Investigating the Pharmacological Mechanisms of Total Flavonoids from Eucommia ulmoides Oliver Leaves for Ischemic Stroke Protection.

The aim of this study was to explore how the total flavonoids from Eucommia ulmoides leaves (EULs) regulate ischemia-induced nerve damage, as well as the protective effects mediated by oxidative stress. The cell survival rate was significantly improved compared to the ischemic group (p < 0.05) after treatment with the total flavonoids of EULs. The levels of reactive oxygen species (ROS), lactate dehydrogenase (LDH), and malondialdehyde (MDA) decreased, while catalase (CAT) and glutathione (GSH) increased, indicating that the total flavonoids of EULs can significantly alleviate neurological damage caused by ischemic stroke by inhibiting oxidative stress (p < 0.01). The mRNA expression level of VEGF increased (p < 0.01), which was consistent with the protein expression results. Meanwhile, the protein expression of ERK and CCND1 increased (p < 0.01), suggesting that the total flavonoids of EULs could protect PC12 cells from ischemic injury via VEGF-related pathways. MCAO rat models indicated that the total flavonoids of EULs could reduce brain ischemia-reperfusion injury. In conclusion, this study demonstrates the potential mechanisms of the total flavonoids of EULs in treating ischemic stroke and their potential therapeutic effects in reducing ischemic injury, which provides useful information for ischemic stroke drug discovery.

Development of machine learning models using multi-source data for geographical traceability and content prediction of Eucommia ulmoides leaves.

Rapid and scientific quality evaluation is a hot topic in the research of food and medicinal plants. With the increasing popularity of derivative products from Eucommia ulmoides leaves, quality and safety have attracted public attention. The present study utilized multi-source data and traditional machine learning to conduct geographical traceability and content prediction research on Eucommia ulmoides leaves. Explored the impact of different preprocessing methods and low-level data fusion strategy on the performance of classification and regression models. The classification analysis results indicated that the partial least squares discriminant analysis (PLS-DA) established by low-level fusion of two infrared spectroscopy techniques based on first derivative (FD) preprocessing was most suitable for geographical traceability of Eucommia ulmoides leaves, with an accuracy rate of up to 100 %. Through regression analysis, it was found that the preprocessing methods and data blocks applicable to the four chemical components were inconsistent. The optimal partial least squares regression (PLSR) model based on aucubin (AU), geniposidic acid (GPA), and chlorogenic acid (CA) had a residual predictive deviation (RPD) value higher than 2.0, achieving satisfactory predictive performance. However, the PLSR model based on quercetin (QU) had poor performance (RPD = 1.541) and needed further improvement. Overall, the present study proposed a strategy that can effectively evaluate the quality of Eucommia ulmoides leaves, while also providing new ideas for the quality evaluation of food and medicinal plants.

Aqueous extract of fermented Eucommia ulmoides leaves alleviates hyperlipidemia by maintaining gut homeostasis and modulating metabolism in high-fat diet fed rats.

BACKGROUND: As a traditional Chinese medicinal herb, the lipid-lowing biological potential of Eucommia ulmoides leaves (EL) has been demonstrated. After fermentation, the EL have been made into various products with lipid-lowering effects and antioxidant activity. However, the anti-hyperlipidemic mechanism of fermented Eucommia ulmoides leaves (FEL) is unclear now. PURPOSE: To evaluate the effects of FEL on hyperlipidemia and investigate the mechanism based on regulating gut homeostasis and host metabolism. METHODS: Hyperlipidemia animal model in Wistar rats was established after 8 weeks high-fat diet (HFD) fed. The administered doses of aqueous extract of FEL (FELE) were 128, 256 and 512 mg/kg/d, respectively. Serum biochemical parameters detection, histopathological sections analysis, 16S rDNA sequencing of gut microbiota and untargeted fecal metabolomics analysis, were performed to determine the therapeutic effects and predict related pathways of FELE on hyperlipidemia. The changes of proteins and genes elated to lipid were detected by Immunofluorescence (IF) and quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: 56 Components in FELE were identified by UPLC-MS, with organic acids, flavonoids and phenolic acids accounting for the majority. The intervention of FELE significantly reduced the body weight, lipid accumulation and the levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein-cholesterol (LDL-C) in hyperlipidemia rats, while increased the level of High-density lipoprotein-cholesterol (HDL-C). Meanwhile, FELE improved the inflammatory makers and oxidative stress factors, which is tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT). These results demonstrated that FETE can effectively reduce blood lipids and alleviate inflammation and oxidative damage caused by hyperlipidemia. Mechanistically, FELE restore the homeostasis of gut microbiota by reducing the Firmicutes/Bacteroidetes ratio and increasing the abundance of probiotics, especially Lactobacillus, Rombousia, Bacteroides, Roseburia, Clostridia_UCG-014_Unclassified, while modulated metabolism through amino acid, bile acid and lipid-related metabolism pathways. In addition, the Pearson correlation analysis found that the upregulated bilirubin, threonine, dopamine and downregulated lipocholic acid, d-sphingosine were key metabolites after FELE intervention. IF and qRT-PCR analysis showed that FELE upregulated the expression of fatty acid oxidation proteins and genes (PPARα, CPT1A), bile acid synthesis and excretion proteins and genes (LXRα, CYP7A1, FXR), and downregulated the expression of adipogenic gene (SREBP-1c) by regulating gut microbiota to improve metabolism and exert a lipid-lowering effect. CONCLUSION: This work filled the lipid-lowering mechanism gap of FEL. FELE can improve HFD-induced hyperlipidemia by regulating the gut microbiota homeostasis and metabolism. Thus, FEL has the potential to develop into the novel raw material of lipid-lowering drugs.

Combination of a Deep Eutectic Solvent and Macroporous Resin for Green Recovery of Iridoids, Chlorogenic Acid, and Flavonoids from Eucommia ulmoides Leaves.

To increase the effectiveness of using typical biomass waste as a resource, iridoids, chlorogenic acid, and flavonoids from the waste biomass of Eucommia ulmoides leaves (EULs) were extracted by deep eutectic solvents (DESs) in conjunction with macroporous resin. To optimize the extract conditions, the experiment of response surface was employed with the single-factor of DES composition molar ratio, liquid-solid ratio, water percentage, extraction temperature, and extraction time. The findings demonstrated that the theoretical simulated extraction yield of chlorogenic acid (CGA), geniposidic acid (GPA), aucubin (AU), geniposide (GP), rutin (RU), and isoquercetin (IQU) were 42.8, 137.2, 156.7, 5.4, 13.5, and 12.8 mg/g, respectively, under optimal conditions (hydrogen bond donor-hydrogen bond acceptor molar ratio of 1.96, liquid-solid ratio of 28.89 mL/g, water percentage of 38.44%, temperature of 317.36 K, and time of 55.59 min). Then, 12 resins were evaluated for their adsorption and desorption capabilities for the target components, and the HPD950 resin was found to operate at its optimum. Additionally, the HPD950 resin demonstrated significant sustainability and considerable potential in the recyclability test. Finally, the hypoglycemic in vitro, hypolipidemic in vitro, immunomodulatory, and anti-inflammatory effects of EUL extract were evaluated, and the correlation analysis of six active components with biological activity and physicochemical characteristics of DESs by heatmap were discussed. The findings of this study can offer a theoretical foundation for the extraction of valuable components by DESs from waste biomass, as well as specific utility benefits for the creation and development of natural products.

An integrated process by ultrasonic enhancement in the deep eutectic solvents system for extraction and separation of chlorogenic acid from Eucommia ulmoides leaves.

This study established an integrated process for the extraction and enrichment of chlorogenic acid(CGA)from Eucommia ulmoides leaves in a deep eutectic solvent system via ultrasonic wave-enhanced adsorption and desorption practices utilizing macroporous resins. Although deep eutectic solvents (DESs) have the advantages of chemical stability, good dissolving capacity, and nonvolatilization, routine solvent recovery operations are not suitable for subsequent separation in this solvent system. Based on the above characteristics, this study integrated the extraction and enrichment processes, in which DESs extracts directly loaded onto the macroporous adsorption resin, avoiding the loss of target components in solvent recovery and redissolution processes. The screening results of solvents and resin types further showed that choline chloride-malic acid (1:1) was the optimal DES, and the NKA-II resin had high adsorption and elution performance for CGA. The viscosities of the DESs were much higher than those of water and conventional organic solvents; thus, the mass transfer resistance was large, which could also affect the adsorption behaviour of the macroporous resin. The thermal and mechanical effects of ultrasound could effectively enhance the efficiency of the mass transfer, adsorption, and desorption in the DES systems. When compared to no sonication treatment, the CGA adsorption at various ultrasonic powers (120-600 W) was examined. At optimal ethanol concentration (60%), the effect of the ultrasonic treatment on the recovery of the DESs (water eluting process) and the desorption capability of CGA were confirmed. The use of three volumes of water elution could recover the DESs without loss of CGA. The adsorption process significantly differed depending on the ultrasonic settings, and the absorption balance time and experimental adsorption capacity at equilibrium were enhanced. Additionally, the adsorption procedure of the NKA-II macroporous resin for CGA under ultrasonic treatment could be clarified by the pseudo second order kinetic equation and the Freundlich isotherm model. Thermodynamic and dynamic parameters indicated that physical adsorption was the main process of the entire procedure, and it was a spontaneous, exothermic, and entropy-reducing physical adsorption process. This study potentially indicates that the use of ultrasonication, as a high-efficiency, environmentally friendly method, can enhance the features of the macroporous resin to better purify target chemicals from a DES extract.

[Assay of amino acids in leaves of Eucommia ulmoides under arbor forest mode and leaf-oriented cultivation mode by pre-column derivatization HPLC].

This study aims to develop the pre-column derivatization high performance liquid chromatography(HPLC) method for the determination of 16 kinds of amino acids in Eucommia ulmoides leaves, and compare the content of amino acids in the leaves harvested at different time and under leaf-oriented cultivation mode(LCM) and arbor forest mode(AFM). The HPLC conditions are as below: phenyl isothiocyanate(PITC) as pre-column derivatization agent, Agilent ZORBAX C_(18 )column(4.6 mm×250 mm, 5 µm), mobile phase A of acetonitrile-water(80∶20), mobile phase B of 0.1 mol·L~(-1) sodium acetate solution-acetonitrile(94∶6), gradient elution, flow rate of 1.0 mL·min~(-1), injection volume of 5 µL, column temperature of 40 ℃, and detection wavelength of 254 nm. The HPLC profile indicated well separation of 16 kinds of amino acids and the amino acid content in E. ulmoides leaves was up to 16.26%. In addition, the amino acid content in leaves of E. ulmoides under LCM was higher than under AFM. The amino acid content varied with the harvesting time. Through orthogonal partial least squares discriminant analysis, the amino acids of E. ulmoides under LCM and AFM were compared, which can distinguish the leaves under LCM from those under AFM. Principal component analysis was applied to comprehensively score the amino acids of E. ulmoides leaves. The results showed that the score of leaves under LCM was higher than that under AFM. Nutritional evaluation results indicated that the proteins in E. ulmoides leaves belonged to high-quality vegetable proteins. The established method for the determination of amino acid content is reliable. With the amino acid content as index, the leaf quality of E. ulmoides under LCM is better than that under AFM. This study lays a theoretical basis for the promotion of LCM for E. ulmoides and the development of medicinal and edible products from E. ulmoides leaves.

Extraction of Eucommia ulmoides gum and microbial lipid from Eucommia ulmoides Oliver leaves by dilute acid hydrolysis.

OBJECTIVES: Eucommia ulmoides gum (EUG) is an important natural biomass rubber material, which is usually extracted from Eucommia ulmoides Oliver (EUO). In the extraction process of EUG, pretreatment is the most important step which can efficiently damage EUG-containing cell wall and improve yield of EUG. RESULTS: The FT-IR, XRD, DSC and TG results showed that the thermal properties and structure of the EUG from the dilute acids hydrolysis residue are similar with that of the EUG directly extracted from EUO leaves (EUGD). EUO leaves hydrolysis with AA had the highest EUG yield (16.1%), which was higher than the EUGD yield (9.5%). In the case of the EUO leaves hydrolysis with 0.33 ~ 0.67 wt% of acetic acid (AA), the total sugar was stable in the range of 26.82-27.67 g/L. Furthermore, the EUO leaves acid hydrolysate (AA as reagent) was used as carbon sources for lipid-producing fermentation by Rhodosporidium toruloides. After 120 h of fermentation, the biomass, lipid content and lipid yield were 12.13 g/L, 30.16% and 3.64 g/L, respectively. The fermentation results indicated organic acids were no toxic for Rhodosporidium toruloides and the AA also could be used as carbon source for fermentation.

Assay of amino acids in leaves of Eucommia ulmoides under arbor forest mode and leaf-oriented cultivation mode by pre-column derivatization HPLC / 中国中药杂志

This study aims to develop the pre-column derivatization high performance liquid chromatography(HPLC) method for the determination of 16 kinds of amino acids in Eucommia ulmoides leaves, and compare the content of amino acids in the leaves harvested at different time and under leaf-oriented cultivation mode(LCM) and arbor forest mode(AFM). The HPLC conditions are as below: phenyl isothiocyanate(PITC) as pre-column derivatization agent, Agilent ZORBAX C_(18 )column(4.6 mm×250 mm, 5 μm), mobile phase A of acetonitrile-water(80∶20), mobile phase B of 0.1 mol·L~(-1) sodium acetate solution-acetonitrile(94∶6), gradient elution, flow rate of 1.0 mL·min~(-1), injection volume of 5 μL, column temperature of 40 ℃, and detection wavelength of 254 nm. The HPLC profile indicated well separation of 16 kinds of amino acids and the amino acid content in E. ulmoides leaves was up to 16.26%. In addition, the amino acid content in leaves of E. ulmoides under LCM was higher than under AFM. The amino acid content varied with the harvesting time. Through orthogonal partial least squares discriminant analysis, the amino acids of E. ulmoides under LCM and AFM were compared, which can distinguish the leaves under LCM from those under AFM. Principal component analysis was applied to comprehensively score the amino acids of E. ulmoides leaves. The results showed that the score of leaves under LCM was higher than that under AFM. Nutritional evaluation results indicated that the proteins in E. ulmoides leaves belonged to high-quality vegetable proteins. The established method for the determination of amino acid content is reliable. With the amino acid content as index, the leaf quality of E. ulmoides under LCM is better than that under AFM. This study lays a theoretical basis for the promotion of LCM for E. ulmoides and the development of medicinal and edible products from E. ulmoides leaves.

Effect of Eucommia ulmoides leaves on hyperuricemia and kidney injury induced by a high-fat/high-fructose diet in rats.

Objectives: To investigate the protective and preventive treatment effects of Eucommia ulmoides leaves on a rat model of high-fat and high-fructose diet (HFFD) induced hyperuricemia and renal injury. Materials and Methods: Network pharmacology and molecular-docking methods were used to predict the effects and action mechanisms of the major components of E. ulmoides leaves on hyperuricemia. Combining literature collection, we used SciFinder and the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) and Analysis Platform to collect E. ulmoides leaf flavonoid and iridoid components. Swiss Target Prediction, Similarity ensemble approach (SEA), GeneCards, and the Online Mendelian Inheritance in Man (OMIM) database were used to obtain core targets, and the Search Tool for Recurring Instances of Neighbouring Genes (STRING) protein database was used as core target for gene ontology enrichment Set and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Molecular docking was applied to predict the pathways regulating the metabolism of uric acid. The selected targets and targeting efficacy were validated using a rat model of hyperuricemia and renal injury induced by a high-fat and high-fructose diet. Results: A total of 32 chemical components with effective targets, which regulated the PI3K-AKT pathway and endocrine resistance, were collected. Molecular docking results showed that iridoids and flavonoids are bound to proteins related to inflammation and uric acid metabolism. In addition, it was verified via animal experiments that an E. ulmoides leaf extract ameliorated hyperuricemia, renal injury, and inflammation, which are closely related to the targets Interleukin- 6 (IL-6), Tumor necrosis factor-α (TNF-α), Toll-Like Receptor 4 (TLR4), and Glucose transporter 9 (GLUT9). Conclusion: E. ulmoides leaf flavonoids and iridoids ameliorate hyperuricemia and uric-acid-induced inflammation through a multi-component, multi-target, and multi-pathway mechanism, which provides a theoretical basis for the development of therapeutics from E. ulmoides leaf components.

Protective effect of active components of Eucommia ulmoides leaves on gastric ulcers in rats: Involvement of the PI3K/Akt/NF-κB pathway.

Eucommia ulmoides leaves are widely developed as food and medicines in China and Japan. Its main components have anti-inflammatory properties against gastric ulcers. The purpose of this study was to assess the protective role of an extract derived from the active components of Eucommia ulmoides leaves (EUL 50) against a gastric ulcer and analyze the underlying antiulcer mechanism. The main components of EUL 50 were identified using an ultra-performance liquid chromatography (UPLC) method. Network pharmacology and molecular docking were performed to predict the possible mechanism of action of EUL 50 in the treatment of gastric ulcers. The rats received EUL 50 intragastric administration twice a day for 3 days. Hydrochloric acid/ethanol (HCl/EtOH) was utilized to induce gastric ulcers, followed by histopathological and histochemical evaluation of the ulcer tissues and determination of the main oxidative stress parameters and inflammatory cytokines. The expression of PI3K/Akt/NF-κB pathway-related proteins was measured. Neochlorogenic acid, chlorogenic acid, rutin, and so on were identified as the major components of EUL 50 by UPLC. The prediction results identified the PI3K/Akt/NF-κB signaling pathway as the main possible protective mechanism against gastric ulcers. Furthermore, in a dose-dependent manner, EUL 50 reduced gastric tissue damage. In addition, the high dose of EUL 50 administration resulted in remarkable reductions in the levels of malondialdehyde (MDA), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and interleukin-1ß (IL-1ß) by 22.64%, 42.61%, 57.78%, and 56.51%, respectively, and suppression of the phosphorylation of Akt, p65, IKKα, and IκBα by 60.87%, 67.65, 74.58%, and 59.57%, respectively, and increased the antioxidant enzyme activity. EUL 50 is rich in flavonoids and organic acids that can act on the PI3K/Akt/NF-κB signaling pathway; as a result, oxidative stress and inflammation are considerably reduced, and gastric ulcers caused by HCl/EtOH are reduced. PRACTICAL APPLICATION: As a medicinal and food substance, Eucommia ulmoides leaves are widely used in the development of health products. EUL 50, a moderately polar part of E. ulmoides leaves, was obtained by extraction and enrichment and was found to have a better protective effect against HCl/EtOH-induced gastric ulcers. This finding can enrich the traditional application of E. ulmoides leaves and provide a basis for their health product development.

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.

Study on enhanced extraction and seasonal variation of secondary metabolites in Eucommia ulmoides leaves using deep eutectic solvents.

It has been considered as a crucial field for the extraction of active ingredients from herbal medicine to use a green and efficient method in the medicinal and food industries. In recent years, deep eutectic solvents (DESs) have been obtaining increase attention in green chemistry area since its sustainability, safety and biodegradability. In this study, an efficient DES composed of choline chloride and L-(+)-ascorbic acid with a molar ratio of 2:1 performed higher efficacy on the extraction of target compounds (including iridoids, phenolic acids and flavonoids) in Eucommia ulmoides leaves than 50% methanol solution. Considering the extraction efficacy and time consumption, microwave-assisted extraction (MAE) was selected and the operational conditions, including power of microwave, liquid/solid ratio and irradiation time were optimized by response surface methodology (RSM). Water was used as anti-solvent to recover ten target analytes from DES with recovery yields of 97.59%, 94.91%, 96.09%, 90.66%, 95.16%, 87.33%, 86.57%, 82.15%, 89.28% and 80.75% for eucommiol (EU), aucubin (AU), geniposidic acid (GA), chlorogenic acid (CA), asperuloside (AP), rutin (RU), kaempferol-3-O-sambubioside (KS), isoquercitrin (IQ), kaempferol-3-O-rutinoside (KR) and astragaline (AS), respectively. By combining the DES-based MAE and quantitative analysis of multi-components by single mark (QAMS) methods, the contents of ten compounds in the leaves of Eucommia ulmoides were determined to clarify the relationship between the accumulation of secondary metabolites and the harvest period. It was found that the contents of main ingredients reached the highest during May to October. The period appears to be the best harvest period for Eucommia ulmoides leaves. This study provides a novel strategy for the harvesting, processing, and quality control of the raw materials from Eucommia ulmoides leaves.

Innovation in sweet rice wine with high antioxidant activity: Eucommia ulmoides leaf sweet rice wine.

The dried leaves of Eucommia ulmoides Oliv., which have a high nutritional value, are mainly used in both medicine and food. In this study, we used Eucommia ulmoides leaf superfine powder as an additive in the fermentation of glutinous rice (Semen Oryzae Glutinosae) to develop a new healthcare product, Eucommia leaf sweet rice wine. The fermentation conditions were optimized, and the nutrient value was evaluated through analyses of metabolites, functional compositions, antioxidant capacity, and antihyperglycemic, antihyperlipidemic, and antihypertensive abilities. The metabolic analysis demonstrated that Eucommia leaf sweet rice wine contained a large number of flavonoids and other metabolites. Eucommia leaf sweet rice wine had higher contents of flavonoid (729.0 ± 0.11 µg/g), free amino acids (55.0 ± 0.37 µg/g), polyphenol (150.0 ± 0.43 µg/g), and polysaccharide (0.25 ± 0.03 µg/g) than traditional sweet rice wine, with increases of 14.7, 2.6, 6.8, and 6.3 times, respectively. In addition, an analysis of antioxidant capacity in vitro revealed that Eucommia leaf sweet rice wine had a high level of activity in scavenging 2, 2-diphenyl-1-picrylhydrazyl (DPPH), superoxide anion, and hydroxyl radicals, as well as in reducing iron, indicating that it was a strong antioxidant. Furthermore, Eucommia leaf sweet rice wine had a high cholate binding capacity and could significantly inhibit α-amylase, α-glucosidase, and angiotensin-converting enzyme (ACE) activity. In conclusion, this study developed a new application of Eucommia leaf in sweet rice wine fermentation and brewed Eucommia leaf sweet rice wine with strong antioxidant activity and positive antihypertensive, antihyperglycemic, and antihyperlipidemic effects in vitro. This study suggests new opportunities for the wider use of Eucommia ulmoides leaves and adds variety to sweet rice wine.

Ultrasonic Microwave-Assisted Micelle Combined with Fungal Pretreatment of Eucommia ulmoides Leaves Significantly Improved the Extraction Efficiency of Total Flavonoids and Gutta-Percha.

A biological pretreatment of Eucommia ulmoides leaf biomass was carried out. Above all, the total flavonoids were isolated from Eucommia ulmoides leaves by the treatment of alkaline solution of sodium dodecyl benzene sulfonate combined with ultrasonic microwave. The extraction parameters were optimized by central composite design (CCD) software and were displayed as follows: surfactant concentration of 1.5%, microwave power of 700 W, extraction time of 30 min, and liquid-solid ratio of 50 mL/g. The actual yield of total flavonoids was 1.45%. The results of Sudan III color development showed that the cuticle of Eucommia ulmoides leaves was completely removed after dilute alkali pretreatment. Then, Eucommia ulmoides leaves were fermented by Trichoderma viride to remove the holocellulose and obtain gutta-percha. The content of cellulose and hemicellulose in Eucommia ulmoides leaves obviously decreased after Trichoderma viride fermentation. The optimum parameters were listed as follows: solid-liquid ratio of 0.06 g/mL, four extraction times, extraction time of 89.72 min, and extraction temperature of 85 °C. The actual yield of gutta-percha was 4.38%. The amount of extraction solvent per unit weight of gutta-percha from untreated Eucommia ulmoides leaves was 2.91 mL/mg, while that from leaves treated by Trichoderma viride was only 0.96 mL/mg. The amount of extraction solvent was grossly reduced, which is beneficial in terms of environmental protection. The characterizations of gutta-percha were verified by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and proton nuclear magnetic resonance (1H NMR). This study laid a certain theoretical and experimental basis for the multi-stage extraction of Eucommia ulmoides leaves and the utilization of Eucommia ulmoides resources.

The Gut Microbiota-Bile Acids-TGR5 Axis Mediates Eucommia ulmoides Leaf Extract Alleviation of Injury to Colonic Epithelium Integrity.

Eucommia ulmoides leaves (EL) are rich in phenolic acids and flavonoids, showing enhancing intestinal health effects. The intestinal microbiota-bile acid axis plays important roles in the occurrence and recovery of inflammatory bowel disease (IBD). However, whether EL extract (ELE) has regulatory effects on the intestinal microbiota, bile acid metabolism, and IBD is still unclear. To fill this gap, 2% dextran sulfate sodium (DSS)-induced mild IBD in a C57BL/6J mouse model that was treated with 200 or 400 mg/kg (intake dose/body weight) ELE was used. Oral ELE supplementation alleviated DSS-induced shortening of colon and colonic epithelial injury. Compared with the DSS group, ELE supplementation significantly decreased Toll-like receptor 4 (TLR4) and interlukin-6 (IL-6) and increased occludin and claudin-1 mRNA expression level in the colon (p < 0.05). Combined 16S rRNA gene sequencing and targeted metabolomic analyses demonstrated that ELE significantly improved the diversity and richness of the intestinal microbiota, decreased the abundance of Bacteroidaceae, and increased Akkermansiaceae and Ruminococcaceae abundance (p < 0.05) compared with DSS-induced IBD mice. Moreover, ELE significantly increased the serum contents of deoxycholic acid (DCA) and tauroursodeoxycholic acid (TUDCA), which were highly positively correlated with Akkermansia and unidentified_Ruminococccaceae relative to the DSS group. We then found that ELE increased Takeda G-protein coupled receptor 5 (TGR5), claudin-1, and occludin mRNA expression levels in the colon. In the Caco-2 cell model, we confirmed that activation of TGR5 improved the reduction in transepithelial electoral resistance (TEER) and decreased the permeability of FITC-dextran on monolayer cells induced by LPS (p < 0.05). siRNA interference assays showed that the decrease in TGR5 expression led to the decrease in TEER, an increase in FITC-dextran permeability, and a decrease in claudin-1 protein expression in Caco-2 cells. In summary, ELE alleviated IBD by influencing the intestinal microbiota structure and composition of bile acids, which in turn activated the colonic TGR5 gene expression in the colon and promoted the expression of tight junction proteins. These findings provide new insight for using ELE as a functional food with adjuvant therapeutic effects in IBD.

Metabolomic and Lipidomic Approaches to Evaluate the Effects of Eucommia ulmoides Leaves on Milk Quality and Biochemical Properties.

Eucommia ulmoides leaves (EUL) contain a variety of natural bioactive compounds including chlorogenic acid, geniposide acid, and aucubin. These bioactive chemicals improve immune function and regulate lipid metabolism. The aim of this study was to investigate the effects of EUL on the biochemical properties of milk. Twenty Holstein dairy cows were randomly allocated to two groups fed a control (CTR, diet without EUL, n = 10) or EUL (diet containing 3% EUL, dry matter, n = 10) diet for 55 d. At the end of the experimental period (d 55), milk samples were collected and analyzed to determine their composition. Though levels of milk fat, protein, lactose, and total milk solids were similar between the groups, small molecules, metabolites, lipids, and cytokines differed. Compared with the CTR group, the EUL group had an improved cluster of differentiation (CD)4/CD8 ratio (P < 0.05) and lower interleukin (IL)-8 and IL-6 content (P < 0.05). Metabolomics analysis identified 14 metabolites including 7Z, 10Z, 13Z, 16Z, 19Z-docosapentaenoic acid (FC = 3.129), adrenic acid (FC = 2.830), and eicosapentaenoic acid (FC=1.685) as having significantly increased in the EUL group (P < 0.05) while 11 metabolites, including indole-2-carboxylic acid (FC = 0.636), cholic acid (FC = 0.430), and creatine (FC = 0.784) had significantly decreased (P < 0.05). Based on a constructed metabolome map, linoleic acid metabolism had the highest impact value for EUL. A total of 21 lipid classes and 1,094 lipid species were detected in the milk by lipidomic analysis, among which 40 differed significantly between the CTR and EUL groups. The present findings showed that the EUL altered milk composition. Correlation analysis showed that 7Z, 10Z, 13Z, 16Z, 19Z-docosapentaenoic acid, adrenic acid, and eicosapentaenoic acid levels were negatively correlated with those of the inflammatory factors IL-6 and IL-8 (P < 0.05), indicating that EUL improved milk quality by reducing inflammatory factors and increasing the CD4/CD8 ratio. Overall, our data demonstrate that EUL had positive effects on milk antioxidant parameters, immune indices, and micro-composition metabolism, thereby improving milk quality.

Synthesis of carbon dots for Al3+ sensing in water by fluorescence assay.

Fifty-four Eucommia ulmoides leaves were subjected to a hydrothermal technique to synthesize carbon dots (CDs) of 3.55 ± 1.45 nm size. The nanomaterial possessed excellent stability and strong fluorescence emission (φf 42.3%). In a neutral buffer solution, the fluorescence signals of CDs solution were enhanced by aluminium ion without interference from other ions. Degree of enhancement correlated linearly with the Al3+ content in the range 0.01-2.5 mM. Response of this method was fast and sensitive (detection limit was 23 nM). The CDs performed successfully as a sensitive sensor for trace Al3+ determination in water samples, and satisfactory results were obtained.

[Possible pharmaceutical effect and active components in different parts of Eucommia ulmoides based on network pharmacology].

The difference in pharmacological activities and active components between leaves, barks and flowers of Eucommia ulmoides(EU) are still unclear. However, clarifying the differences in pharmacological effects of different parts of EU is of great significance for the development of EU products, and their corresponding active components provide basis for quality control of different parts of EU. Based on the chemical compositions of different parts of EU, integrated strategy of target prediction and target analysis of the compounds was used to investigate the difference in the pharmacological effects of leaves, barks and followers. The "component-target-function" association network was constructed to mine the specific material basis corresponding to specific efficacy of different parts of EU. In this study, the author found that EU may have the activities of anti-oxidation, neuromodulation, blood pressure regulation, myo-cardial expansion, and anti-apoptosis according to target prediction and function analysis. However, the effects of different parts of EU were different. Leaves were involved in the process of bone development such as osteoblast differentiation and bone mineralization in a specific way. In addition, the leaves may affect the process of bone development by regulating the metabolism of vitamin D and affecting the absorption of calcium. Leaves may also specifically act on estrogen and estradiol response processes where estrogen receptors were involved. Regarding its protective function for the liver, leaves may play a role by regulating vitamin A-related pathways. As compared with leaves, the specific pharmacological effects of barks may be related to the development of the urinary system. Flowers specifically participate in functions related to pain sensation, glutamate signaling pathway, and excitatory postsynaptic potential. Based on the hie-rarchical network of "component-target-pathway", we further found that specific activities of different parts of EU were inseparable from its specific chemical compositions. Phenylpropanoids, terpenoids and rings, iridoids, flavonoids and other components which are specific in leaves can target the specific effects of leaves, while the flavonoids in barks and the quinones in flowers may be the material basis for their respective specific effects. The prediction of the activities of different parts of EU provides a new basis for the focuses and differences in subsequent Eucommia product development. At the same time, the material basis research based on differential efficacy also provides a basis for the quality control of Eucommia differentiated products.

Manufacture and antibacterial characteristics of Eucommia ulmoides leaves vinegar.

In this work, the fermentation conditions and the antibacterial characteristics of Eucommia ulmoides leaves vinegar (EV) were studied. By single factor orthogonal test, it was found that under optimal fermentation conditions (bran addition 10%, sugar addition 8%, leaven addition 0.3% and acetic acid bacteria solution 12%), the acetic acid content and CA content of EV were 45.5 ± 2.8 mg/mL and 0.98 ± 0.08 mg/mL, respectively. Then, by the disc diffusion method, it was concluded that the antibacterial effect of EV was significantly higher than that of Eucommia ulmoides leaves enzymatic hydrolysate and Zhenjiang aromatic vinegar (P<0.05). An investigation into action mode of EV against Bacillus subtilis indicated that, under the combined action of CA and acetic acid, EV exerted its antibacterial effect by damaging bacterial cell wall and cell membrane, increasing the cell permeability which resulted in the structural lesions and release of cell components, thus led to cell death.

Geographic Authentication of Eucommia ulmoides Leaves Using Multivariate Analysis and Preliminary Study on the Compositional Response to Environment.

To explore the influences of different cultivated areas on the chemical profiles of Eucommia ulmoides leaves (EUL) and rapidly authenticate its geographical origins, 187 samples from 13 provinces in China were systematically investigated using three data fusion strategies (low, mid, and high level) combined with two discrimination model algorithms (partial least squares discrimination analysis; random forest, RF). RF models constructed by high-level data fusion with different modes of different spectral data (Fourier transform near-infrared spectrum and attenuated total reflection Fourier transform mid-infrared spectrum) were most suitable for identifying EULs from different geographical origins. The accuracy rates of calibration and validation set were 92.86% and 93.44%, respectively. In addition, climate parameters were systematically investigated the cluster difference in our study. Some interesting and novel information could be found from the clustering tree diagram of hierarchical cluster analysis. The Xinjiang Autonomous Region (Region 5) located in the high latitude area was the only region in the middle temperate zone of all sample collection areas in which the samples belonged to an individual class no matter their distance in the tree diagram. The samples were from a relatively high elevation in the Shennongjia Forest District in Hubei Province (>1200 m), which is the main difference from the samples from Xiangyang City (78 m). Thus, the sample clusters from region 9 are different from the sample clusters from other regions. The results would provide a reference for further research to those samples from the special cluster.