Discovery of the covalent SARS-CoV-2 Mpro inhibitors from antiviral herbs via integrating target-based high-throughput screening and chemoproteomic approaches.

The main proteases (Mpro ) are highly conserved cysteine-rich proteins that can be covalently modified by numerous natural and synthetic compounds. Herein, we constructed an integrative approach to efficiently discover covalent inhibitors of Mpro from complex herbal matrices. This work begins with biological screening of 60 clinically used antiviral herbal medicines, among which Lonicera japonica Flos (LJF) demonstrated the strongest anti-Mpro effect (IC50 = 37.82 µg/mL). Mass spectrometry (MS)-based chemical analysis and chemoproteomic profiling revealed that LJF extract contains at least 50 constituents, of which 22 exhibited the capability to covalently modify Mpro . We subsequently verified the anti-Mpro effects of these covalent binders. Gallic acid and quercetin were found to potently inhibit severe acute respiratory syndrome coronavirus 2 Mpro in dose- and time- dependent manners, with the IC50 values below 10 µM. The inactivation kinetics, binding affinity and binding mode of gallic acid and quercetin were further characterized by fluorescence resonance energy transfer, surface plasmon resonance, and covalent docking simulations. Overall, this study established a practical approach for efficiently discovering the covalent inhibitors of Mpro from herbal medicines by integrating target-based high-throughput screening and MS-based assays, which would greatly facilitate the discovery of key antiviral constituents from medicinal plants.

Chemical profiling and mechanistic studies of Zhi-Shang-Feng granules against influenza virus by high-performance liquid chromatography coupled with Q exactive focus hybrid quadrupole orbitrap high-resolution mass spectrometry in combination with network pharmacology analysis.

Zhi-Shang-Feng Granules are used in the clinical treatment of influenza to relieve headaches, chills and fever, bronchitis, nasal congestion, neuralgia and other symptoms. To decipher the components responsible for therapeutic effects of Zhi-Shang-Feng g ranules against influenza virus, an analytical method based on high-performance liquid chromatography coupled with Q exactive focus hybrid quadrupole orbitrap high resolution mass spectrometry was developed and the chemical profile of Zhi-Shang-Feng granules was characterized. Then, the identified components were used to conduct network pharmacological analysis and determine the potential mechanism of Zhi-Shang-Feng Granules. As a result, 177 compounds were putatively identified through comprehensive analysis by liquid chromatography coupled with high-resolution mass spectrometry, of which 23 compounds were unambiguously confirmed with reference standards. Components in Zhi-Shang-Feng Granules were found to specifically act on different enzymes, G-protein-coupled receptors, ion channels and transporters in the immune, endocrine, nervous, and circulatory systems. The potential mechanism was related to several biological processes, including cell growth and death, pattern recognition receptor signalling, signalling by interleukins, and lipid metabolism. The combination of chemical profile characterization and network construction provided useful insight into the overall chemical composition of Zhi-Shang-Feng granules and revealed their potential anti-infection, anti-inflammatory and immunoregulatory mechanisms against influenza virus infected disease.

[Effects of Renshenjian Decoction on blood and urine metabonomics in type 2 diabetes rats based on ~1H-NMR].

Proton nuclear magnetic resonance(~1H-NMR) is used to investigate the effect of Renshenjian Decoction on serum and urine metabolism of type 2 diabetic rats with insulin resistance induced by high-sugar and high-fat diet combined with low-dose streptozotocin(STZ). After the successful establishment of the insulin resistance model of type 2 diabetes, administration for 35 days, the serum and urine of rats were taken. Once the ~1H-NMR data have been collected and processed, PCA and OPLS-DA were used to analyze them. The results show that: compared with the blank group, the contents of methionine, taurine, α-glucose and ß-glucose in the serum of the model group increased significantly(P<0.001), while the contents of 3-hydroxybutyric acid, lactic acid and unsaturated fatty acids decreased significantly(P<0.01). In the model group, the contents of trimethylamine oxide, glycine, α-glucose, ß-glucose, taurine and phosphocholine in urine increased significantly(P<0.05), while the contents of creatine, lactic acid, acetic acid and citric acid decreased significantly(P<0.05). Compared with the model group, the contents of 3-hydroxybutyric acid and unsaturated fatty acids in serum of rats in the treatment group increased significantly(P<0.05), while the contents of taurine, α-glucose and ß-glucose decreased significantly(P<0.01). In the treatment group, the contents of lactic acid, taurine and creatine in urine increased significantly(P<0.05), while the contents of trimethylamine oxide, glycine, α-glucose, ß-glucose and phosphocholine decreased significantly(P<0.01). The results show that Renshenjian Decoction can regulate metabolic disorder and promote the metabolic phenotype to return to the normal range. It displayed therapeutic effect on type 2 diabetic rats with insulin resistance and provided a certain scientific basis for the biological basic research of Renshenjian Decoction by improving insulin resistance in diabetes mellitus.

Effect of enzymatic degraded polysaccharides from Enteromorpha prolifera on the physical and oxidative stability of fish oil-in-water emulsions.

To explore the potential application of enzymatic degraded polysaccharides from Enteromorpha prolifra (EEP) as antioxidant in fish oils, a stable fish oil emulsion system incorporating EEP was established. Effects of emulsifier (Tween 80, gum arabic and lecithin) and EEP concentration on the physical characteristics of fish oil emulsions were investigated. The results indicated that Tween 80 was the best choice, and 1% (w/w) of EEP was the optimum concentration for the preparation of fish oil emulsions. Influence of EEP on the oxidative stability and physical stability of fish oil emulsions was compared with that of antioxidants VE and TBHQ by determining the physical properties, lipid hydroperoxide formation, secondary reaction products formation, pH and long chain polyunsaturated fatty acid content, during storage at 45 °C. The results indicated that the fish oil emulsion system (5% oil, 1% EEP and 1% Tween 80, w/w) possessed good physical and oxidative stabilities.

[Advances on network pharmacology in ethnomedicine research].

Ethnomedicine is the precious wealth left by ethnic minorities in their struggle against diseases. It is similar to traditional Chinese medicine in a narrow sense and has the characteristics of multi-component,multi-target and multi-channel synergy. Under the guidance of the theory of ethnomedicine,the combination of ethnomedicine and network pharmacology will help to understand the essence of the prevention and treatment of ethnomedicines in a dynamic and holistic manner. This paper reviews the research progress of network pharmacology applied in ethnomedicine,analyses the problems and challenges existing in the application of network pharmacology in ethnomedicine research at present,such as inaccurate data and information,lack of network analysis platform for effective analysis of dose-effect relationship of chemical constituents and weak basic research of ethnomedicine,and puts forward corresponding prospects.

Metabolism and pharmacokinetics of major polyphenol components in rat plasma after oral administration of total flavonoid tablet from Anemarrhenae Rhizoma.

Total flavonoid tablet from Anemarrhenae Rhizoma (Zhimu tablet), which was made of total polyphenol components extracted from the dried rhizome of Anemarrhena asphodeloides Bge. (Zhimu in Chinese), is a novel traditional Chinese medicine prescribed for the treatment of diabetes. Mangiferin (MF) and neomangiferin (NMF) are the two main components detected and determined in Zhimu tablet, accounting for 8.9% of the total weight of each tablet. In the present study, high performance liquid chromatography (HPLC) coupled with time-of-flight (TOF) tandem mass spectrometry (MS) was applied to characterize the metabolites of MF and NMF in rat plasmas collected at different time points after oral administration of Zhimu tablet at a dose of 3.63g/kg (corresponding to 270mg/kg MF). Accurate mass measurement was used to determine the elemental composition of metabolites and thus to confirm the proposed structures of identified metabolites. Time points of appearance of some metabolites, such as isomers, were also taken into account during the structure confirmation. A total of 21 potential metabolites were found in rat plasma at different time points, and the metabolic pathways in vivo were involved in hydrolysis, methylation, glucuronide conjugation, glycoside conjugation, sulphation, dehydration and isomerisation. Furthermore, a selective and accurate LC-MS assay method was developed and validated for the quantification of MF in plasma. Semi-quantification of main conjugated metabolites was also performed in order to describe the dynamic metabolism profiles of polyphenol components in Zhimu tablet. MF concentration in plasma reached 1.36±0.47µgmL(-1) about 5.0h after oral administration of Zhimu tablet, which showed a 3.24- and 4.91-fold increase in plasma maximum concentration and area under the concentration-time curve (AUC) from 0 to 24h of MF compared with those for rats administered with free MF, respectively. The results indicated that the pharmacokinetic processes and bioavailability of MF in rats would be affected by other components in Zhimu tablet.

Hexadentate 3-hydroxypyridin-4-ones with high iron(III) affinity: design, synthesis and inhibition on methicillin resistant Staphylococcus aureus and Pseudomonas strains.

A range of hexadentate 3-hydroxypyridin-4-ones have been synthesized. These compounds were found to possess a high affinity for iron(III), with logK1 values of about 34 and pFe values over 30. Antimicrobial assays indicated that they can inhibit the growth of three clinical isolates of methicillin resistant Staphylococcus aureus (MRSA) and three clinical isolates of Pseudomonas, suggesting that hexadentate 3-hydroxypyridin-4-ones have potential application in the treatment of wound infections.

Integrating qualitative and quantitative characterization of traditional Chinese medicine injection by high-performance liquid chromatography with diode array detection and tandem mass spectrometry.

The present study aims to describe and exemplify an integrated strategy of the combination of qualitative and quantitative characterization of a multicomponent mixture for the quality control of traditional Chinese medicine injections with the example of Danhong injection (DHI). The standardized chemical profile of DHI has been established based on liquid chromatography with diode array detection. High-performance liquid chromatography coupled with time-of-flight mass spectrometry and high-performance liquid chromatography with electrospray multistage tandem ion-trap mass spectrometry have been developed to identify the major constituents in DHI. The structures of 26 compounds including nucleotides, phenolic acids, and flavonoid glycosides were identified or tentatively characterized. Meanwhile, the simultaneous determination of seven marker constituents, including uridine, adenosine, danshensu, protocatechuic aldehyde, p-coumaric acid, rosmarinic acid, and salvianolic acid B, in DHI was performed by multiwavelength detection based on high-performance liquid chromatography with diode array detection. The integrated qualitative and quantitative characterization strategy provided an effective and reliable pattern for the comprehensive and systematic characterization of the complex traditional Chinese medicine system.

[Urine metabonomic study of intervention effects of Morinda officinalis how. on 'kidney-yang deficiency syndrome'].

To investigate the intervention effects of Morinda officinalis How. on 'Kidney-yang deficiency syndrome' induced by hydrocortisone in rats, the metabolic profiles of rat urine were characterized using proton nuclear magnetic resonance and principal component analysis (PCA) was applied to study the trajectory of urinary metabolic phenotype of rats with 'Kidney-yang deficiency syndrome' under administration of M. officinalis at different time points. Meanwhile, the intervention effects of M. officinalis on urinary metabolic potential biomarkers associated with 'Kidney-yang deficiency syndrome' were also discussed. The experimental results showed that in accordance to the increased time of administration, an obvious tendency was observed that clustering of the treatment group moved gradually closed to that of the control group. Eight potential biomarkers including citrate, succinate, alpha-ketoglutarate, lactate, betaine, sarcosine, alanine and taurine were definitely up- or down-regulated. In conclusion, the effectiveness of M. oficinalis on 'Kidney-yang deficiency syndrome' is proved using the established metabonomic method and the regulated metabolic pathways involve energy metabolism, transmethylation and transportation of amine. Meanwhile, the administration of M. officinalis can alleviate the kidney impairment induced by 'Kidney-yang deficiency syndrome'.

Comparative evaluation of cultivars of Chrysanthemum morifolium flowers by HPLC-DAD-ESI/MS analysis and antiallergic assay.

A multicomponent quantification fingerprint based on HPLC coupled with diode array detection and electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI/MS) technique has been established for the analysis of phenolic compounds in 12 samples originated from 5 different cultivars of Chrysanthemum morifolium flowers in China. Four caffeoylquinic acids and 15 flavonoids in the capitulum were identified by comparing the retention times and ultraviolet spectra as well as the mass spectrum and/or matching the empirical molecular formula with that of reference compounds, and the contents of these compounds have been determined simultaneously. The samples from three medicinal cultivars significantly differed in the quality and quantity of flavonoid aglycones and glycosides compared with those from two edible cultivars, which allows the possibility of showing the chemical distinctness of these cultivars and may be useful in their standardization. Moreover, the antiallergic effects of these cultivars were comparatively assayed for the first time. A representative medicinal cultivar, 'huaiju', showed potential activity on the inhibition of antigen-induced degranulation from RBL-2H3 cells and compound 48/80-induced scratching in mice, whereas the in vitro and in vivo antiallergic activities of two edible cultivars were weak. The results suggested that the quality and quantity of some active flavonoid aglycones should be responsible for the pharmacological profiles of these cultivars.

Steaming-induced chemical transformations and holistic quality assessment of red ginseng derived from Panax ginseng by means of HPLC-ESI-MS/MS(n)-based multicomponent quantification fingerprint.

The purpose of this study is to evaluate the steaming-induced chemical transformation of red ginseng manufactured from fresh ginseng by means of simultaneous quantitative and qualitative analyses with a combinative high-performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS(n)) technique. Thirty-six ginsenosides were identified in red ginseng and white ginseng by comparing the mass spectrum and/or matching the empirical molecular formula with that of known published compounds, and 11 of them were determined to be newly generated during the red ginseng preparatory process. The mechanisms involved were further deduced to be hydrolysis, dehydration, isomerization, and decarboxylation at C-20, and hydrolysis also occurs at C-3 or C-6 of the original ginsenosides through the mimic process of steaming and heating in laboratory. The multicomponent quantification fingerprint of ginseng was also established by HPLC-UV method, and the contents of 12 ginsenosides in red and white ginsengs from different sources were determined simultaneously. The ratio of the total content of determined malonyl ginsenosides to the corresponding neutral ginsenosides (T(m-PPD)/T(PPD)) in white ginseng ranged from 0.46 to 0.62 and from 0 to 0.19 in red ginseng. The validated method is expected to provide an effective approach to standardize the processing procedures of ginseng products and regulate the usage of ginseng in Traditional Chinese Medical prescription.

[Comparative study on chemical quality of main species of epimedium].

This study was to comprehensively evaluate the chemical quality of main species of epimedium planted in China. The contents of 5 marker compounds, epimedin A, epimedin B, epimedin C, icariin and baohuoside I, as well as total flavonoids of 22 samples of 8 officinal species of Epimedium were determined by HPLC and UV, separately. Some physical and chemical tests (H2O, total ash, acid-insoluble ash and EtOH extract) were also carried out to investigate their chemical qualities. There were significant differences in types and contents of prenyl-flavonoid glycosides such as epimedin A, epimedin B, epimedin C, icariin and baohuoside I in different species, meanwhile, the physical and chemical parameters results also showed that there were obvious differences in chemical quality among different species of epimedium herb. The results provide theoretical and experimental basis for the establishment of comprehensive quality assessment system of epimedium in China.

Effective components screening and anti-myocardial infarction mechanism study of the Chinese medicine NSLF6 based on "system to system" mode.

BACKGROUND: Shuanglong formula (SLF), a Chinese medicine composed of panax ginseng and salvia miltiorrhiza exhibited significant effect in the treatment of myocardial infarction (MI) in clinical. Because of the complex nature and lack of stringent quality control, it's difficult to explain the action mechanism of SLF. METHOD: In this study, we present a "system to system" (S2S) mode. Based on this mode, SLF was simplified successively through bioactivity-guided screening to achieve an optimized minimal phytochemical composition (new formula NSLF6) while maintaining its curative effect for MI. RESULTS: Pharmacological test combining with the study of systems biology show that NSLF6 has activity for treatment MI through synergistic therapeutic efficacies between total ginsenosides and total salvianolic acids via promoting cardiac cell regeneration and myocardial angiogenesis, antagonistic myocardial cell oxidative damage. CONCLUSIONS: The present S2S mode may be an effective way for the discovery of new composite drugs from traditional medicines.

A novel spinosin derivative from Semen Ziziphi Spinosae.

A novel spinosin derivative, 6‴-(4‴'-O-ß-D-glucopyranosyl)-vanilloyl spinosin (1) was isolated from the methanol extract of Semen Ziziphi Spinosae, together with five known flavonoids, swertish, spinosin, 6‴-feruloylspinosin, isospinosin and kaempferol 3-O-α-L-rhamnopyranosyl-(1 → 2)-O-[O-α-L-rhamnopyranosyl-(1 → 6)]-ß-D-glucopyranoside, and two alkanoids, zizyphusine and 6-(2',3'-dihydroxyl-4'-hydroxymethyl-tetrahydro-furan-1'-yl)-cyclopentene[c]pyrrole-1,3-diol. The structure of compound 1 was elucidated by spectroscopic methods including UV, IR, ESI-TOF-MS, 1D, and 2D NMR techniques.

[Tissue distribution of arsenic of liushen pills and realgar].

This study is to report the tissue distribution of arsenic after giving different doses of realgar and Liushen pills to Beagle dogs, in order to provide basis for the safety evaluation of Liushen pills. ICP-MS was used to measure arsenic concentration, and HPLC-ICP-MS was used to analyze arsenic speciation. The concentration of total arsenic and As(III) + DMA (arsenite + dimethylarsenic acid) increased with dosing of realgar. Total arsenic concentration in most tissues and As(III) + DMA concentration in all tissues of Liushen pills group are lower than that of realgar group, but AsB concentration in liver, spleen and kidney of Liushen pills group increased. The concentration of total arsenic showed a dose-dependent manner with dosage administered. It was indicated that components in Liushen pills can reduce solubility of arsenic in realgar, which may decrease toxicity of realgar.

Anti-inflammatory effects of rhynchophylline and isorhynchophylline in mouse N9 microglial cells and the molecular mechanism.

Excessive production of nitric oxide (NO) and proinflammatory cytokines from activated microglia contributes to human neurodegenerative disorders. Our previous study demonstrated the potent inhibition of lipopolysaccharide (LPS)-induced NO production in rat primary microglial cells by rhynchophylline (RIN) and isorhynchophylline (IRN), a pair of isomeric alkaloids of Uncaria rhynchophylla (Miq.) Jacks. that has been used in China for centuries as a "cognitive enhancer" as well as to treat strokes. We further investigated whether RIN and IRN effectively suppress release of proinflammatory cytokines in LPS-activated microglial cells and the underling molecular mechanism for the inhibition of microglial activation. RIN and IRN concentration-dependently attenuated LPS-induced production of proinflammatory cytokines such as TNF-alpha and IL-1beta as well as NO in mouse N9 microglial cells, with IRN showing more potent inhibition of microglial activation. The western blotting analysis indicated that the potential molecular mechanism for RIN or IRN-mediated attenuation was implicated in suppressions of iNOS protein level, phosphorylation of ERK and p38 MAPKs, and degradation of IkappaBalpha. In addition, the differential regulation of the three signaling pathways by two isomers was shown. Our results suggest that RIN and IRN may be effective therapeutic candidates for use in the treatment of neurodegenerative diseases accompanied by microglial activation.

Inhibitory activity of isoflavones of Pueraria flowers on nitric oxide production from lipopolysaccharide-activated primary rat microglia.

Microglial activation plays an important role in alcohol-induced neuroinflammation. In search for natural medicines that may be of therapeutic potential for alcoholism, two new natural isoflavone glycosides, 6-hydroxybiochanin A-6,7-di-O-beta-d-glucopyranoside (1) and 6-hydroxygenistein-7-O-beta-d-glucopyranoside (2), were isolated from the ethanolic extract of the flowers of Pueraria thomsonii Benth., together with the seven known isoflavones, genistein (3), tectorigenin (4), irisolidone (5), genistin (7), tectoridin (8), tectorigenin-7-O-beta-d-xylosyl-(1 --> 6)-beta-d-glucopyranoside (9), and 6-hydroxygenistein-6,7-di-O-beta-d-glucopyranoside (11). Moreover, gehuain (6) and kakkalide (10) were obtained from the flowers of Pueraria lobata (Willd.) Ohwi. The structures of the new compounds were elucidated by UV, IR, HR-MS, and 1D and 2D NMR spectroscopic methods. Compounds 3-5 substantially inhibited the lipopolysaccharide-induced nitric oxide release from primary cultured rat cortical microglia (IC50: 1.3-9.3 microM). The inhibitory effects of compounds 6, 8, 9, and 11 (IC50: 38-62 microM) were significant but weaker than the above aglycones. However, compounds 1, 2, 7, and 10 showed little inhibitory activity. With regard to the structure-activity relationships of the isoflavonoids for the inhibition of microglial activation, the glycosylation at the C-7 hydroxyl group reduces the inhibitory activity. The methoxylation of 4'-hydroxyl group of 7-glycosylated isoflavonoids reduces the inhibitory activity, while the methoxyl group at the 6-position enhances the activity. The results suggest that isoflavonoids of Pueraria flowers may be of therapeutic potential in alcoholism related to microglial activation.

Cytotoxic activity of flavonoids from the flowers of Chrysanthemum morifolium on human colon cancer Colon205 cells.

A new p-hydroxyphenylacetyl flavonoid, diosmetin 7-(6''-O-p-hydroxyphenylacetyl)-O-beta-d-glucopyranoside (1), was isolated from the flowers of Chrysanthemum morifolium Ramat. 'huaiju' cv. nov. (Compositae), together with five known flavonoids, luteolin (2), diosmetin (3), diosmetin 7-O-beta-d-glucopyranoside (4), diosmin (5), and scolimoside (6), and four known caffeoylquinic acid derivatives, macranthoin F (7), 3,5-dicaffeoylquinic acid (8), 1,3-dicaffeoyl-epi-quinic acid (9), and chlorogenic acid (10). The structure of 1 was elucidated by UV, IR, ESI-TOF-MS, 1D, and 2D NMR spectroscopic methods. Cytotoxic activity of compounds 1-5 against human colon cancer cell Colon205 was investigated using MTT assays. Compounds 2 and 3 showed significant cytotoxicities against Colon205, with their IC(50) values being 96.9 and 82.9 microM, respectively. However, compounds 1, 4, and 5 showed little cytotoxic activity.

The antihyperglycaemic activity of berberine arises from a decrease of glucose absorption.

The mechanism of action of berberine as an antihyperglycaemic agent was investigated in the Caco-2 cell line. Berberine was found to effectively inhibit the activity of disaccharidases in Caco-2 cells. It also decreased sucrase activity after preincubation with Caco-2 cells for 72 hours. However gluconeogenesis and glucose consumption of Caco-2 cells were not influenced. 2-Deoxyglucose transporting through Caco-2 cell monolayers was decreased by berberine but the effect was not statistically significant. These results suggest that the antihyperglycaemic activity of berberine is at least partly due to its ability to inhibit alpha-glucosidase and decrease glucose transport through the intestinal epithelium.