatogram, D. hercoglossum is divided into two classes, each with different flavone peaks. These flavone peaks were identified using high-performance liquid chromatography coupled with electrospray ionization multistage tandem mass spectrometry. Among them, the acylated (3-hydroxy-3-methylglutaryl, p-coumaroyl, feruloyl, or sinapoyl) flavones-C-glycosides are first found in D. hercoglossum in this study. In addition, one unique band was found in D. hercoglossum by thin-layer chromatography, which can be used to distinguish it from other Dendrobium species as a characteristic marker of this plant. Combining the high-performance liquid chromatography characteristic chromatogram and high-performance liquid chromatography coupled with electrospray ionization multistage tandem mass spectrometry, the unique band was identified as 4,5-dihydroxy-3,3'-dimethoxybibenzyl. These analysis methods can be applied for the quality control and identification of D. hercoglossum as well as providing reference for the identification of similar constituents in other Dendrobium species.

Positive detection of SARS-CoV-2 combined HSV1 and HHV6B virus nucleic acid in tear and conjunctival secretions of a non-conjunctivitis COVID-19 patient with obstruction of common lacrimal duct.

BACKGROUND: The current outbreak of COVID-19 has spread rapidly all over the world. Respiratory droplets and contaction with infected patients are the two major transmission routes. However, the value of tear virus nucleic acid is still not clear. We dynamic detected the SARS-CoV-2 in eye sample of one COVID-19 patient with obstruction of common lacrimal ducts. METHODS: Besides the routine examination, nasopharyngeal and eye swab were continuously measured by polymerase chain reaction assay and next-generation sequencing (NGS). Gene detection was performed for drug use guidance, and flow cytometry was performed to analyse the lymphocyte subsets. RESULTS: Nasopharyngeal swabs were positive for 22 days, but eye swabs were still continuously positive for 2 weeks after nasopharyngeal swabs turned negative. The low level of lymphocyte and the high level IL-6 lasted for almost 4 weeks, then became near normal. Next-generation sequencing (NGS) confirmed the existing of SARS-CoV-2, HSV1 and HHV6B virus nucleic acid. The gene detection for drug use guidance showed the genetic locus ABCB1 (3435T>C) rs1045642 belonged to type CC and it mean the efficiency of lopinavir-ritonavir would be significantly decreased. The flow cytometry of lymphocyte subsets showed PD-1+  CD95+ cells was accounting for 94.8% in CD3+  CD8+ T subset and for 94.8% in CD3+  TCRγδ+ T subset. CONCLUSIONS: As obstruction of common lacrimal duct, positively detection in one eye for 2 weeks more after nasopharyngeal swab became negative. More eye swabs should be collected from COVID-19 patients, especially from those immunocompromised, those with eye symptoms and those had a history of ocular diseases.

Identification of Differentially Expressed miRNAs in the Response of Spleen CD4+ T Cells to Electroacupuncture in Senescence-Accelerated Mice.

Immunological aging impairs immune system protection in the body and is associated with high morbidity and mortality in aged people. Electroacupuncture (EA) has been proven to boost immunity. The purpose of this study was to identify the effect of EA on miRNA expression in the immune system of senescence-accelerated mouse P8 (SAMP8) mice. We utilized SAMP8 mice as an aging model and detected the altered expression of miRNAs in CD4+ T cells after EA stimulation by deep sequencing. Differentially expressed miRNAs in different groups were identified using Venn diagrams and functional analysis was performed. The effect of EA on the expression of the identified miRNAs was investigated in natural-aged C57BL/6J mice and the biological functions of miR-301a-3p and miR-181a-1-3p in CD4+ T cells were identified. Four upregulated and two downregulated miRNAs were identified in group I (EA-SAMP8 vs. shEA-SAMP8); 41 upregulated and nine downregulated miRNAs were identified in group II (EA-SAMP8 vs. SAMP8); 42 upregulated and eight downregulated miRNAs were identified in group III (shEA-SAMP8 vs. SAMP8). The three groups shared four overlapping differentially expressed miRNAs, and 10 miRNAs were only found in group II. Gene Ontology enrichment analysis of these 14 miRNAs revealed that their target genes were enriched in 229 "biological process" categories. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the targets were significantly mapped in 76 pathways. Furthermore, five significant pathways were involved in T cell differentiation. MiRNA-gene-net showed that miR-582-5p, miR-17-5p, miR-144-3p, miR-451a, and miR-301a-3p regulated the most important target genes in these pathways. The expression of these miRNAs was also regulated by EA in aged C57BL/6J mice. In addition, miR-301a-3p was involved in regulating the expression of inflammatory factors by mediating the differentiation of CD4+ T cells in C57BL/6J mice. Analysis of miRNAs indicated that EA contributes to maintaining the balance of CD4+ T cell differentiation in the aging immune system. These results provide novel insights into the effect of EA in immunological aging.

Identification of C-glycosyl flavones by high performance liquid chromatography electrospray ionization mass spectrometry and quantification of five main C-glycosyl flavones in Flickingeria fimbriata.

Flickingeria fimbriata is commonly applied in China as a traditional Chinese medicine (TCM), however the quality control of it is incomplete. In this work, we aim to identify and quantify the structures of C-glycosyl flavones in F. fimbriata. High performance liquid chromatography-diode array detector (HPLC-DAD) and High performance liquid chromatography-electrospray ionization-multiple stage tandem mass spectrometry (HPLC-ESI-MSn) methods were combined to identify C-glycosyl flavones and determine their contents. Twenty acylated C-glycosyl flavones and ten non-acylated C-glycosyl flavones were identified for the first time in F. fimbriata on systematic MSn analysis via HPLC-ESI-MSn. The aglycones of all of these compounds were apigenin or chrysoeriol and were acylated with p-coumaric, ferulic, 3,4-dimethoxycinnamic or 3,4,5-trimethoxycinnamic acids. Furthermore, the quantification result suggest that two C-glycosyl flavones (vicenin-I and vicenin-III) with relative high contents were revealed to be more strongly acylated in F. fimbriata. The method is sufficiently precise, accurate, and sensitive for the qualitative and quantitative analysis of C-glycosyl flavones, which is expected to establish a standard for quality control and identification in this plant.