Shouhui Tongbian Capsule in treatment of constipation: Treatment and mechanism development.

Constipation is common in the diseases of the digestive system in clinics. With the change in diet structure and the increase in life pressure, the prevalence rate increases year by year. In traditional Chinese medicine (TCM), the location of the disease of constipation is in the large intestine, which is related to the dysfunction of lung, spleen, liver, kidney and other viscera. Its pathogenesis is conductive dysfunction of large intestine. Based on the theory, Shouhui Tongbian Capsule (SHTB) is composed of eight traditional Chinese medicines, including Polygoni multiflori Radix (Heshouwu in Chinese), Aloe (Luhui in Chinese), Cassiae Semen (Juemingzi in Chinese), Ginseng Radix et Rhizoma (Renshen in Chinese), Lycii Fructus (Gouqizi in Chinese), Asini Corii Colla (Ejiao in Chinese), Aurantii Fructus Immaturus (Zhishi in Chinese), and Atractylodis Macrocephalae Rhizoma (Baizhu in Chinese), which could help to release excessive turbid, and nourishing yin and supplementing qi in the treatment. This study has been carried out to review the latest advances of SHTB in the treatment of constipation. The results showed that significant effect of SHTB was found in the treatment of constipation, such as functional constipation, and constipation associated with tumor chemotherapy, colitis, type 2 diabetes and chronic cardiac failure. Besides, obvious adverse reactions were not observed. SHTB could effectively treat five types of constipation, provide direction for the future exploration of SHTB in the treatment of other types of constipation.

Simultaneous enrichment and speciation of lead and mercury by magnetic solid-phase extraction coupled to HPLC-ICP-MS based on magnetic hydrazine-linked covalent organic frameworks.

BACKGROUND: Trace levels of organic and inorganic lead and mercury species in the environment, including divalent lead (Pb2+), trimethyllead (TML), divalent mercury (Hg2+), monomethylmercury (MeHg), and ethylmercury (EtHg), are highly toxic to humans and ecology. It is of great importance for speciation of lead and mercury to evaluate the toxicity of lead and mercury and their biogeochemistry in the environment. However, simultaneous multi-elemental enrichment and speciation at trace level remains a challenge. There are few reports of simultaneous magnetic solid-phase extraction (MSPE) of organic and inorganic lead and mercury species at trace level in the real water. RESULTS: In this work, a novel core-shell magnetic hydrazine-linked covalent organic frameworks (Fe3O4@COF-TCH) was prepared for the first time by grafting hydrazine-linked COFs on the Fe3O4 nanoparticles. Fe3O4@COF-TCH with abundant thione and imino groups has strong adsorption for lead and mercury species. Based on it, a simple and practical magnetic solid-phase extraction high-performance liquid chromatography-inductively coupled plasma mass spectrometry (MSPE-HPLC-ICP-MS) method was developed for extraction and determination of trace lead and mercury species, including Hg2+, MeHg, EtHg, Pb2+ and TML. The limits of detection (3δ) of the developed method were 0.08, 0.81, 0.90, 0.56 and 0.88 ng L-1 with the enrichment factors (EFs) of 384, 376, 379, 389 and 360-fold for Pb2+, TML, Hg2+, MeHg and EtHg, respectively. The high accuracy and reproducibility have been proved by the spiked recoveries (94.4-103 %) in real samples. SIGNIFICANCE: The proposed method with simple operation and high sensitivity has been successfully applied to simultaneous speciation of lead and mercury at trace levels in the water samples with complicated matrices, including underground water, surface water, sea water. Meanwhile, it has the advantages of cost-saving, labor-saving and time-saving and is suitable for the investigation and risk assessment in water. The development of MSPE-HPLC-ICP-MS method provides ideas and guidance for the simultaneous multi-elemental enrichment and speciation.

In vitro anti-Helicobacter pylori activity and antivirulence activity of cetylpyridinium chloride.

The primary treatment method for eradicating Helicobacter pylori (H. pylori) infection involves the use of antibiotic-based therapies. Due to the growing antibiotic resistance of H. pylori, there has been a surge of interest in exploring alternative therapies. Cetylpyridinium chloride (CPC) is a water-soluble and nonvolatile quaternary ammonium compound with exceptional broad-spectrum antibacterial properties. To date, there is no documented or described specific antibacterial action of CPC against H. pylori. Therefore, this study aimed to explore the in vitro activity of CPC against H. pylori and its potential antibacterial mechanism. CPC exhibited significant in vitro activity against H. pylori, with MICs ranging from 0.16 to 0.62 µg/mL and MBCs ranging from 0.31 to 1.24 µg/mL. CPC could result in morphological and physiological modifications in H. pylori, leading to the suppression of virulence and adherence genes expression, including flaA, flaB, babB, alpA, alpB, ureE, and ureF, and inhibition of urease activity. CPC has demonstrated in vitro activity against H. pylori by inhibiting its growth, inducing damage to the bacterial structure, reducing virulence and adherence factors expression, and inhibiting urease activity.

Development and Validation of Analytical Procedure for Elemental Impurities in Rosuvastatin Calcium Tablets by ICP-MS and Microwave Digestion.

Rosuvastatin calcium is a widely used 3-hydroxy-3-methylglutaryl coenzyme A-reductase inhibitor developed for the treatment of dyslipidemia. To establish a control strategy for the elemental impurities, a new digestion method combined with an inductively coupled plasma-mass spectrometer (ICP-MS) was developed and validated by our team to determine elements Cd, Pb, As, Hg, Co, V, and Ni in rosuvastatin calcium tablets, which digest the sample perfectly even in the presence of a large number of excipients, especially titanium dioxide. The measurement mode was collision cell mode with kinetic energy discrimination (KED). 209Bi+, 115In+, and 89Y+ were chosen as internal standard elements. The recoveries of the limit of quantitation (LOQ) ranged from 90.5% to 106.4%, concentrations of the abovementioned elements in LOQ were 0.25 µg·L-1, 0.25 µg·L-1, 0.75 µg·L-1, 1.5 µg·L-1, 2.5 µg·L-1, 5 µg·L-1, and 8 µg·L-1 , respectively, linear correlation coefficients were above 0.9997, the recoveries in accuracy item ranged from 91.8% to 103.6%, and relative standard deviations (RSDs) of recovery in precision were not more than 1.8%, reflecting a reliable method of high sensitivity, strong anti-interference capacity, and good precision, and that it was suitable for the determination of elemental impurities in drugs.

A sensitive, portable, and smartphone-based whole-cell biosensor device for salicylic acid monitoring.

Considerable effort has been invested in developing salicylic acid (SA) biosensors for various application purposes. Here, by engineering the sensing modules and host cell chassis, we have gradually optimized the NahR-Psal/Pr-based SA biosensor, increasing the sensitivity and maximum output by 17.2-fold and 9.4-fold, respectively, and improving the detection limit by 800-fold, from 80 µM to 0.1 µM. A portable SA sensing device was constructed by embedding a gelatin-based hydrogel containing an optimized biosensor into the perforations of tape adhered to glass slide, which allowed good determination of SA in the range of 0.1 µM-10 µM. Then, we developed a customized smartphone App to measure the fluorescence intensity of each perforation and automatically calculate the corresponding SA concentration so that we could detect SA concentrations in real cosmetic samples. We anticipate that this smartphone-based imaging biosensor, with its compact size, higher sensitivity, cost-effectiveness, and easy data transfer, will be useful for long-term monitoring of SA.

Development of a Universal One-Step Purification and Activation Method to Engineer Protein-Glutaminase through Rational Design.

Cytotoxic enzymes often exist as zymogens containing prodomains to keep them in an inactive state. Protein-glutaminase (PG), which can enhance various functional characteristics of food proteins, is an enzyme containing pro-PG and mature-PG (mPG). However, poor activity and stability limit its application while tedious purification and activation steps limit its high-throughput engineering. Here, based on structural analysis, we replaced the linker sequence between pro-PG and mPG with the HRV3C protease recognition sequence and then coexpressed it with HRV3C protease in Escherichia coli to develop an efficient one-step purification and activation method for PG. We then used this method to obtain several mutants designed by a combination of computer-aided approach and beneficial point mutations. The specific activity (131.6 U/mg) of the best variant D1 was 4.14-fold that of the wild type, and t1/2 and T5010 increased by 13 min and 7 °C, respectively. D1 could effectively improve the solubility and emulsification of wheat proteins, more than twice the effect of the wild type. We also discussed the mechanism underlying the improved properties of D1. In summary, we not only provide a universal one-step purification and activation method to facilitate zymogen engineering but also obtain an excellent PG mutant.

N-terminal domain truncation yielded a unique dimer of polysaccharide hydrolase with enhanced enzymatic activity, stability and calcium ion independence.

Domain engineering, including domain truncation, fusion, or swapping, has become a common strategy to improve properties of enzymes, especially glycosyl hydrolases. However, there are few reports explaining the mechanism of increased activity from a protein structure perspective. Amy703 is an alkaline amylase with a unique N-terminal domain. Prior studies have shown that N-Amy, a mutant without an N-terminal domain, exhibits improved activity, stability, and calcium ion independence. In this study, we have used X-ray crystallography to determine the crystal structure of N-Amy and used AlphaFold2 to model the Amy703 structure, respectively. We further used size exclusion chromatography to show that Amy703 existed as a monomer, whereas N-Amy formed a unique dimer. It was found that the N-terminus of one monomer of N-Amy was inserted into the catalytic domain of its symmetrical subunit, resulting in the expansion of the catalytic pocket. This also significantly increased the pKa of the hydrogen donor Glu350, thereby enhancing substrate binding affinity and contributing to increased N-Amy activity. Meanwhile, two calcium ions were found to bind to N-Amy at different binding sites, which also contributed to the stability of protein. Therefore, this study provided new structural insights into the mechanisms of various glycosyl hydrolases.

A combinatorial strategy for HRV 3C protease engineering to achieve the N-terminal free cleavage.

Human rhinovirus 3C protease (HRV 3CP) has a high specificity against the substrate of LEVLFQ↓G at P1' site, which plays an important role in biotechnology and academia as a fusion tag removal tool. However, a non-ignorable limitation is that an extra residue of Gly would remain at the N terminus of the recombinant target protein after cleavage with HRV 3CP, thus potentially causing protein mis-functionality or immunogenicity. Here, we developed a combinatorial strategy by integrating structure-guided library design and high-throughput screening of eYESS approach for HRV 3CP engineering to expand its P1' specificity. Finally, a C3 variant was obtained, exhibiting a broad substrate P1' specificity to recognize 20 different amino acids with the highest activity against LEVLFQ↓M (kcat/KM = 3.72 ± 0.04 mM-1∙s-1). Further biochemical and NGS-mediated substrate profiling analysis showed that C3 variant still kept its substrate stringency at P1 site and good residue tolerance at P2' site, but with an expanded P1' specificity. Structural simulation of C3 indicated a reconstructed S1' binding pocket as well as new interactions with the substrates. Overall, our studies here prompt not only the practical applications and understanding of substrate recognition mechanisms of HRV 3CP, also provide new tools for other enzyme engineering.

[Analysis of signal peptides and secreted proteins in the whole proteome of Pichia pastoris].

The signal peptide is a key factor that affects the efficiency of protein secretion in Pichia pastoris. Currently, the most used signal peptide is the α-mating factor (MFα) pre-pro leader from Saccharomyces cerevisiae. This exogenous signal peptide has been successfully utilized to express and secret many heterologous proteins. However, MFα is not suitable for the secretory expression of all heterologous proteins. Many typical signal peptides are present in the secretory proteins of P. pastoris, which provides more options besides MFα. Therefore, it is necessary to analyze and identify more efficient endogenous signal peptides that can guide the secretion of heterologous proteins in P. pastoris. In this study, we employed bioinformatics tools such as SignalP, TMHMM, Phobius, WoLF PSORT, and NetGPI to predict endogenous signal peptides from the entire proteome of P. pastoris GS115 (ATCC 20864). Moreover, we analyzed the distribution, length, amino acid composition, and conservation of these signal peptides. Additionally, we screened 69 secreted proteins and their signal peptides, and through secretome validation, we identified 10 endogenous signal peptides that have potential to be used for exogenous protein expression. The endogenous signal peptides obtained in this study may serve as new valuable tools for the expression and secretion of heterologous proteins in P. pastoris.

In vitro anti-Helicobacter pylori activity and the underlining mechanism of an empirical herbal formula - Hezi Qingyou.

Background: Helicobacter pylori (H. pylori) is thought to primarily colonize the human stomach and lead to various gastrointestinal disorders, such as gastritis and gastric cancer. Currently, main eradication treatment is triple or quadruple therapy centered on antibiotics. Due to antibiotic resistance, the eradication rate of H. pylori is decreasing gradually. Therefore, searching for anti-H. pylori drugs from herbal sources has become a strategy for the treatment. Our team proposed a Hezi Qingyou Formula (HZQYF), composed of Chebulae Fructus, Ficus hirta Vahl and Cloves, and studied its anti-H. pylori activity and mechanism. Methods: Chemical components of HZQYF were studied using UHPLC-MS/MS and HPLC. Broth microdilution method and agar dilution method were used to evaluate HZQYF's antibacterial activity. The effects of HZQYF on expression of adhesion genes (alpA, alpB, babA), urease genes (ureE, ureF), and flagellar genes (flaA, flaB) were explored using Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) technology. Effects on morphology and permeability of the extracellular membrane were studied using scanning electron microscopy (SEM) and N-phenylnaphthalen-1-amine (NPN) uptake. Effect on urease activity was studied using a urease kinetics analysis in vitro. Immunofluorescence staining method was used to examine the effect on adhesion. Western blot was used to examine the effect on cagA protein. Results: Minimum inhibitory concentration (MIC) values of the formula against H. pylori clinical strains and standard strains were 80-160 µg/mL, and minimum bactericidal concentration (MBC) values were 160-320 µg/mL. The formula could down-regulate the expression of adhesion genes (alpA, alpB, babA), urease genes (ureE, ureF) and flagellar genes (flaA, flaB), change the morphology of H. pylori, increase its extracellular membrane permeability, and decrease its urease activity. Conclusion: Present studies confirmed that HZQYF had promising in vitro anti-H. pylori activities and demonstrated its possible mechanism of action by down-regulating the bacterial adhesion, urease, and flagellar gene expression, which provided scientific bases for further clinical investigations.

1,3,6-Trigalloylglucose: A Novel Potent Anti-Helicobacter pylori Adhesion Agent Derived from Aqueous Extracts of Terminalia chebula Retz.

1,3,6-Trigalloylglucose is a natural compound that can be extracted from the aqueous extracts of ripe fruit of Terminalia chebula Retz, commonly known as "Haritaki". The potential anti-Helicobacter pylori (HP) activity of this compound has not been extensively studied or confirmed in scientific research. This compound was isolated using a semi-preparative liquid chromatography (LC) system and identified through Ultra-high-performance liquid chromatography-MS/MS (UPLC-MS/MS) and Nuclear Magnetic Resonance (NMR). Its role was evaluated using Minimum inhibitory concentration (MIC) assay and minimum bactericidal concentration (MBC) assay, scanning electron microscope (SEM), inhibiting kinetics curves, urea fast test, Cell Counting Kit-8 (CCK-8) assay, Western blot, and Griess Reagent System. Results showed that this compound effectively inhibits the growth of HP strain ATCC 700392, damages the HP structure, and suppresses the Cytotoxin-associated gene A (Cag A) protein, a crucial factor in HP infection. Importantly, it exhibits selective antimicrobial activity without impacting normal epithelial cells GES-1. In vitro studies have revealed that 1,3,6-Trigalloylglucose acts as an anti-adhesive agent, disrupting the adhesion of HP to host cells, a critical step in HP infection. These findings underscore the potential of 1,3,6-Trigalloylglucose as a targeted therapeutic agent against HP infections.

Enhancing the activity of zearalenone lactone hydrolase toward the more toxic α-zearalanol via a single-point mutation.

Zearalenone (ZEN) and its derivatives are estrogenic mycotoxins known to pose significant health threats to humans and animals. Especially, the derivative α-zearalanol (α-ZAL) is over 10 times more toxic than ZEN. Simultaneous degradation of ZEN and its derivatives, especially α-ZAL, using ZEN lactone hydrolases (ZHDs) is a promising solution to eliminate their potential hazards to food safety. However, most available ZHDs exhibit limited activity toward the more toxic α-ZAL compared to ZEN. Here, we identified a broad-substrate spectrum ZHD, named ZHDAY3, from Exophiala aquamarina CBS 119918, which could not only efficiently degrade ZEN but also exhibited 73% relative activity toward α-ZAL. Through rational design, we obtained the ZHDAY3(N153H) mutant, which exhibited the highest specific activity (253.3 ± 4.3 U/mg) reported so far for degrading α-ZAL. Molecular docking, structural comparative analysis, and kinetic analysis collectively suggested that the shorter distance between the side chain of the catalytic residue His242 and the lactone bond of α-ZAL and the increased binding affinity to the substrate were mainly responsible for the improved catalytic activity of ZHDAY3(N153H) mutant. This mechanism was further validated through additional molecular docking of 18 mutants and experimental verification of six mutants.IMPORTANCEThe mycotoxins zearalenone (ZEN) and its derivatives pose a significant threat to food safety. Here, we present a highly promising ZEN lactone hydrolase (ZHD), ZHDAY3, which is capable of efficiently degrading both ZEN and the more toxic derivative α-ZAL. Next, the ZHDAY3(N153H) mutant obtained by single-point mutation exhibited the highest specific activity for degrading α-ZAL reported thus far. We further elucidated the molecular mechanisms underlying the enhanced hydrolytic activity of ZHDAY3(N153H) toward α-ZAL. These findings represent the first investigation on the molecular mechanism of ZHDs against α-ZAL and are expected to provide a significant reference for further rational engineering of ZHDs, which will ultimately contribute to addressing the health risks and food safety issues posed by ZEN-like mycotoxins.

Site-directed display of zearalenone lactonase on spilt-intein functionalized nanocarrier for green and efficient detoxification of zearalenone.

In this study, we prepared a functional organic-inorganic hybrid nanoflower (InHNF) via split intein moiety in a biomineralization process without using organic solvents. InHNF could specifically bind the target enzymes from crude cell lysates within seconds and site-directedly display them on the surface by forming a peptide bond with enzyme's terminal amino acid residue. This unique feature enabled InHNF to increase the specific activity of zearalenone detoxifying enzyme ZHD518 by 40 âˆ¼ 60% at all tested temperatures and prevented enzyme denaturation even under extreme pH conditions (pH 3-11). Furthermore, it exhibited excellent operational stability, with a residual activity of over 70% after eight reaction cycles. Strikingly, InHNF-ZHD518 achieved above 50% ZEN degradation despite the near inactivation of free ZHD518 in beer sample. Overall, InHNF nanocarriers can achieve environmentally friendly, purification-free, and site-directed immobilization of food enzymes and enhance their catalytic properties, making them suitable for a wide range of industrial applications.

Icaritin Sensitizes Thrombin- and TxA2-Induced Platelet Activation and Promotes Hemostasis via Enhancing PLCγ2-PKC Signaling Pathways.

BACKGROUND: Vascular injury results in uncontrollable hemorrhage in hemorrhagic diseases and excessive antithrombotic therapy. Safe and efficient hemostatic agents which can be orally administered are urgently needed. Platelets play indispensable roles in hemostasis, but there is no drug exerting hemostatic effects through enhancing platelet function. METHODS: The regulatory effects of icaritin, a natural compound isolated from Herba Epimedii, on the dense granule release, thromboxane A2 (TxA2) synthesis, α-granule release, activation of integrin αIIbß3, and aggregation of platelets induced by multiple agonists were investigated. The effects of icaritin on tail vein bleeding times of warfarin-treated mice were also evaluated. Furthermore, we investigated the underlying mechanisms by which icaritin exerted its pharmacological effects. RESULTS: Icaritin alone did not activate platelets, but significantly potentiated the dense granule release, α-granule release, activation of integrin αIIbß3, and aggregation of platelets induced by thrombin and U46619. Icaritin also shortened tail vein bleeding times of mice treated with warfarin. In addition, phosphorylated proteome analysis, immunoblotting analysis, and pharmacological research revealed that icaritin sensitized the activation of phospholipase Cγ2 (PLCγ2)-protein kinase C (PKC) signaling pathways, which play important roles in platelet activation. CONCLUSION: Icaritin can sensitize platelet activation induced by thrombin and TxA2 through enhancing the activation of PLCγ2-PKC signaling pathways and promote hemostasis, and has potential to be developed into a novel orally deliverable therapeutic agent for hemorrhages.

Rapid, easy and catalyst-free preparation of magnetic thiourea-based covalent organic frameworks at room temperature for enrichment and speciation of mercury with HPLC-ICP-MS.

The complex and cumbersome preparation of magnetic covalent organic frameworks (COFs) nanocomposites on a small scale limits their application. Herein, a rapid and easy route was employed for the preparation of magnetic thiourea-based COFs nanocomposites. COFs were coated on Fe3O4 nanoparticles at room temperature without a catalyst within approximately 30 min. This method is suitable for the large-scale preparation of magnetic adsorbent. Using the as-prepared magnetic adsorbent (Fe3O4@COF-TpTU), we developed a simple, efficient, and sensitive magnetic solid-phase extraction-high performance liquid chromatography-inductively coupled plasma-mass spectrometry (MSPE-HPLC-ICP-MS) for the enrichment and determination of mercury species, including Hg2+, methylmercury (MeHg), and ethylmercury (EtHg). The effects of the experimental parameters on the extraction efficiency, including solution pH, adsorption and desorption time, composition and volume of the elution solvent, salinity, coexisting ions, and dissolved organic matter, were comprehensively investigated. Under optimised conditions, the limits of detection in the developed method were 0.56, 0.34, and 0.47 ng L-1 with enrichment factors of 190, 195, and 180-fold for Hg2+, MeHg, and EtHg, respectively. The satisfactory spiked recoveries (97.0-103%) in real water samples and high consistency between the certified and determined values in a certified reference material demonstrate the high accuracy and reproducibility of the developed method. The as-proposed method with simple operation, high sensitivity, and excellent anti-matrix interference performance was successfully applied to the enrichment and determination of trace levels of mercury species in the natural samples with complicated matrices, such as underground water, surface water, seawater and biological samples.

The Effect of Solvents on the Crystal Morphology of Isosorbide Mononitrate and Its Molecular Mechanisms.

In this work, the modified attachment energy model was used to predict the crystal morphology of isosorbide mononitrate (ISMN) in the dichloromethane (CH2Cl2) solvent system and dichloromethane-n-hexane (CH2Cl2-C6H14) mixed solvent system. The solvent effect can significantly affect the crystal morphology, which can profoundly impact both the drug's physicochemical properties and the subsequent technological treatment process. In addition, the interactions between solvent molecules and crystal faces were investigated using molecular dynamics simulation, and radial distribution function (RDF) analysis was performed to determine the types of interactions. The structural parameter S was introduced to characterize the roughness of each crystal surface; the change in the CH2Cl2 diffusion coefficient before and after the addition of C6H14 was analyzed using mean square displacement (MSD). The calculation results of the modified attachment energy from the two solvent systems revealed that C6H14 could accelerate crystal growth, while the crystal morphology was not greatly affected, which is of some significance as a guide for the industrial crystallization process.

UHPLC-MS/MS Method Development and Validation for the Genotoxic Impurities Trimethyl Phosphate and Triisopropyl Phosphate of Elagolix Sodium.

Elagolix sodium is a gonadotropin-releasing hormone (GnRH) receptor antagonist that inhibits endogenous GnRH signaling by competitively binding to GnRH receptors in the pituitary gland to treat moderate to severe pain associated with endometriosis. To keep the safety and quality of the drug, a fast quantitative method by reversed-phase ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry has been developed and validated for the identification, assay and estimation of potential genotoxic impurities trimethyl phosphate and triisopropyl phosphate in commercial batches of this active pharmaceutical ingredient in accordance with International Conference on Harmonization guidelines Q2 and M7. The method was validated by assessing specificity, sensitivity, linearity, the limit of quantification and detection, accuracy, precision and robustness for above analytes at a very low concentration, whose quantification and detection limits reached to 24 and 4.8 pg/ml, respectively, and the total run time for a single injection was 6 min.

Synergistic and attenuated effects and molecular biological mechanisms of Shouhui Tongbian capsule in the treatment of slow transit constipation based on UPLC-MS/MS, network pharmacology and animal experimental validation.

BACKGROUND: Shouhui Tongbian capsule (SHTB) has been widely used for the treatment of constipation. There are few studies on SHTB at present. The current study aimed to explore the effects of multi-components compatibility of SHTB for efficacy enhancement and toxicity reduction and evaluate its molecular biological mechanisms in the treatment of slow transit constipation (STC). METHODS: Ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to quantify 17 anthraquinone components in different compatible systems of SHTB. Network pharmacological analysis was used to probe the potential mechanisms of SHTB in treating STC. In addition, an animal experiment combined with western blot analysis was performed to further validate the predicted results. RESULTS: After compatibility, the dissolution of 13 components with good effects in treating constipation increased, while the dissolution of 3 components with hepatotoxicity decreased. Overall, 145 common targets of 13 synergistic components and constipation were identified. A synergistic component-target-disease network showed that chrysoobtusin, obtusifolin, emodin, obtusin and 2-hydroxyl emodin-1-methyl ether were the potential key synergistic components. A protein-protein interaction network analysis identified 91 targets, and an analysis of topological characteristics was conducted to confirm the core targets. Gene Ontology function revealed that the 13 synergistic components for the treatment of STC mainly played roles via protein phosphorylation, positive regulation of phosphorylation, phosphotransferase activity, kinase activity and protein kinase activity, and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that these components were enriched in pathways in cancer, MAPK signaling pathway, IL-17 signaling pathway, NF-κB signaling pathway, etc. The results of animal experimental validation showed that SHTB significantly reduced the expression levels of p-p38 and p-ERK proteins in the colon tissue of the STC rats. CONCLUSION: This study preliminarily demonstrated that efficacy enhancement and toxicity reduction of SHTB could be achieved after compatibility, which expounded the connotation of compatibility theory of traditional Chinese medicine from the perspective of chemical composition, reflecting the rationality and scientificity of compatibility theory. Meanwhile, the study also revealed the core targets and potential molecular biological mechanisms of SHTB in the treatment of STC, which may serve as a reference for subsequent studies and clinical applications of SHTB.

Engineering polysaccharide hydrolases in the product-releasing cleft to alter their product profiles.

Polysaccharide hydrolases are enzymes capable of hydrolyzing polysaccharides to generate oligosaccharides that have diverse applications in the food, feed and pharmaceutical industries. However, the detailed mechanisms governing the compositions of their hydrolysates remain poorly understood. Previously, we identified a novel neopullulase Amy117, which exclusively converts pullulan to panose by specifically cleaving α-1,4-glycosidic bonds. Yet, several enzymes with high homology to Amy117 produce a mixture of glucose, maltose and panose during pullulan hydrolysis. To explore this particular phenomenon, we compared the sequences and structures between Amy117 and the maltose amylase ThMA, and identified a specific residue Thr299 in Amy117 (equivalent to His294 in ThMA) within the product-releasing cleft of Amy117, which might be responsible for this characteristic feature. Using structure-based rational design, we have successfully converted the product profiles of pullulan hydrolysates between Amy117 and ThMA by simply altering this key residue. Molecular docking analysis indicated that the key residue at the product-releasing outlet altered the product profile by affecting the panose release rate. Moreover, we modeled the long-chain pullulan substrate G8 to examine its potential conformations and found that G8 might undergo a conformational change in the narrow cleft that allows the Amy117 variant to specifically recognize α-1,6-glycosidic bonds.

Network pharmacology and experimental verification to explore the anti-superficial thrombophlebitis mechanism of Mailuo shutong pill.

ETHNOPHARMACOLOGICAL RELEVANCE: Mailuo shutong pill (MLST) has been widely used in clinical treatment of superficial thrombotic phlebitis (STP). Nevertheless, the major active components of MLST and the mechanism of synergistic action have not been reported. AIM OF THE STUDY: The present study aimed to evaluate the improving effects and the underlying mechanism of MLST on mannitol-induced STP in rabbits. MATERIAL AND METHODS: In this study, Ultrahigh-performance liquid chromatography electrospray ionization quadrupole-exactive orbitrap mass spectrometry (UHPLC-ESI-Q-Exactive-Orbitrap-MS) was used to analyze and identify the chemical composition of MLST and the prototype components absorbed into the blood. Then, according to the prototype components in serum, the targets and mechanisms of MLST were explored by applying network pharmacology. The rabbit model of STP was established by injecting 20% mannitol into bilateral auricular vein. The pathological changes of rabbit ear tissues, inflammatory factors, coagulation function and hemorheology were detected. In addition, molecular docking verified the interaction between the main active ingredient and the key target. Finally, the PI3K/AKT pathway and its regulated downstream pathways were verified by Western blot. RESULTS: A total of 96 MLST components and 53 prototypical components absorbed into the blood were successfully identified. Based on network pharmacology, PI3K/AKT pathway and 10 chemical components closely related to this pathway were obtained. Hematoxylin-eosin (HE) staining results indicated that MLST effectively improved of the pathological damage of ear tissues. MLST decreased levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6 and C-reactive protein (CRP). The expression of platelets (PLT) and fibrinogen concentration (FIB) was decreased, while prothrombin time (PT) and activated partial thromboplastin time (APTT) were prolonged. In addition, the plasma viscosity and whole blood viscosity in the MLST groups were significantly decreased. The more important discovery was that the expressions of P-PI3K, VEGF, P-AKT, P-IκB-α, P-NF-κB, NLRP3, ASC, Cleaved IL-1ß and Cleaved Caspase-1 were effectively reversed after treatment with MLST. CONCLUSIONS: This study comprehensively analyzed and characterized the chemical composition of MLST and the prototypical components absorbed into the blood. This study strongly confirmed the pharmacodynamic effect of MLST on STP. More importantly, this pharmacodynamic effect was achieved through inhibition of the PI3K/AKT pathway and its regulated NF-κB and NLRP3 pathways.