Quality Marker Discovery and Quality Evaluation of Eucommia ulmoides Pollen Using UPLC-QTOF-MS Combined with a DPPH-HPLC Antioxidant Activity Screening Method.

Pollen, as an important component of Eucommia ulmoides (EUP), is rich in nutrients and is receiving increasing attention. At present, there are no reports on research related to the chemical composition and quality standards of EUP, and there are significant quality differences and counterfeit phenomena in the market. This study used a UPLC-QTOF-MS system to identify 49 chemical components in EUP for the first time. In the second step, 2,2-diphenyl-1-picrylhydrazyl (DPPH)-HPLC antioxidant activity screening technology was used to identify the main active components of EUP, quercetin-3-O-sophoroside (QSH), quercetin-3-O-sambubioside (QSB), and quercetin 3-O-neohesperidoside (QNH), and their purification, preparation, and structure identification were carried out. Third, molecular docking was used to predict the activity of these components. Fourth, the intracellular ROS generation model of RAW264.7 induced by H2O2 was used to verify and evaluate the activity of candidate active ingredients to determine their feasibility as Q-markers. Finally, a quality control method for EUP was constructed using the three selected components as Q-markers. The identification of chemical components and the discovery, prediction, and confirmation of characteristic Q-markers in EUP provide important references for better research on EUP and the effective evaluation and control of its quality. This approach provides a new model for the quality control of novel foods or dietary supplements.

[Regulatory effect of eight Chinese herbal medicines on glucose and lipid metabolism and their potential active components based on HCBP6 target].

With the improvement of living standards and changes in working style, the prevalence of abnormal glucose and lipid metabolism in humans is increasing in modern society. Clinically, the related indicators are often improved by changing the lifestyle and/or taking hypoglycemic and lipid-lowering drugs, but there are no therapeutic drugs for disorders of glucose and lipid metabolism at present. Hepatitis C virus core protein binding protein 6(HCBP6) is a newly discovered target that can regulate triglyceride and cholesterol content according to level oscillations in the body, thereby regulating abnormal glucose and lipid metabolism. Relevant studies have shown that ginsenoside Rh_2 can significantly up-regulate the expression of HCBP6, but there are few studies on the effect of Chinese herbal medicines on HCBP6. Moreover, the three-dimensional structural information of HCBP6 has not been determined and the discovery of potential active components acting on HCBP6 is not rapidly advanced. Therefore, the total saponins of eight Chinese herbal medicines commonly used to regulate abnormal glucose and lipid metabolism were selected as the research objects to observe their effect on the expression of HCBP6. Then, the three-dimensional structure of HCBP6 was predicted, followed by molecular docking with saponins in eight Chinese herbal medicines to quickly find potential active components. The results showed that all total saponins tended to up-regulate HCBP6 mRNA and protein expression, where gypenosides showed the optimum effect on up-regulating HCBP6 mRNA and ginsenosides showed the optimum effect on up-regulating HCBP6 protein expression. Reliable protein structures were obtained after the prediction of protein structures using the Robetta website and the evaluation of the predicted structures by SAVES. The saponins from the website and literature were also collected and docked with the predicted protein, and the saponin components were found to have good binding activity to the HCBP6 protein. The results of the study are expected to provide ideas and methods for the discovery of new drugs from Chinese herbal medicines to regulate glucose and lipid metabolism.

Recent Developments in Nanoparticle-Based Photo-Immunotherapy for Cancer Treatment.

Phototherapy is an emerging approach for cancer treatment that is effective at controlling the growth of primary tumors. In the presence of light irradiation, photothermal and photodynamic agents that are delivered to tumor sites can induce local hyperthermia and the production of reactive oxygen species, respectively, that directly eradicate cancer cells. Nanoparticles, characterized by their small size and tunable physiochemical properties, have been widely utilized as carriers for phototherapeutic agents to improve their biocompatibility and tumor-targeted delivery. Nanocarriers can also be used to implement various codelivery strategies for further enhancing phototherapeutic efficiency. More recently, there has been considerable interest in augmenting the immunological effects of nanoparticle-based phototherapies, which can yield durable and systemic antitumor responses. This review provides an overview of recent developments in using nanoparticle technology to achieve photo-immunotherapy.

Mechanism by which Eucommia ulmoides leaves Regulate Nonalcoholic fatty liver disease based on system pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Eucommia ulmoides (E. ulmoides) leaves are included in the Chinese Pharmacopoeia, and are traditionally used to treat hypertension, obesity, diabetes, and other diseases. Numerous pharmacological studies have shown that E. ulmoides has a good effect on lowering blood lipids and can improve obesity and nonalcoholic fatty liver. AIM: To study the mechanism of E. ulmoides leaves in regulating nonalcoholic fatty liver disease by combining prediction and validation. METHODS: Using network pharmacology, and molecular docking to predict E. ulmoides in regulating the action mechanism and potential active ingredients of nonalcoholic fatty liver, large hole adsorption resin enrichment active sites, in vitro experiments were performed to verify its fat-lowering effect and mechanism. RESULTS: The major components of E. ulmoides leaves exhibited good combination with lipid metabolism-regulating core proteins, particularly flavonoids. EUL 50 significantly reduced lipid accumulation, and increased PPARγ. Compared with the control group, the autophagy level increased after the administration of EUL 50. PPARγ decreased significantly after the addition of chloroquine (CQ, autophagy inhibitor). CONCLUSION: The active ingredients in E. ulmoides leaves regulating nonalcoholic fatty liver disease are mainly flavonoids and phenolics. EUL 50 may play a role in lowering lipids by regulating PPARγ expression through inducing autophagy.

[Rapid discovery of tyrosinase inhibitors from Sophora flavescens:significance for comprehensive utilization of its non-medicinal resources].

Sophorae Flavescentis Radix,derived from the root of Sophora flavescens in the Leguminosae family,has been widely used in the medicine,agriculture,animal husbandry,and daily chemical industry. A pharmacophore model-based method for rapid discovery of tyrosinase inhibitors( TIs) from S. flavescens was established by molecular docking under Lipinski rules,and verified by enzyme assays. Briefly,the chemical constituent database of S. flavescens( CDSF) was established based on the previous papers. Theoptimal pharmacophore model( OPM) was constructed by DS 2019 on the basis of known active TIs. Eighty-three hits predominated by flavonoids having higher fitting scores with OPM than the positive control were screened out,and subjected to molecular docking based on the three-dimensional structure of tyrosinase crystal protein. The potential TIs such as kurarinone and nor-kurarinone were rapidly discovered from the compounds with higher docking scores than the positive control under the Lipinski rules. The results were verified by the in vitro enzyme assays. The inhibition activities of tyrosinase from non-medicinal parts of S. flavescens were also tested to explore the relationship between the inhibition activity and chemical compositions. This study is expected to provide data support for the comprehensive application and development of S. flavescens and also a new method for the rapid discovery of active substances or functional constituents in the complex systems.

[Determination of ß-nicotinamide mononucleotide and nicotinamide adenine dinucleotide in Dendrobium officinale and congeneric species by UPLC-MS/MS].

As anti-aging ingredients, ß-nicotinamide mononucleotide(NMN) and nicotinamide adenine dinucleotide(NAD~+) have attracted worldwide attention in recent years. After oral administration, NMN can be converted into NAD~+ in vivo and the latter is the actual ingredient which exerts anti-aging effect. In order to explore the "rejuvenating and anti-aging" effect of Dendrobium officinale, which was firstly recorded in Shennong's Herbal Classic of Materia Medica, this study established the quantitative method of UPLC-MS/MS for simultaneous determination of NMN and NAD~+ in D. officinale and the congeneric species for the first time, and 34 batches of samples were detected. UPLC conditions are as follows: ACQUITY UPLC HSS T3 column(2.1 mm × 100 mm, 1.8 µm), gradient elution with acetonitrile-0.1% formic acid in water at the flow rate of 0.3 mL·min~(-1), and column temperature of 40 ℃. MS conditions were scanned electrospray ionization source and multiple reaction monitoring mode. The method was verified by systematic methodology. The mean recoveries of NMN and NAD~+ were 77.58% and 80.70%, respectively, with RSD of 3.6% and 4.3%, separately. All results showed that the content of NMN was higher in D. officinale than in the other congeneric species. Particularly, the content in fresh D. officinale stems was as high as 0.931 9 µg·g~(-1). NAD~+ was only found in D. officinale and the content was three times higher than that of NMN. This may be the reason that D. officinale topped the "nine famous anti-aging herbs". In addition, processing method influences the content of NMN and NAD~+ in Dendrobium. Specifically, the content of NMN and NAD~+ was in the order of fresh Dendrobium stems > dried Dendrobium stem segments > spiral or spring-like dried Dendrobium stems.

Discovery of potential pharmacodynamic ingredients of Dang-Gui-Si-Ni decoction based on absorbed ingredients and molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: The Dang-Gui-Si-Ni (DGSN) decoction as a classic prescription has been widely used for thousands of years in the clinical practice of traditional Chinese medicine (TCM). Especially in recent years, the potential efficacy of TCM for the treatment of Raynaud's syndrome has attracted great attention as there are still no specific remedies for this disease. However, the active constituents and underlying mechanisms responsible for the therapeutic benefits are not well understood, which makes it difficult to ensure quality control or to design research and drug development strategies. To identify the potential pharmacodynamic ingredients (PPIs) of TCM will help to achieve suitable process control procedures for industrial production and large-scale manufacturing. AIM OF THE STUDY: In the present study, we propose a multi-dimensional qualitative analysis method combining water-decoction spectra, in-vitro intestinal absorption spectra, in-vivo plasma spectra, and molecular docking of components to quickly identify the PPIs for the DGSN decoction of TCM. MATERIALS AND METHODS: Water-based decoctions of DGSN were prepared in accordance with the clinical use registered in ancient books. Ultra-high-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UHPLC-Q/TOF-MS) coupled with computerized modelling activity screening was used to quickly identify the PPIs of the DGSN decoction. Bioactive compounds absorbed in vitro were identified using the everted intestinal sac model from rats and compounds absorbed in vivo were confirmed in portal vein blood samples obtained following oral administration in rats. Molecular docking validation experiments were adopted to predict the binding activity to coagulation factors I, II, VII, X, and IX. The active components were further confirmed by pharmacodynamics analysis. The anticoagulant activity of the DGSN decoction was verified using rat models. RESULTS: Thirty-one compounds were identified in the DGSN decoction. According to the in vivo experiments, 22 compounds that could be absorbed in vivo were detected by the everted intestinal sac model in rats. This model greatly reduces the scope of PPIs and is easy to perform. Ten compounds were detected in the portal vein blood in rats. The compounds detected in plasma provide stronger evidence supporting the PPIs. Molecular docking in vitro experiments indicated that 7 compounds exhibited better binding activity with coagulation factors I, II, VII, X, and IX. The animal experiments confirmed that the DGSN decoction could improve the microcirculation, providing indirect proof of anticoagulant activity suggested by the molecular docking studies. Finally, based on the multi-dimensional methods, 9 potential compounds present in the DGSN decoction were identified as PPIs (i.e., ferulic acid, paeoniflorin, albiflorin, chlorogenic acid, cryptochlorogenic acid, liquiritin, liquiritin apioside, cinnamaldehyde and glycyrrhizic acid). CONCLUSION: Overall, this study combined the water-decoction spectra, intestinal absorption spectra in vitro, plasma spectra in vivo, and molecular docking studies to establish a multi-dimensional qualitative analysis method of the DGSN decoction. Meanwhile, 9 compounds in DGSN decoction were identified as PPIs using this method, and are proposed for application as quality standards for complex TCM prescriptions.

[Optimization of determination of astragaloside â £ in Astragali Radix by continuous single-factor method].

This paper aims to solve the problems of complicated-unstable test solution preparation process and insufficient extraction of the active ingredient astragaloside Ⅳ in the legal method for the determination of astragaloside Ⅳ in Astragali Radix. The continuous single-factor analysis of seven main factors affecting the content of astragaloside Ⅳ was carried out by HPLC-ELSD, and then the pre-paration method of test solution was optimized. This optimized method exhibited excellent performance in precision, repeatability and stability. The average recovery rate of astragaloside Ⅳ was 99.65% with RSD 2.2%. Astragaloside Ⅳ showed a good linearity between the logarithm of peak area and the logarithm of injection quantity in the range of 0.46-9.1 µg(r=0.999 6). The contents of astragaloside Ⅳ in 29 batches of Astragali Radix were determined by the new and the legal methods. The results showed that the average content of astragaloside Ⅳ in these Astragali Radix samples determined by the former method was 1.458 times than that of the latter one, indicating the new method was simple, reliable and more adequate to extract target compound. According to the results, it is suggested to improve the content standard of astragaloside Ⅳ in Astragali Radix in the new edition of Chinese Pharmacopeia.

[Scientificity of traditional commodity grade of Chinese medicinal materials--taking Lonicerae Japonicae Flos as example].

Taking Lonicerae Japonicae Flos as an example, the method of "expert consensus of different regions" was used to screen the representative samples and evaluate their commodity grades. The correlation analysis, hierarchical cluster analysis and partial least squares discriminant analysis(PLS-DA) of "commodity grade-appearance characteristic-component content" were carried out to reveal the scientificity of traditional commodity grade of Chinese medicinal material. By referring to the existing literature and the grade investigation from the sample collection regions, 78 "initial grade" samples were screened out from 118 collected samples. Authoritative experts from four regions(n=4) including Linyi(Shangdong province), Bozhou(Anhui province), Anguo(Hebei province) and Beijing were organized to evaluate their commodity grades, separately. Based on the grade consistency rate(R_i≥70%), 69 "local grade" samples were screened out from the "initial grade" samples. Based on the average grade consistency rate ■ "authoritative grade" samples were screened out from the "local grade" samples, including15 first-grade samples, 9 second-grade samples, 11 third-grade samples and 17 fourth-grade samples. For these "authoritative grade" samples, the main appea-rance characteristics were quantified and the contents of 13 components were determined by ultra performance liquid chromatography(UPLC). Furthermore, the total contents of 6 phenolic acids, 4 flavonoids and 3 iridoids were calculated, respectively. The results of correlation analysis showed that 4 appearance characteristics indices were correlated with the commodity grades: color, rate of yellow bars(including blooming flowers), rate of black heads(including black bars), and rate of stems and leaves(including bud debris). Five component content indices were correlated with the commodity grades: chlorogenic acid, isochlorogenic acid C, sweroside, loganin and the total contents of six phenolic acids. Furthermore, chlorogenic acid, loganin and the total contents of six phenolic acids showed significantly negative correlation with the main appearance characteristics, indicating that the appearance characteristics of Lonicerae Japonicae Flos can reflect its internal quality, and these 3 indices can be used as quality markers(Q-markers). The results of hierarchical cluster analysis showed that the samples of four grades were classified into four categories, and the samples with the same grades and the same categories accounted for 80.8% of the total samples, while the samples with the different grades were obviously classified into different categories. The results of PLS-DA analysis showed that the samples of different grades showed obvious intragroup aggregation and intergroup dispersion. The above results indicated that it was feasible to evaluate the traditional commodity grade of Lonicerae Japonicae Flos by the method of "expert consensus of different regions". For the evaluation of traditional commodity grade of Chinese medicinal material, the samples should be representative, expert conclusions should have enough consensuses, and grade determination should be authoritative. As the crystallization of clinical experience, traditional commodity grade can scientifically reflect the internal quality of Chinese medicinal material.

Microenvironment Remodeling Micelles for Alzheimer's Disease Therapy by Early Modulation of Activated Microglia.

Current strategies for Alzheimer's disease (AD) treatments focus on pathologies in the late stage of the disease progression. Poor clinical outcomes are displayed due to the irreversible damages caused by early microglia abnormality which triggers disease development before identical symptoms emerge. Based on the crosstalk between microglia and brain microenvironment, a reactive oxygen species (ROS)-responsive polymeric micelle system (Ab-PEG-LysB/curcumin (APLB/CUR)) is reported to normalize the oxidative and inflammatory microenvironment and reeducate microglia from an early phase of AD. Through an ß-amyloid (Aß) transportation-mimicked pathway, the micelles can accumulate into the diseased regions and exert synergistic effects of polymer-based ROS scavenging and cargo-based Aß inhibition upon microenvironment stimuli. This multitarget strategy exhibits gradual correction of the brain microenvironment, efficient neuroprotection, and microglia modulation, leading to decreased Aß plaque burdens and consequently enhanced cognitive functions in APPswe/PSEN1dE9 model mice. The results indicate that microglia can be exploited as an early target for AD treatment and their states can be controlled via microenvironment modulation.

Titanium Dioxide Nanoparticle Ingestion Alters Nutrient Absorption in an In Vitro Model of the Small Intestine.

Ingestion of titanium dioxide (TiO2) nanoparticles from products such as agricultural chemicals, processed food, and nutritional supplements is nearly unavoidable. The gastrointestinal tract serves as a critical interface between the body and the external environment, and is the site of essential nutrient absorption. The goal of this study was to examine the effects of ingesting the 30 nm TiO2 nanoparticles with an in vitro cell culture model of the small intestinal epithelium, and to determine how acute or chronic exposure to nano-TiO2 influences intestinal barrier function, reactive oxygen species generation, proinflammatory signaling, nutrient absorption (iron, zinc, fatty acids), and brush border membrane enzyme function (intestinal alkaline phosphatase). A Caco-2/HT29-MTX cell culture model was exposed to physiologically relevant doses of TiO2 nanoparticles for acute (four hours) or chronic (five days) time periods. Exposure to TiO2 nanoparticles significantly decreased intestinal barrier function following chronic exposure. Reactive oxygen species (ROS) generation, proinflammatory signaling, and intestinal alkaline phosphatase activity all showed increases in response to nano-TiO2. Iron, zinc, and fatty acid transport were significantly decreased following exposure to TiO2 nanoparticles. This is because nanoparticle exposure induced a decrease in absorptive microvilli in the intestinal epithelial cells. Nutrient transporter protein gene expression was also altered, suggesting that cells are working to regulate the transport mechanisms disturbed by nanoparticle ingestion. Overall, these results show that intestinal epithelial cells are affected at a functional level by physiologically relevant exposure to nanoparticles commonly ingested from food.