Effect and mechanism of C-terminal cysteine on the properties of HEV p222 protein.

Preliminary investigations have demonstrated that the cysteines located at the C-terminus of HEV ORF2 protein exhibits disulfide bonding capability during virus-like particles (VLPs) assembly. However, the effect and mechanism underlying the pairing of disulfide bonds formed by C627, C630, and C638 remains unclear. The p222 protein encompasses C-terminus and serves as a representative of HEV ORF2 to investigate the specific impacts of C627, C630, and C638. The three cysteines were subjected to site-directed mutagenesis and expressed in prokaryotes; Both the mutated proteins and p222 underwent polymerization except for p222A; Surprisingly, only p222 was observed as abundant spherical particles under transmission electron microscope (TEM); Stability and immunogenicity of the p222 exhibited higher than other mutated proteins; LC/MS/MS analysis identified four disulfide bonds in the p222. The novel findings suggest that the three cysteines contribute to structural and functional properties of ORF2 protein, highlighting the indispensability of each cysteine.

Inhibiting the NLRP3 Inflammasome with MCC950 Alleviates Neurological Impairment in the Brain of EAE Mice.

Multiple sclerosis (MS) is a chronic disease that is characterized by demyelination and neuronal damage. Experimental autoimmune encephalomyelitis (EAE) mice are used to model the disease progression of MS and mirror MS-like pathology. Previous researches have confirmed that inhibition of NLRP3 inflammasome significantly alleviated the severity of EAE mice and the demyelination of spinal cord, but its effect on neuronal damage and oligodendrocyte loss in the brain remains unclear. In this study, female C57BL/6 mice were immunized with MOG35-55 and PTX to establish experimental autoimmune encephalomyelitis (EAE) model. MCC950, a selective NLRP3 inflammasome inhibitor, was used to investigate the effect of NLRP3 inflammasome on the pathological changes and glial cell activation in the brain of EAE mice by immunohistochemistry. Our results demonstrated that MCC950 ameliorated the neuronal damage, demyelination, and oligodendrocyte loss in the brain of EAE mice. This protective effect of MCC950 may be attributed to its ability to suppress the activation of glial cells and prevents microglia polarization to M1 phenotype. Our work indicates that inhibition of NLRP3 inflammasome has the therapeutic effects of neuroprotection through immunomodulation and is a promising therapeutic strategy for MS.

Brassica oleracea L. extract ameliorates isoproterenol-induced myocardial injury by regulating HIF-1α-mediated glycolysis.

Brassica oleracea L. (BO) is an important vegetable with proven health benefits. This study aimed to elucidate the constituents of BO leaf extract (BOE) and evaluate its effect on myocardial injury. For this purpose, the constituents of BOE were identified using ultra-high performance liquid chromatography with quadrupole time-of- flight mass spectrometry, and 26 compounds were determined, including glucosinolates, sulfur compounds, alkaloids, phenolic acids, flavones, and two other kinds of compounds. The effects of BOE on myocardial cells were evaluated using isoproterenol (ISO)-treated H9C2 cells and Wistar rats, and the results revealed that BOE could inhibit cardiomyocyte hypertrophy and reduce the levels of B-type natriuretic peptide, nitric oxide, reactive oxygen species, lactic acid, and pyruvic acid. Meanwhile, BOE could increase the levels of mitochondrial membrane potential. Moreover, BOE could reduce the levels of apoptosis- and glycolysis-related proteins. Taken together, our data demonstrated that BOE treatment could alleviate ISO-induced myocardial cell injury by downregulating apoptosis and glycolysis signals.

Widely Targeted Metabolomics Reveals the Effects of Soil on the Metabolites in Dioscorea opposita Thunb.

Chinese yam (Dioscorea opposita Thunb. cv. Tiegun), a type of homologous medicinal plant, mainly grows in sandy soil (SCY) and loessial soil (LCY). However, the effects of the soil on the metabolites in SCY and LCY remain unclear. Herein, this study aims to comprehensively elucidate the metabolites in SCY and LCY. A UPLC-MS/MS-based, widely targeted metabolomics approach was adapted to compare the chemical composition of SCY and LCY. A total of 988 metabolites were detected, including 443 primary metabolites, 510 secondary metabolites, and 35 other compounds. Notably, 177 differential metabolites (classified into 12 categories) were identified between SCY and LCY; among them, 85.9% (152 differential metabolites) were upregulated in LCY. LCY significantly increased the contents of primary metabolites such as 38 lipids and 6 nucleotides and derivatives, as well as some secondary metabolites such as 36 flavonoids, 28 phenolic acids, 13 alkaloids, and 6 tannins. The results indicate that loessial soil can improve the nutritional and medicinal value of D. opposita.

Aniline-induced production of aniline-containing polyketides and related bicyclic polyketides by the Yellow River wetland-derived fungus Talaromyces funiculosus.

Introduction and Methods: Silencing gene activation can effectively enrich the diversity of fungal secondary metabolites. Results and Discussion: Cultivation of the Yellow River wetland-derived fungus Talaromyces funiculosus HPU-Y01 with aniline led to the isolation of one new aniline-containing polyketide tanicutone A (1), two new bicyclic polyketides tanicutones B-C (2-3), a new related trienoic acid 8-methyldeca-2,4,6-trienoic acid (5), and a known compound 4. The planar structures and configurations of 1-5 were determined by NMR, MS, and ECD calculations. Compound 2 featured a key aldehyde group and showed promising inhibitory activity against Vibrio parahaemolyticus with a minimum inhibitory concentration (MIC) value of 0.17 µg/mL. This is a rare report of aniline-induced fungal production of tetrahydronaphthone polyketides.

Production of Multiple Talaroenamines from Penicillium malacosphaerulum via One-Pot/Two-Stage Precursor-Directed Biosynthesis.

Nineteen new talaroenamine derivatives, talaroenamines F1-F19 (1-19), were isolated from the Yellow River wetland derived Penicillium malacosphaerulum HPU-J01 by use of a one-pot/two-stage precursor-directed biosynthesis approach. During this approach, the initial precursor p-methylaniline was first used as a carrier to capture the biologically synthesized cyclohexanedione to produce talaroenamine F, and then the other aniline derivatives were employed to replace the p-methylaniline fragment of talaroenamine F to generate the final products. LC-MS analysis showed that only four compounds (2, 8, 10, and 12) could be produced by the traditional precursor-directed biosynthesis in which the aniline precursors were added simultaneously. Compound 14 was cytotoxic against the K562 cell line with an IC50 value of 2.2 µM. This work demonstrated the one-pot/two-stage precursor-directed biosynthesis could improve substrate acceptance leading to the production of diverse talaroenamines.

Precursor-Directed Biosynthesis of Talaroenamine Derivatives Using a Yellow River Wetland-Derived Penicillium malacosphaerulum.

Precursor-directed biosynthesis was used to introduce selected aniline derivatives into the talaroenamine pathway, which had recently been defined from a Yellow River wetland-derived Penicillim malacosphaerulum HPU-J01. The known talaroenamine B (1) and six previously undescribed talaroenamine derivatives, talaroenamines F-K (2-7), were generated and structurally characterized. The aniline derivatives are introduced via nonenzymatic addition to the reactive intermediate cyclohexanedione. Compound 2 was active against Bacillus cereus with an MIC value of 0.85 µg/mL.

Rapid identification of chemical compositions in callicarpa kwangtungensis Chun by ultra-high-performance liquid chromatography with Q Exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometry.

Callicarpa kwangtungensis Chun is a traditional Chinese medicine that has various therapeutic effects. Despite its wide use in Chinese medicine, the study is still quite limited, especially its chemical compositions. In this research, an ultra-high-pressure liquid chromatography coupled with Q Exactive hybrid quadrupole-orbitrap high-resolution accurate mass spectrometry tandem mass spectrometry method was utilized to analyze its chemical compositions for the first time. As a result, a total of 124 compounds, including 20 phenylethanoid glycosides, 31 flavonoids, 36 organic acids, 26 terpenoids and 11 phenols, were identified or tentatively characterized in 30 min. Among them, 49 compounds, including 5 phenylethanoid glycosides, 12 flavonoids, 16 organic acids, 12 terpenoids, and 4 phenols, were identified in Callicarpa kwangtungensis Chun for the first time. Besides, the fragmentation pathways were also discussed. This research established a rapid and reliable method to analyze the chemical compositions of complicated herb without the process of isolation, and provide abundant information on the chemical material basis for further bioactivity and quality control studies.

Three new labdane-type diterpenoids from Callicarpa macrophylla Vahl.

Three new labdane-type diterpenoids, callicapene M3-M5 (1-3) were isolated from the Callicarpa macrophylla Vahl. Their structures were identified by spectroscopic method. The isolated compounds were evaluated for inhibitory activity on NO production in LPS-activated RAW 264.7 macrophage cells by using MTT assays. Compounds 1-3 showed potent inhibitory activity, with IC50 value of 48.15, 46.31 and 38.72 µM respectively.

Quantitative fingerprinting based on the limited-ratio quantified fingerprint method for an overall quality consistency assessment and antioxidant activity determination of Lianqiao Baidu pills using HPLC with a diode array detector combined with chemometric methods.

Lianqiao Baidu pills are widely used herbal medicinal preparation that were analyzed to develop a quality consistency technique. The characteristic fingerprints of 28 batches of Lianqiao Baidu pill samples were established at five wavelengths and simultaneously assessed by using a limited-ratio quantified fingerprint method using 15 marker compounds. The principal component analysis and fingerprinting results were compared, and the qualitative classification of the samples by principal component analysis agreed with their quantitative evaluation by the limited-ratio quantified fingerprint method. Furthermore, the antioxidant activities of the samples were surveyed and determined using a 2,2-diphenyl-1-picrylhydrazyl radical-scavenging approach. A relationship between the common peaks in the fingerprints and the antioxidant activities was established using a partial least squares model. The relationship can be used both to determine the antioxidant activities of the Lianqiao Baidu pill preparations in vitro and as a reference for the selection of active constituents for sample quality classification. The classification results for the samples based on principal component analysis agreed with the quantitative evaluation by the limited-ratio quantified fingerprint method, which demonstrated that the method can be applied to the holistic quality control of traditional Chinese medicine and herbal preparations.

Comprehensively Evaluating the Quality Consistency of Liuwei Dihuang Pill by Five-Wavelength Overall Fused HPLC Fingerprints Combined with Simultaneous Determination and Antioxidant Activity.

A combination method of five-wavelength overall fused fingerprints and multi-component quantification by high-performance liquid chromatography coupled with diode array detection was developed to monitor the quality consistency of a Traditional Chinese Medicine (TCM), Liuwei Dihuang Pill (LDP). The validation results demonstrated that this method met the requirements of fingerprint analysis and quantification analysis with desired linearity, precision, accuracy, LOD and LOQ. In the fingerprint assessments, the five-wavelength overall fused fingerprints were estimated by the quantified ratio fingerprint method from qualitative and quantitative perspectives, achieved by an in-house developed software "Digitized Evaluation System for Super-Information Characteristics of TCM/HM Fingerprints 4.0". In order to holistically reflect the quality of LDP, simultaneous analysis of the content of five marker compounds was also carried out. The correlation results between quantified fingerprints and quantitative determination demonstrated that multi-component quantification could be replaced by quantified fingerprints. In addition, the fingerprint-efficacy was also established and confirmed through the partial least squares to visualize the quality information of LDP. The study demonstrated that five-wavelength overall fused fingerprints combined with antioxidant activity assay provided a reliable and efficient method for the assessment of quality of TCM and herbal preparations.

Micellar electrokinetic capillary chromatography fingerprints combined with multivariate statistical analyses to evaluate the quality consistency and predict the fingerprint-efficacy relationship of Salviae miltiorrhizae Radix et Rhizoma (Danshen).

Micellar electrokinetic capillary chromatography fingerprinting combined with quantification was successfully established and applied to evaluate the quality consistency of Danshen, which is a medicinal herb used to treat various diseases, especially coronary cerebrovascular diseases. A background electrolyte composed of 20 mmol/L sodium tetraborate, 90 mmol/L orthoboric acid, 25 mmol/L sodium phosphate monobasic dehydrate, and 65 mmol/L sodium dodecyl sulfate was used to separate compounds. To optimize micellar electrokinetic capillary chromatography conditions, a response surface strategy was set up for orthogonal experimental design. In fingerprint assessments, a systematic quantified fingerprint method was established for integrated quality assessment of Danshen samples from qualitative and quantitative perspectives, by which the quality of 30 samples was well differentiated. The principal component analysis coupled with quantitative determination of two components was applied to explain that the quality consistency of the medicinal herb was relatively good within one harvest season, but poor among harvest seasons for the Danshen samples. In addition, the fingerprint-efficacy relationship between the chemical fingerprints and antioxidant activities was investigated utilizing orthogonal projection to latent structures, which provided important medicinal efficacy information for quality control. This work offered an efficient, holistic, and powerful approach to evaluate the quality consistency of Danshen samples.

Micellar electrokinetic chromatography fingerprinting combined with chemometrics as an efficient strategy for evaluating the quality consistency and predicting the antioxidant activity of Lianqiao Baidu pills.

An approach combining micellar electrokinetic chromatography fingerprinting with chemometrics was developed to evaluate the quality consistency of Lianqiao Baidu pills, which are traditional Chinese patent medicines composed of 19 herbs used mainly to treat skin ulcers, common cold, rheumatism, herpes, and constipation. The triangle optimization method was employed to choose a satisfactory background electrolyte, with the information index, I, as an objective function for assessing the capillary electrophoresis conditions. Then, under the optimal conditions, the micellar electrokinetic chromatography fingerprints of 28 batches of samples were established, and five marker compounds were quantitatively determined simultaneously. A limited-ratio quantified fingerprint method was introduced to evaluate the chromatographic fingerprints both qualitatively and quantitatively. Principle component analysis revealed that the 28 batches of samples can be clustered according to different manufacturers. Moreover, the relationship between the fingerprint and the antioxidant activity was explored by orthogonal partial least-squares regression, which provided critical medicinal efficacy information for quality control. The present study establishes a powerful and reliable method for monitoring the quality consistency of Lianqiao Baidu pill.

Monitoring the Consistency Quality and Antioxidant Activity of Da Hong Pao Teas by HPLC Fingerprinting.

In the present study, a high performance liquid chromatography (HPLC) method was developed and validated for fingerprint profiling to evaluate the quality consistency of the Da Hong Pao tea. The chromatographic separation was performed on a USA Waters C18 column (4.6 mm × 250 mm, 5 µm, Waters-XBridge Shield RP 18) with gradient elution. The column temperature was maintained at 30°C and the detection wavelength was set at 278 nm. The results demonstrated that the developed HPLC method achieved the desired linearity, precision, accuracy and limit of quantification. The fingerprints were developed and evaluated qualitatively using the ratio quantitative fingerprint method. The fingerprint analysis of the reference standards (caffeine, catechin, epicatechin, epigallocatechin, epicatechingallate, Epigallocatechingallate, theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate and theaflavin-3, 3'-gallate) was found to reflect the content of the reference standards in the tea samples. Furthermore, the fingerprint-efficacy relationship among the components as well as the antioxidant activity in vitro were investigated. The selected samples showed good antioxidant activity based on the results. Our study demonstrated that the method of HPLC fingerprint profiling combine the antioxidant activity assay offering a reliable and efficient approach to quantitatively evaluate the quality consistency of the traditional Chinese medicine and other herbal preparations.

Capillary electrophoresis fingerprints combined with chemometric methods to evaluate the quality consistency and predict the antioxidant activity of Yinqiaojiedu tablet.

The quality consistency of Yinqiaojiedu tablets (YQJDTs) was monitored by extracting their electrophoretic fingerprint and marker compound data that were obtained using capillary electrophoresis. To select the suitable background electrolyte, wing-shape method was proposed. A background electrolyte composed of 103.1 mM boric acid, 51.6 mM sodium borate, 9.8 mM disodium hydrogen phosphate, and 15.6 mM sodium dihydrogen phosphate was used to separate compounds. Under the optimized conditions, the content of three marker compounds was determined in 25 YQJDT samples. The capillary electrophoresis fingerprints were developed simultaneously, then 25 samples from two manufacturers were clearly divided into two clusters based on the principal component analysis. In fingerprint assessments, systematic quantified fingerprint method was adopted for integrative quality discrimination of YQJDTs from both qualitative and quantitative perspectives, by which the qualities of 25 samples were well differentiated. In addition, partial least squares model was established to explore fingerprint-efficacy relationship between the fingerprints and the antioxidant activities in vitro, providing clinically useful information for quality control. The proposed method was reliable and comprehensive, which could be used for a valuable reference to monitor the quality consistency of traditional Chinese medicines.

Microemulsion Electrokinetic Chromatography in Combination with Chemometric Methods to Evaluate the Holistic Quality Consistency and Predict the Antioxidant Activity of Ixeris sonchifolia (Bunge) Hance Injection.

In this paper, microemulsion electrokinetic chromatography (MEEKC) fingerprints combined with quantification were successfully developed to monitor the holistic quality consistency of Ixeris sonchifolia (Bge.) Hance Injection (ISHI). ISHI is a Chinese traditional patent medicine used for its anti-inflammatory and hemostatic effects. The effects of five crucial experimental variables on MEEKC were optimized by the central composite design. Under the optimized conditions, the MEEKC fingerprints of 28 ISHIs were developed. Quantitative determination of seven marker compounds was employed simultaneously, then 28 batches of samples from two manufacturers were clearly divided into two clusters by the principal component analysis. In fingerprint assessments, a systematic quantitative fingerprint method was established for the holistic quality consistency evaluation of ISHI from qualitative and quantitative perspectives, by which the qualities of 28 samples were well differentiated. In addition, the fingerprint-efficacy relationship between the fingerprints and the antioxidant activities was established utilizing orthogonal projection to latent structures, which provided important medicinal efficacy information for quality control. The present study offered a powerful and holistic approach to evaluating the quality consistency of herbal medicines and their preparations.

Holistic Evaluation of Quality Consistency of Ixeris sonchifolia (Bunge) Hance Injectables by Quantitative Fingerprinting in Combination with Antioxidant Activity and Chemometric Methods.

A widely used herbal medicine, Ixeris sonchifolia (Bge.) Hance Injectable (ISHI) was investigated for quality consistency. Characteristic fingerprints of 23 batches of the ISHI samples were generated at five wavelengths and evaluated by the systematic quantitative fingerprint method (SQFM) as well as simultaneous analysis of the content of seven marker compounds. Chemometric methods, i.e., support vector machine (SVM) and principal component analysis (PCA) were performed to assist in fingerprint evaluation of the ISHI samples. Qualitative classification of the ISHI samples by SVM was consistent with PCA, and in agreement with the quantitative evaluation by SQFM. In addition, the antioxidant activities of the ISHI samples were determined by both the off-line and on-line DPPH (2, 2-diphenyl-1-picryldrazyl) radical scavenging assays. A fingerprint-efficacy relationship linking the chemical components and in vitro antioxidant activity was established and validated using the partial least squares (PLS) and orthogonal projection to latent structures (OPLS) models; and the online DPPH assay further revealed those components that had position contribution to the total antioxidant activity. Therefore, the combined use of the chemometric methods, quantitative fingerprint evaluation by SQFM, and multiple marker compound analysis in conjunction with the assay of antioxidant activity provides a powerful and holistic approach to evaluate quality consistency of herbal medicines and their preparations.

Monitoring the quality consistency of Weibizhi tablets by micellar electrokinetic chromatography fingerprints combined with multivariate statistical analyses, the simple quantified ratio fingerprint method, and the fingerprint-efficacy relationship.

Micellar electrokinetic chromatography fingerprinting combined with quantification was successfully developed and applied to monitor the quality consistency of Weibizhi tablets, which is a classical compound preparation used to treat gastric ulcers. A background electrolyte composed of 57 mmol/L sodium borate, 21 mmol/L sodium dodecylsulfate and 100 mmol/L sodium hydroxide was used to separate compounds. To optimize capillary electrophoresis conditions, multivariate statistical analyses were applied. First, the most important factors influencing sample electrophoretic behavior were identified as background electrolyte concentrations. Then, a Box-Benhnken design response surface strategy using resolution index RF as an integrated response was set up to correlate factors with response. RF reflects the effective signal amount, resolution, and signal homogenization in an electropherogram, thus, it was regarded as an excellent indicator. In fingerprint assessments, simple quantified ratio fingerprint method was established for comprehensive quality discrimination of traditional Chinese medicines/herbal medicines from qualitative and quantitative perspectives, by which the quality of 27 samples from the same manufacturer were well differentiated. In addition, the fingerprint-efficacy relationship between fingerprints and antioxidant activities was established using partial least squares regression, which provided important medicinal efficacy information for quality control. The present study offered an efficient means for monitoring Weibizhi tablet quality consistency.

[Establishment of the mathematical model for the inhibition on alpha-glycosidase by chemical compositions from Terminalia chebula Retz. based on the liquid chromatographic information].

The extracts from Chinese herbs are complex compounds with uncertain mechanism, which affects the target with all ingredients. Therefore, it is of great importance to evaluate the bioactivity of these extracts. In this study, 29 samples which had continuous variation of constituents were obtained from the alcohol extract of Terminalia chebula Retz. by a Sephadex LH-20 size exclusion chromatographic column. After detecting the chromatographic information of detection wavelength at 254 nm and the activity information of these samples, the mathematical relationship equation was established to determine the impact on the active components. Stepwise regression method was used to research the relationship between the peak areas (V(i), independent variables) of the compositions in the samples and activities (according to the enzyme inhibition (W), dependent variables). The results showed that there were 55 chromatographic peaks in the samples and the mathematical relationship between the compositions of Terminalia chebula Retz. extract (V(i), i = 1, 2, ..., 55) and activities was certain. The expression was: W = V(7) x (-0.034 +/- 0.013) + V18 x (-0.155 +/- 0.051) + V29 x (-0.142 +/- 0.028) + V4 x (0.079 +/- 0.020) + V11 x (0.074 +/- 0 028) + V36 x (-0.117 +/- 0.053) + 85.669 +/- 4.476, multiple correlation coefficient R = 0.854, significance level (Sig. F change) = 0.037. The steadiness of model was favorable with new digital information. The compounds corresponding to 18th, 29th, 36th, 4th, 11th and 7th peaks were the main active compounds. The method can be also applied to the relationship between a wide variety of components of traditional Chinese medicine resources and different kinds of activities. It is significant to the research and development of traditional Chinese medicine.

[Evaluation of Renshen Guipi Wan quality by linearly quantified fingerprint method based on three-wavelength high performance liquid chromatographic fingerprints].

The theoretical model of linearly quantified fingerprint method (LQFM) for qualitative and quantitative control traditional Chinese medicine (TCM) has been developed based on the relationship of linear function between a sample and the reference fingerprint vector, as the concepts of correlation coefficient r, slope a, intercept b, linearly qualitative similarity S(L), linearly quantitative similarity M(L) and so on were introduced into the study. The high performance liquid chromatography coupled with diode array detector (DAD) was used to simultaneously obtain chromatograms of ten batches of Renshen Guipi Wan (RSGPW) at 203, 228 and 326 nm, separately. The model of LQFM was applied to evaluate the three-wavelength fingerprints for the quality control of RSGPW, it could obviously distinguish RSGPW from different resources and the results showed the quality was almost consistent from the same manufacture. The results of LQFM were compared with those of the systematically quantified fingerprint method (SQFM), a known method for quality control of TCM. It proved that LQFM can evaluate qualitatively and quantitatively the complex system of TCM which is composed of diverse chemical components. The quality control can be better and more reliable by multiple-wavelength fingerprints which give full consideration of the ultraviolet absorption characteristics of different components.