Analyzing chemical composition of Sargentodoxae caulis water extract and their hypouricemia effect in hyperuricemic mice.

Hyperuricemia (HUA) is a metabolic disease characterized by the increase of serum uric acid (UA) level. Sargentodoxae Caulis (SC) is a commonly used herbal medicine for the treatment of gouty arthritis, traumatic swelling, and rheumatic arthritis in clinic. In this study, a total of fifteen compounds were identified in SC water extract using UHPLC-Q-TOF-MS/MS, including three phenolic acids, seven phenolic glycosides, four organic acids, and one lignan. Then, to study the hypouricemia effect of SC, a HUA mouse model was induced using a combination of PO, HX, and 20% yeast feed. After 14 days of treatment with the SC water extract, the levels of serum UA, creatinine (CRE), blood urea nitrogen (BUN) were reduced significantly, and the organ indexes were restored, the xanthine oxidase (XOD) activity were inhibited as well. Meanwhile, SC water extract could ameliorate the pathological status of kidneys and intestine of HUA mice. Additionally, quantitative real-time PCR (qRT-PCR) and western blotting results showed that SC water extract could increase the expression of ATP binding cassette subfamily G member 2 (ABCG2), organic cation transporter 1 (OCT1), organic anion transporter 1 (OAT1) and organic anion transporter 3 (OAT3), whereas decrease the expression of glucose transporter 9 (GLUT9). This study provided a data support for the clinical application of SC in the treatment of HUA.

Screening and characterization of potential anti-gout components from Polygonum cuspidatum by integration off-line two-dimensional liquid chromatography-mass spectrometry with affinity ultrafiltration and on-line HPLC-ABTS.

Polygonum cuspidatum (P. cuspidatum) is a traditional herbal medicine with a long history and proven efficacy in treating gout. However, due to the complexity of composition and extensive content distribution, the substance basis of its anti-gout effectiveness is still unclear. A strategy was proposed via integrating off-line two-dimensional liquid chromatography (2D-LC) and targeted rapid screening technology based on ultrafiltration-liquid chromatography-mass spectrometry (UF-LC/MS) and on-line high-performance liquid chromatography-2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (HPLC-ABTS) to accomplish high coverage and high throughput screening of anti-gout components from P. cuspidatum. As a result, twenty components were screened from P. cuspidatum extract with both xanthine oxidase (XOD) inhibitory activity and free radical scavenging activity, then were preliminarily identified by high-resolution electrospray ionization-quadrupole-time-of-flight mass spectrometer (ESI-Q-TOF/MS). The screened results were verified by the in vitro assays. Meanwhile, molecular docking further elucidated that the screened bioactive ingredients had favourable binding capabilities with XOD. The performance of this study can achieve high efficiency and high coverage screening of the anti-gout components from P. cuspidatum, which provides methodology and strategy support for the rapid screening of bioactive ingredients from complex medicinal plants.

Identification of different degrees of processed ginger using GC-IMS combined with machine learning.

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Prediction of quality markers in Maren Runchang pill for constipation using machine learning and network pharmacology.

Maren Runchang pill (MRRCP) is a Chinese patent medicine used to treat constipation in clinics. It has multi-component and multi-target characteristics, and there is an urgent need to screen markers to ensure its quality. The aim of this study was to screen quality markers of MRRCP based on a "differential compounds-bioactivity" strategy using machine learning and network pharmacology to ensure the effectiveness and stability of MRRCP. In this study, UPLC-Q-TOF-MS/MS was used to identify chemical compounds in MRRCP and machine learning algorithms were applied to screen differential compounds. The quality markers were further screened by network pharmacology. Meanwhile, molecular docking was used to verify the screening results of machine learning and network pharmacology. A total of 28 constituents in MRRCP were identified, and four differential compounds were screened by machine learning algorithms. Subsequently, a total of two quality markers (rutin and rubiadin) in MRRCP. Additionally, the molecular docking results showed that quality markers could spontaneously bind to core targets. This study provides a reference for improving the quality evaluation method of MRRCP to ensure its quality. More importantly, it provided a new approach to screen quality markers in Chinese patent medicines.

Structure and properties of acidic polysaccharides isolated from Massa Medicata Fermentata: Neuroprotective and antioxidant activity.

Massa Medicata Fermentata (MMF) is a fermented food with therapeutic effects. Previous studies suggested that after stir-frying, the uronic acid content in MMF crude polysaccharides increases, and the pH value decreases, which is caused by the change in acidic polysaccharides. However, the detailed physicochemical properties and structure-activity correlation of the acidic polysaccharides in MMF have not been fully explored. In this study, two acidic polysaccharides (SMMFAP and CMMFAP) were isolated from the MMF and its stir-fried product, respectively. Their structural characteristics and bioactivities were comparatively studied, and the structure-activity correlation was examined. Our findings revealed that the SMMFAP had a higher average Mw and higher Gal and Man content than the CMMFAP. Both the SMMFAP and CMMFAP were mainly composed of Xyl, Man, and Gal residues, whereas the CMMFAP had fewer linkage types. Additionally, the CMMFAP exhibited stronger neuroprotective activity than the SMMFAP owing to its higher content of 1,6-linked-Galp, while the SMMFAP exhibited better antioxidant activity, which might be related to its higher average Mw. Our findings suggest that acidic polysaccharides may be the active substances that cause differences in effectiveness between the sheng and chao MMF. Furthermore, the research qualified the SMMFAP and CMMFAP with different potential applications.

Isolation, characterization, trypsin inhibition, liver protective and antioxidant activities of arabinoxylan from Massa Medicata Fermentata and its processed products.

Massa Medicata Fermentata (MMF) is a traditional Chinese medicine widely used in feed additives and human medicine. In this study, two neutral polysaccharides (SMMFP-1 and CMMFP-1) were isolated from two forms of MMF (sheng and chao MMF), and their structural characteristics and bioactivities were studied. The results showed that CMMFP-1 had higher average Mw compared with that of SMMFP-1. SMMFP-1 had a lower proportion of Ara, Xyl, GalA, and GlcA, but higher levels of Fuc, Gal, Man, and GulA. Compared with CMMFP-1, SMMFP-1 had a triple helix structure. SMMFP-1 had a layered structure, whereas CMMFP-1 had a curly layered structure. More glycosidic linkage types were found in SMMFP-1 than in CMMFP-1, and SMMFP-1 had a greater number of side chains. More importantly, SMMFP-1 showed better trypsin inhibition activity in vitro, liver-protective activity in vivo, and stronger antioxidant activity in vivo than CMMFP-1. Thus, arabinoxylans may be one of the active substances for different efficacies between MMF and its processed product. The results of this study facilitate the exploration of the correlation between the structural characteristics and biological functionalities of MMF arabinoxylans. Moreover, a theoretical basis is established for further study of the unique properties of arabinoxylans and their applications.

Subcritical water extraction of polysaccharides from Gastrodiae Rhizoma: optimization, characterization and in vitro hepatoprotective activity.

Subcritical water extraction (SWE) is an efficient and eco-friendly technology that rapidly extracts valuable compounds from natural materials. In this study, response surface methodology (RSM) was utilized to determine the optimal extraction conditions for Gastrodiae Rhizoma using SWE (GRP-S). The optimum conditions were found to be 161 °C extraction temperature, 41 min extraction time, and a solid-to-liquid ratio of 1.55 mg/mL. Under these optimal conditions, the experimental yield of GRP-S was 66.32% ± 0.10% (n = 3), demonstrating a significant increase compared to hot water reflux extraction (HWE) in the extraction yield of polysaccharides. Characterization studies employing SEM, FT-IR, and HPAEC-PAD confirmed the differences between GRP-S and GRP-H (GRP obtained by HWE). Furthermore, both GRP-S and GRP-H exhibited a significant ability to protect HepG2 cells from ethanol-induced damage, with GRP-S showcasing a superior effect. The widespread adoption of SWE technology can lead to high GRP content in extracts and promote the green and sustainable development of natural products extraction processes.

Integration of metabolomics and network pharmacology to reveal the protective mechanism underlying Wogonoside in acute myocardial ischemia rats.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional medicine, both Scutellaria baicalensis Georgi (SBG) and the traditional formulas composed of it have been used to treat a wide range of diseases, including cancer and cardiovascular. Wogonoside (Wog) is the biologically active flavonoid compound extracted from the root of SBG, with potential cardiovascular protective effects. However, the mechanisms underlying the protective effect of Wog on acute myocardial ischemia (AMI) have not yet been clearly elucidated. AIM OF THE STUDY: To explore the protective mechanism of Wog on AMI rats by comprehensively integrating traditional pharmacodynamics, metabolomics, and network pharmacology. METHODS: The rat was pretreatment with Wog at a dose of 20 mg/kg/d and 40 mg/kg/d once daily for 10 days and then ligated the left anterior descending coronary artery of rats to establish the AMI rat model. Electrocardiogram (ECG), cardiac enzyme levels, heart weight index (HWI), Triphenyltetrazolium chloride (TTC) staining, and histopathological analyses were adopted to evaluate the protective effect of Wog on AMI rats. Moreover, a serum metabolomic-based UHPLC-Q-Orbitrap MS approach was performed to find metabolic biomarkers and metabolic pathways, and network pharmacology analysis was applied to predict targets and pathways of Wog in treating AMI. Then, the network pharmacology and metabolomic results were integrated to elucidate the mechanism of Wog in treating AMI. Finally, RT- PCR was used to detect the mRNA expression levels of PTGS1, PTGS2, ALOX5, and ALOX15 to validate the result of integrated metabolomics and network analysis. RESULTS: Pharmacodynamic studies suggest that Wog could effectively prevent the ST-segment of electrocardiogram elevation, reduce the myocardial infarct size, heart weight index, and cardiac enzyme levels, and alleviate cardiac histological damage in AMI rats. Metabolomics analysis showed that the disturbances of metabolic profile in AMI rats were partly corrected by Wog and the cardio-protection effects on AMI rats involved 32 differential metabolic biomarkers and 4 metabolic pathways. In addition, the integrated analysis of network pharmacology and metabolomics showed that 7 metabolic biomarkers, 6 targets, and 6 crucial pathways were the main mechanism for the therapeutic application of Wog for AMI. Moreover, the results of RT-PCR showed that PTGS1, PTGS2, ALOX5, and ALOX15 mRNA expression levels were reduced after treatment with Wog. CONCLUSION: Wog exerts cardio-protection effects on AMI rats via the regulation of multiple metabolic biomarkers, multiple targets, and multiple pathways, our current study will provide strong scientific evidence supporting the therapeutic application of Wog for AMI.

A 13-LOX participates in the biosynthesis of JAs and is related to the accumulation of baicalein and wogonin in Scutellaria baicalensis.

Although baicalein and wogonin contents in Scutellaria baicalensis, a traditional Chinese herb, are known to be regulated by jasmonic acid, the exact mechanism by which jasmonic acid regulates the accumulation of baicalein and wogonin remains unclear. In this study, we discovered SbLOX3, a gene encoding 13-lipoxygenase from the roots of S. baicalensis, which plays an important role in the biosynthesis of jasmonic acid. The contents of methyl jasmonate, baicalin, wogonin, and three metabolic intermediates of methyl jasmonate, 13-HPOT, OPDA, and OPC-8, were downregulated in the hair roots of the SbLOX3 RNAi lines. We confirmed that SbLOX3 was induced by drought stress simulated by PEG and Fusarium oxysporum, which subsequently led to changes in the content of MeJA, baicalin, and wogonin. Taken together, our results indicate that a 13-LOX is involved in the biosynthesis of jasmonic acid, and regulates the accumulation of baicalein and wogonin in S. baicalensis roots.

An efficient Pt@MXene platform for the analysis of small-molecule natural products.

Small-molecule (m/z<500) natural products have rich biological activity and significant application value thus need to be effectively detected. Surface-assisted laser desorption/ionization mass spectrometry (SALDI MS) has become a powerful detection tool for small-molecule analysis. However, more efficient substrates need to be developed to improve the efficiency of SALDI MS. Thus, platinum nanoparticle-decorated Ti3C2 MXene (Pt@MXene) was synthesized in this study as an ideal substrate for SALDI MS in positive ion mode and exhibited excellent performance for the high-throughput detection of small molecules. Compared with using MXene, GO, and CHCA matrix, a stronger signal peak intensity and wider molecular coverage was obtained using Pt@MXene in the detection of small-molecule natural products, with a lower background, excellent salt and protein tolerance, good repeatability, and high detection sensitivity. The Pt@MXene substrate was also successfully used to quantify target molecules in medicinal plants. The proposed method has potentially wide application.

Baicalein alleviates non-alcoholic fatty liver disease in mice by ameliorating intestinal barrier dysfunction.

Non-alcoholic fatty liver disease (NAFLD) is the main cause of chronic liver disease, and its pathological development is closely related to the gut-liver axis. The intestinal barrier, an important component of the gut-liver axis, can prevent gut microbes and endotoxins from entering the liver. Intestinal barrier function is impaired in patients with NAFLD. Baicalein, which is the main flavonoid in Scutellariae Radix, can improve NAFLD. However, whether baicalein alleviates NAFLD by ameliorating intestinal barrier dysfunction remains unclear. In this study, a methionine-choline deficient (MCD) diet-induced NAFLD mouse model is used. The effects of baicalein on lipid accumulation, inflammation and the intestinal barrier in MCD-fed mice were evaluated by detecting blood lipid levels, lipid accumulation, liver pathological changes, inflammatory factors, inflammatory signaling pathways, the three main short-chain fatty acids (acetate, propionate and butyrate), intestinal permeability and intestinal tight junction protein expression. Compared with the MCD-only group, baicalein intake decreased the serum and liver lipid levels. Moreover, the accumulation of lipid droplets and steatosis in the liver were also alleviated; all these results demonstrated that baicalein could alleviate NAFLD. Meanwhile, the levels of inflammatory cytokines decreased in the baicalein group. Further investigation of the mucosal permeability to 4 kDa fluorescein isothiocyanate-dextran, concentrations of short-chain fatty acids in feces, and the expression of intestinal zonula occluden 1 and claudin-1 indicated that a baicalein diet could decrease the intestinal permeability caused by a MCD diet. Moreover, the protein levels of p-NF-κB p65 and the ratio of p-NF-κB p65/NF-κB p65 increased, and IκB-α and PPARα decreased in NAFLD mice, while the administration of baicalein could alleviate these changes. The above results indicated that the mechanism of baicalein in the alleviation of NAFLD lies in the regulation of the intestinal barrier.

Preparative separation of eight phenolic acids from Echinacea purpurea L. Moench using pH-zone-refining counter-current chromatography and evaluation of their immunomodulatory effects and synergistic potential.

Phenolic acids headed by cichoric acid in Echinacea purpurea L. Moench show remarkable immunomodulatory effects. In this study, pH-zone-refining counter-current chromatography with a two-phase solvent system composed of EtOAc-ACN-H2O (4 : 1 : 5, v/v/v) (TFA (10 mM) in the upper phase and NH3·H2O (30 mM) in the lower phase) was applied to the pre-segmentation enrichment of 3.5 g of a crude sample of Echinacea purpurea L. Moench. Then two fractions of highly concentrated samples were further separated using EtOAc-H2O (1 : 1, v/v) and EtOAc-n-BuOH-ACN-H2O (3 : 1 : 1 : 5, v/v/v/v) solvent systems (TFA (10 mM) in the upper phase and NH3·H2O (10 mM) in the lower phase), separately. As a result, eight phenolic acids including cichoric acid were isolated successfully. Moreover, the immunomodulatory effects of the isolated compounds and the synergy of the binary and ternary compound combinations were first studied using CompuSyn software. And the ternary combination (caffeoyl tartaric acid + feruloyl tartaric acid + cichoric acid) showed a 94.91% synergistic inhibition rate of NO production released by LPS-induced RAW 264.7 cells. This study developed a strategy for the rapid preparative separation and purification of phenolic acids with complex components and large differences in content and provided a theoretical basis for the synergistic use of monomeric compounds in Echinacea purpurea L. Moench.

Comparison of Physicochemical and Bioactive Properties of Polysaccharides from Massa Medicata Fermentata and Its Processed Products.

Two polysaccharides were separately extracted and purified from different types of medicinal slices of Massa Medicata Fermentata (Sheng Massa Medicata Fermentata and Chao Massa Medicata Fermentata). The physicochemical properties of these polysaccharides were studied, including the molecular weight, monosaccharide composition, and glycosidic linkage. Moreover, inhibition of trypsin, α-amylase, and α-glucosidase by the polysaccharides and their antioxidant activity were investigated. Compared with polysaccharides from Sheng Massa Medicata Fermentata, polysaccharides from Chao Massa Medicata Fermentata had a lower molecular weight, higher uronic acid content, and a lower proportion of side chains. Polysaccharides from Sheng Massa Medicata Fermentata displayed stronger trypsin, α-amylase, and α-glucosidase inhibition activity, whereas the antioxidant activity of the polysaccharides from Chao Massa Medicata Fermentata was higher. These results indicated that stir-frying changes the physicochemical properties of the polysaccharides significantly, leading to reduced enzyme inhibition activity and an increase in antioxidant activity. This research provides a guide for the selective application of Massa Medicata Fermentata.

[Application of bioassay in quality evaluation of Chinese medicine:a review].

The quality of Chinese medicine is the foundation of the clinical effects and industrial development. Component analysis ensures the consistency and stability of medicinals, but fails to evaluate the clinical efficacy. Bioassay is an analytical method to evaluate the effect of a substance on living organisms, tissues, or cells, which is an optimal option for assessing the quality of Chinese medicine. Bioassay of Chinese medicine starts early but progresses slowly. At the moment, it has attracted the interest of scholars. However, no systematic research is available. This study aims to summarize the research on the application of bioassay in quality evaluation of Chinese medicine, focusing on the application of key techniques and experimental systems in bioassay in heat-clearing and blood-activating and stasis-eliminating Chinese medicine and the common problems. Meanwhile, suggestions were proposed in terms of the association with clinical efficacy and chemical analysis and the status quo of biological assay. This study is expected to promote the study and application of bioassay.

MeJA regulates the accumulation of baicalein and other 4'-hydroxyflavones during the hollowed root development in Scutellaria baicalensis.

The dried roots of Scutellaria baicalensis are important traditional Chinese medicine used to treat liver and lung inflammation. An anomalous structure, hollowed root, was discovered in perennial cultivated Scutellaria baicalensis. The presence of the hollow may change the contents of bioactive metabolites, such as baicalein, and other 4'-hydroxyflavones in Scutellaria baicalensis roots, but the relationship between the hollowed root and bioactive metabolite contents is poorly understood. In this study, we identified the anatomical structure of the hollowed root and detected differentially accumulating flavonoid metabolites and enzymes related to 4'-hydroxyflavone biosynthesis in 3-year-old roots with a hollow. We confirmed that methyl jasmonate (MeJA) induced the accumulation of 4'-hydroxyflavones and the expression of enzymes related to 4'-hydroxyflavone biosynthesis in hydroponically cultured Scutellaria baicalensis roots. The development of the hollowed root were divided into 4 stages. The 4'-hydroxyflavone contents and expression of enzymes related to 4'-hydroxyflavone biosynthesis increased synchronously with the content of MeJA during the development of hollowed root. Pathogen and programed-cell-death related genes were induced during hollowed root development. Taken together, our results provide novel insight into the importance of MeJA in the development of hollowed root and the accumulation of 4'-hydroxyflavones in Scutellaria baicalensis roots. Our results suggest that a pathogen and senescence are the two major causes for the development of hollowed root in Scutellaria baicalensis roots.

Structural characterization and antioxidant activities of one neutral polysaccharide and three acid polysaccharides from the roots of Arctium lappa L.: A comparison.

In this work, we comparatively analyzed the structure and antioxidant activities of different polysaccharide fractions from Arctium lappa L. A total of four water-soluble polysaccharide fractions (ALP-1, ALP-2, ALP-3 and ALP-4) were obtained from the roots of Arctium lappa L. They differed in monosaccharide composition, molecular weight and linkage mode. ALP-1 and ALP-2 mainly consisted of fructose, with average molecular weights of 2.676 × 103 and 2.503 × 104 g/mol, respectively. ALP-3 and ALP-4 were mainly composed of fructose, arabinose and galactose, with average molecular weights of 9.709 × 104 and 6.790 × 104 g/mol, respectively. Furthermore, Fourier transform infrared spectrometry, methylation analysis and nuclear magnetic resonance spectroscopy suggested that the main polysaccharide ALP-1 had a linear chain of (1 → 2)-linked ß-D-Fructofuranosyl backbone (n ≈ 15) linked to a terminal (1 → 2)-linked α-d-Glucopyranosyl at the non-reducing end. All five polysaccharides displayed high antioxidant ability, especially ALP-4 in H2O2-induced HepG2 cell model and ALP-1 in metronidazole [MET]-induced zebrafish model. These findings provided comparative information on the structure and biological activity of different burdock polysaccharides and highlighted their potential as antioxidants in functional foods.

Purification of hypocrellins from Shiraia bambusicola by coordinated high-speed countercurrent chromatography using cupric chloride as a complexing agent.

Hypocrellins are anthraquinone that can act as excellent photosensitizers for photodynamic therapy. In the present work, we found that high-speed countercurrent chromatography using cupric chloride as a complexing agent effectively separated hypocrellins from Shiraia bambusicola extract. The optimal two-phase solvent system consisted of petroleum ether/ethyl acetate/methanol/water (7:3:5.5:4.5, v/v/v/v), with 0.01 mol/L cupric chloride in the lower phase at pH of 2.45. This lower phase served as the mobile phase, whereas the upper phase acted as the stationary phase. Employing a continuous separation mode, three continuous injections were found to allow the purification of 1.2 g of crude extract in approximately 12 h. Hypocrellin B (10.8 mg), hypocrellin A (16.2 mg), and hypocrellin C (15.6 mg) were obtained from this process. Simulation of complexation of hypocrellin A with divalent copper ion by computational chemistry calculations indicated that three pairs of hydroxyl and carbonyl groups in hypocrellin A had similar binding energies, and demonstrated that hypocrellin A and B owned different metal-to-ligand ratios as compared to hypocrellin C. These factors could modify the partitioning of these compounds in two-phase solvent system, and resulting in a suitable separation factor. This method would also be used to purify other anthraquinones from natural products.

Eight-gene metabolic signature related with tumor-associated macrophages predicting overall survival for hepatocellular carcinoma.

BACKGROUND: In recent years, the relationship between tumor-associated macrophages (TAMs) and solid tumors has become a research hotspot. This study aims to explore the close relationship of TAMs with metabolic reprogramming genes in hepatocellular carcinoma (HCC) to provide new methods of treatment for HCC. METHODS: The study selected 343 HCC patients with complete survival information (survival time > = 1 month) in the Cancer Genome Atlas (TCGA) as study subjects. Kaplan-Meier survival analysis assisted in determining the relationship between macrophage infiltration and overall survival (OS), and Pearson correlation tests were used to identify metabolic reprogramming genes (MRGs) associated with tumor macrophage abundance. Lasso regression algorithms were used on prognosis-related MRGs identified by Kaplan-Meier survival analysis and univariate Cox regression analysis to construct a risk score; another independent cohort (including 228 HCC patients) from the International Cancer Genome Consortium (ICGC) was used to verify prognostic signature externally. RESULTS: A risk score composed of 8 metabolic genes could accurately predict the OS of a training cohort (TCGA) and a testing cohort (ICGC). The risk score could be widely used for people with different clinical characteristics, and it is a predictor that is independent of other clinical factors that affect prognosis. As expected, compared with the low-risk group, the high-risk group exhibited an obviously higher macrophage abundance, together with a positive correlation between the risk score and the expression levels of three commonly used immune checkpoints (PD1, PDL1, and CTLA4). CONCLUSION: Our study constructed and validated a novel eight-gene signature for predicting HCC patient OS, which may contribute to clinical treatment decisions.

Lipotoxic hepatocytes promote nonalcoholic fatty liver disease progression by delivering microRNA-9-5p and activating macrophages.

M1-polarized macrophages are involved in chronic inflammatory diseases, including nonalcoholic fatty liver disease (NAFLD). However, the mechanisms responsible for the activation of macrophages in NAFLD have not been fully elucidated. This study aimed at investigating the physiological mechanisms by which extracellular vesicles (EVs)-encapsulated microRNA-9-5p (miR-9-5p) derived from lipotoxic hepatocytes might activate macrophages in NALFD. After blood sample and cell collection, EVs were isolated and identified followed by co-culture with macrophages. Next, the palmitic acid-induced cell and high fat diet-induced mouse NALFD models were established to explore the in vitro and in vivo effects of EVs-loaded miR-9-5p on NAFLD as evidenced by inflammatory cell infiltration and inflammatory reactions in macrophages. Additionally, the targeting relationship between miR-9-5p and transglutaminase 2 (TGM2) was identified using dual-luciferase reporter gene assay. miR-9-5p was upregulated in the NAFLD-EVs, which promoted M1 polarization of THP-1 macrophages. Furthermore, miR-9-5p could target TGM2 to inhibit its expression. Downregulated miR-9-5p in NAFLD-EVs alleviated macrophage inflammation and M1 polarization as evidenced by reduced levels of macrophage inflammatory factors, positive rates of CD86+ CD11b+, and levels of macrophage surface markers in vitro. Moreover, the effect of silencing of miR-9-5p was replicated in vivo, supported by reductions in TG, TC, AST and ALT levels and attenuated pathological changes. Collectively, lipotoxic hepatocytes-derived EVs-loaded miR-9-5p downregulated the expression of TGM2 and facilitated M1 polarization of macrophages, thereby promoting the progression of NAFLD. This highlights a potential therapeutic target for treating NAFLD.

Extraction of Flavonoids from Scutellariae Radix using Ultrasound-Assisted Deep Eutectic Solvents and Evaluation of Their Anti-Inflammatory Activities.

Deep eutectic solvents (DESs) play important roles in the extraction of active constituents in traditional Chinese medicine. Ultrasound-assisted DES has been used to extract flavonoids from Scutellaria baicalensis. Using the contents of scutellarin, baicalin, baicalein, wogonoside, wogonin, and oroxylin A as quantitative indices, different kinds of DESs have been optimized for extraction and betaine/acetic acid has shown the highest yield. The Box-Behnken response surface method (RSM) was utilized to select the extraction conditions with the highest yields. The optimal extraction conditions were as follows: the molar ratio of betaine/acetic acid was 1:4, the water content was 40%, the solid/liquid ratio was 1:100 g/mL, the extraction temperature was 52 °C, and the extraction time was 23 min. Compared with traditional reflux extraction using 70% ethanol as the solvent, ultrasound-assisted DES has a shorter extraction time and higher yields. Furthermore, anti-inflammatory activities of the two extracts by ultrasound-assisted DES and reflux were compared using RAW264.7 cells and the methyl thiazolyl tetrazolium (MTT) method, and they showed equal anti-inflammatory activities. The results demonstrated that the ultrasound-assisted DES method for extraction of flavonoids from scutellariae radix is simple, green, efficient, and reproducible. This research provides good method guides for the rapid and efficient extraction of flavonoids from natural sources.