Study on Synergistic Anti-Inflammatory Effect of Typical Functional Components of Extracts of Ginkgo Biloba Leaves.

There are some differences in the anti-inflammatory activities of four typical components in EGB (extracts of ginkgo biloba leaves), and there is also a synergistic relationship. The order of inhibiting the NO-release ability of single functional components is OA > GF > OPC > G. Ginkgolide (G), proanthocyanidins (OPC), and organic acids (OA) all have synergistic effects on ginkgo flavonoids (GF). GF:OA (1:9) is the lowest interaction index among all complexes, showing the strongest synergy. The anti-inflammatory mechanism of the compound affects the expression of p-JNK, p-P38, and p-ERK1/2 proteins by inhibiting the expression of iNOS and COX2 genes on NFKB and MAPK pathways. This also provides a research basis for the development of anti-inflammatory deep-processing products of EGB.

Study on Synergistic Antioxidant Effect of Typical Functional Components of Hydroethanolic Leaf Extract from Ginkgo Biloba In Vitro.

The predicted anti-oxidation is related to apoptosis, proliferation, lipid metabolism, cell differentiation, and immune response. There are some differences in the antioxidant capacity of the four typical components of ginkgo biloba extract (EGb) including ginkgo flavone (GF), ginkgolide (G), procyanidins (OPC), and organic acids (OA), and any two members of them can exhibit apparent synergistic effects. The order of DPPH scavenging ability was: OPC > GF > OA > G. The scavenging ability of procyanidins was close to that of VC; the scavenging capacity of ABTS was GF > OPC > OA > G. The GF:OPC (1:9) showed the best synergism in scavenging DPPH and ABTS radicals. The 193 kinds of small molecules reported in EGb were obtained by analyzing the properties of EGb. In order to construct a corresponding biological activity target set, molecular docking and the network pharmacology method were employed to build the molecular action mechanism network of a compound target, and the main biological functions and signaling pathways involved with their antioxidant activities were predicted. The results displayed that the top ten compounds which belonged to the two broad categories, ginkgo flavonoids and proanthocyanidins, could interact closely with several important target proteins (CASP3, SOD2, MAPK1, HSPA4, and NQO1). This would be expected to lay a theoretical foundation for the deep development of Ginkgo biloba extract.

Isovitexin Inhibits Ginkgolic Acids-Induced Inflammation Through Downregulating SHP2 Activation.

It has been reported that Celtis sinensis Pers. is employed as a folk medicine for the treatment of inflammatory diseases. But the mechanism supporting its use as anti-inflammatory remains unclear. To investigate the anti-inflammatory of Celtis sinensis Pers. ICR mice were provided Celtis sinensis Pers. leaf extract (CLE) at 100, 200 mg/kg after ginkgolic acids (GA) sensitization. Our data showed that CLE and the main flavonoid isovitexin in CLE could ameliorate GA-induced contact dermatitis in mice. Ear swelling, inflammatory cell infiltration and splenomegaly were inhibited significantly by isovitexin, while the weight loss of mice in the isovitexin-treated group was much better than that in the dexamethasone-treated group (positive control drug). It has been reported in previous research that GA-induced inflammation is closely related to the T cell response. Therefore, T cells were the focus of the anti-inflammatory effect of isovitexin in this paper. The in vivo results showed that isovitexin (10, 20 mg/kg) inhibited the expression of proinflammatory cytokines (TNF-α, IFN-γ, IL-2 and IL-17A) in lymph nodes, inhibited the secretion of cytokines into the serum from mice with contact dermatitis and promoted the expression of apoptosis-related proteins. In vitro, isovitexin also induced apoptosis and inhibited proinflammatory cytokine expression in Con A-activated T cells. Further study showed that the MAPK and STAT signaling pathways and the phosphorylation of SHP2 were inhibited by isovitexin. Both molecular docking and biological experiments indicated that SHP2 may be an anti-inflammatory target of isovitexin in T cells. Taken together, isovitexin can serve as a potential natural agent for the treatment or prevention of GA-induced inflammatory problems.

Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), Polysaccharides Suppressed Adipogenesis and Stimulated Lipolysis in HPA-v and 3T3-L1 Adipocytes.

Ganoderma lucidum polysaccharides (GLP) are one of the major bioactive components with many beneficial properties. In the present study we aimed to systematically evaluate the effects of GLP on lipid metabolism in human (HPA-v) and murine adipocytes (3T3-L1). Cell viability was assessed by MTT assay. Lipid accumulation in mature adipocytes were evaluated by ORO staining and quantified using the triglyceride (TG) assay. Lipolysis was investigated by measuring the free glycerol released in the cell culture medium after treatments. The mRNA and protein levels of key genes regulating lipid metabolism were determined by qRT-PCR and western blotting in HPA-v cells. ORO staining showed that GLP suppressed lipid accumulation similarly in both HPA-v and 3T3-L1 cells. TG assay confirmed that GLP significantly inhibited cell differentiation (p < 0.001). The lipolysis assay showed that GLP enhanced triglyceride hydrolysis in both adipocytes (p < 0.05). GLP stimulated AMPK phosphorylation, which promoted the phosphorylation of ACC1, its downstream target. qRT-PCR and western blotting showed that the genes encoding transcription factors for adipocyte differentiation (PPARγ, C/EBPα, and SREBPlc) and certain adipogenic genes (ACC1, PLIN1, and FASN) were downregulated (p < 0.05). The lipolytic gene HSL was upregulated and highly phosphorylated (activated) at mRNA and protein levels, respectively, upon GLP treatment. These results suggested that GLP possessed beneficial antiadipogenic effects and can potentially be developed into antiobesity products.

Structural Optimization of Caffeoyl Salicylate Scaffold as NO Production Inhibitors.

Chlorogenic acid (CGA) has been considered as one of important active components in a number of medicinal herbs. Recently our group demonstrated that caffeoyl salicylate scaffold derived from CGA can be employed for the development of novel anti-inflammatory agents. The most active compound D104 can be a very promising starting point for the further structural optimization. A series of novel caffeoyl salicylate analogs were designed, synthesized, and evaluated by preliminary biological evaluation. The obtained results showed that the two compounds B12 and B13 can not only inhibit production of nitric oxide (NO) in RAW264.7 cells induced by lipopolysaccharides (LPS) effectively, but also have high safety in in vitro cytotoxic test, which could be comparable with D104. Molecular docking study on the peroxisome proliferator-activated receptor γ (PPARγ) protein revealed that compounds B12 and B13 can follow the same binding mode with D104, and the carboxyl group of caffeoyl salicylate scaffold might play a key role in the interaction with protein target, which implied the carboxyl group should be retained in the further optimization.

Design, synthesis, and anti-inflammatory activity of caffeoyl salicylate analogs as NO production inhibitors.

Chlorogenic acid (CGA) has been reported to exhibit potent anti-inflammatory activity. However, the development of anti-inflammatory agent based on CGA has not been investigated. In this paper, a series of caffeoyl salicylate compounds derived from CGA were designed, synthesized, and evaluated by LPS-induced nitric oxide synthase inhibition and QRT-PCR technique. Most compounds showed modest activity to inhibit production of nitric oxide (NO) in RAW 264.7 cells induced by lipopolysaccharides (LPS). Among these compounds, QRT-PCR and western blotting results indicated that compounds 6b, 6c, 6f, 6g and D104 that possess 5-member ring or 6-member ring caused a significant inhibition against expression of the iNOS2 in LPS-induced macrophages. In addition, cytotoxic assay displayed most derivatives have good safety in vitro. This new promising scaffold could be further exploited for the development of anti-inflammatory agent in the future.

Improvement of Animal Feed Additives of Ginkgo Leaves through Solid-state Fermentation using Aspergillus niger.

To improve the quality of Ginkgo biloba leaves as biological feed additives, twelve Aspergillus niger strains were evaluated for their growth in the moisture ginkgo leaf meal media through solid-state fermentation. The results relating to flavor, flavonoids, enzymes, crude protein, and reducing sugars showed A. niger Gyx086 strain was capable of efficiently fermenting ginkgo leaves. The optimal cultural conditions were three loops of spores inoculation to every 75 g medium containing 60 % water, grew at 28˚C for 48 h. The Gyx086 grew well in the medium. The fermented leaves generated a strong sweet-smelling odor, could be identified by electronic nose equipment using a cluster analysis, other than the original offensive smell from non-fermented ginkgo leaves. Each gram dried culture with Gyx086 showed 2.83 × 109 CFU of A. niger; 3.19 ± 0.37 FPU of acid-resistant filter paper activity. Its total contents of flavonoids, reducing sugars, and crude proteins were 19.95 ± 0.23 mg, 24.28 ± 2.35 mg, and 162.81 ± 3.46 mg in each gram of leaves, 26.03 %, 62.73 %, and 14.58 % higher than the controls, respectively. The essential amino acids and total amino acids contents were 96.41 % and 16.49 % higher than the controls.

Enrichment and Purification of Total Ginkgo Flavonoid O-Glycosides from Ginkgo Biloba Extract with Macroporous Resin and Evaluation of Anti-Inflammation Activities In Vitro.

In the present study, the performance and separation characteristics of six macroporous resins for the enrichment and purification of total ginkgo flavonoid O-glycosides (TGFs) (quercetin (I), kaempferol (II), isorhamnetin (III)) from Ginkgo Biloba extracts (EGB) are evaluated. The adsorption and desorption properties of TGFs are studied on macroporous resins, including D101, D201, AB-8, HPD400, D301, and D311. Along with the results, AB-8 resin exhibits the best adsorption and desorption capacity for these three ginkgo flavonoid O-glycosides among the six resins. Adsorption isotherms are created on AB-8 resin and fit well to the Langmuir (R² > 0.96) and Freundlich (R² > 0.92, 0.3 < 1/n < 0.7) models. After the treatment with gradient elution on AB-8 resin packed chromatography column, the contents of the three main ginkgo flavonoid O-glycosides (I, II, and III) increase from 8.93%, 9.88%, and 6.11% in the extracts to 30.12%, 35.21%, and 14.14%, respectively, in the product. The recoveries of compounds I, II, and III are 88.76%, 93.78%, and 60.90%, respectively. Additionally, the anti-inflammatory effects of TGFs are evaluated in LPS-treated RAW 264.7 macrophages, and the result demonstrates that TGFs could significantly inhibit LPS-induced NO release in vitro in a dose-dependent manner compared with the control group. These findings suggest that TGFs could potentially be natural antioxidants and anti-inflammatory ingredients that could be used in pharmaceutical products and functional food additives.

Antitumor, antioxidant and anti-inflammatory activities of kaempferol and its corresponding glycosides and the enzymatic preparation of kaempferol.

Kaempferol (kae) and its glycosides are widely distributed in nature and show multiple bioactivities, yet few reports have compared them. In this paper, we report the antitumor, antioxidant and anti-inflammatory activity differences of kae, kae-7-O-glucoside (kae-7-O-glu), kae-3-O-rhamnoside (kae-3-O-rha) and kae-3-O-rutinoside (kae-3-O-rut). Kae showed the highest antiproliferation effect on the human hepatoma cell line HepG2, mouse colon cancer cell line CT26 and mouse melanoma cell line B16F1. Kae also significantly inhibited AKT phosphorylation and cleaved caspase-9, caspase-7, caspase-3 and PARP in HepG2 cells. A kae-induced increase in DPPH and ABTS radical scavenging activity, inhibition of concanavalin A (Con A)-induced activation of T cell proliferation and NO or ROS production in LPS-induced RAW 264.7 macrophage cells were also seen. Kae glycosides were used to produce kae via environment-friendly enzymatic hydrolysis. Kae-7-O-glu and kae-3-O-rut were hydrolyzed to kae by ß-glucosidase and/or α-L-rhamnosidase. This paper demonstrates the application of enzymatic catalysis to obtain highly biologically active kae. This work provides a novel and efficient preparation of high-value flavone-related products.

[Comparison of three different thermophilic beta-glucosidases for baicalin transformation].

Three different beta-glycosidase sequences of Ttebgl3, Tpebgl1 and Tpengl3 from Thermotoga thermarum DSM 5069 and Thermotoga petrophila RKU-1 were analyzed. Also, the influence of temperature, pH, concentration of DMSO, metal ions and kinetic constant on catalytic conversion of baicalin had been compared. The results indicated that the optimal pH and optimum temperature for transformation of baicalin was 4.5 85 °C, 5.0 80 °C and 5.5 80 °C, respectively. The family GH3 beta-glycosidase Ttebgl3 and Tpebgl3 had the better DMSO tolerance. The activation effect of the metal ions on the catalytic conversion of baicalin was not obvious, and the inhibition of the GH3 family beta glucosidase was significantly stronger than that of the GH1 family. The kinetic constants of three different beta-glucosidases catalyzed baicalin were significantly different. The Km and Vmax values of Tpebgl1, Tpebgl3 and Ttebgl3 were 0.029 2 mmol·L⁻¹ 4.85 U·mg⁻¹, 0.268 6 mmol·L⁻¹ 121.04 U·mg⁻¹ and 0.391 8 mmol·L⁻¹ 308.90 U·mg⁻¹, respectively. Family GH3 beta-glycosidase converted more baicalin than family GH1 with the optimal conditions, 0.02 g baicalin, and the conversion rate was 68%, 97.3%, 97.31% respectively. The results of the study provided a guarantee for the transformation of baicalin.

Identification of Human Acetylcholinesterase Inhibitors from the Constituents of EGb761 by Modeling Docking and Molecular Dynamics Simulations.

AIM AND OBJECTIVE: EGb761, a standardized and well-defined product extract of Ginkgo biloba leaves, has beneficial role in the treatment of multiple diseases, particularly Alzheimer's disease (AD). Identification of natural acetylcholinesterase (AChE) inhibitors from EGb761 would provide a novel therapeutic approach against the Alzheimer's disease. MATERIAL AND METHOD: A series of 21 kinds of promising EGb761 compounds were selected, and subsequently evaluated for their potential ability to bind AChE enzyme by molecular docking and a deep analysis of protein surface pocket features. RESULTS: Docking results indicated that these compounds can bind tightly with the active site of human AChE, with favorable distinct interactions around several important residues Asp74, Leu289, Phe295, Ser293, Tyr341, Trp286 and Val294 in the active pocket. Most EGB761 compounds could form the hydrogen bond interactions with the negatively charged Asp74 and Phe295 residues. Among these compounds, diosmetin is the one with the best-predicted docking score while three key hydrogen bonds can be formed between small molecule and corresponding residues of the binding site. Besides, other three compounds luteolin, apigenin, and isorhamnetin have better predicted docking scores towards AChE than other serine proteases, i.e. Elastase, Tryptase, Factor XA, exhibiting specificity for AChE inhibition. The RMSD and MM-GBSA results from molecular dymamic simulations indicated that the docking pose of diosmetin-AChE complex displayed highly stable, which can be used for validating the accuracy of molecular docking study. Subsequently, the AChE inhibitory activities of these compounds were evaluated by the Ellman's colorimetric method. CONCLUSION: The obtained results revealed that all the four compounds exhibited modest AChE inhibitory activity, among which Diosmetin manifested remarkable anti-AChE activity, comparable with the reference compound, Physostigmine. It can be deduced that these EGB761 compounds can be regarded as a promising starting point for developing AChE inhibitors against AD.

Structures and bioactivities of seven flavonoids from Osmanthus fragrans 'Jinqiu' essential oil extraction residues.

Osmanthus fragrans are well-known for their fragrance, but it is wasteful if to discard O. fragrans flower after extracting their essential oils. In this paper, we found that O. fragrans flower residues were rich in flavonoids. Six flavonoids and one phenylethanoid glycoside were isolated from the ethanol extract of O. fragrans flower residues, identified as quercetin (1), rutin (2), verbascoside (3), genistin (4), kaempferol (5), isorhamnetin (6) and naringin (7). In bioactivity study, kaempferol (IC50 = 1.43 µg/mL) showed the best anti-inflammatory activity. Isorhamnetin, quercetin, kaempferol, verbascoside and rutin (the values of IC50 were 18.30, 11.05, 16.88, 20.21 and 22.76 µg/mL, respectively) showed excellent DPPH free radical scavenging activity. Verbascoside performed relatively well at inhibiting the growth of both CT26 colonic carcinoma cells (IC50 = 46.87 µg/mL) and HepG2 hepatocarcinoma cells (IC50 = 30.58 µg/mL). In addition, quercetin and kaempferol showed strong anti-proliferation activity against HepG2 cells.

Seasonal variation of pheophorbide a and flavonoid in different organs of two Carpinus species and its correlation with immunosuppressive activity.

The genus Carpinus of Betulaceae is the most widely distributed in the European landscape. This study reports a comparative study based on the pheophorbide a and flavonoid content from the two main species of the genus Carpinus, Carpinus betulus and Carpinus turczaninowii, respectively, in Nanjing, China. The pheophorbide a and flavonoid content depends on the organ, species, and season. HPLC analysis showed that the pheophorbide a and flavonoid levels were the highest in May and June, respectively, from the leaves of C. betulus 'Fastigiata.' In contrast, the content of pheophorbide a and flavonoid in the stems of C. betulus 'Fastigiata' or in other species was low. The immunosuppressive effects of the ethyl acetate extracts and methanol extracts from the two Carpinus species were also evaluated. The ethyl acetate extracts of C. betulus 'Fastigiata' in May and the methanol extracts of C. betulus 'Fastigiata' in June showed better immunosuppressive activity than in other seasons, which coincided with the content of pheophorbide a and flavonoid, respectively. Our findings indicated that C. betulus 'Fastigiata' can serve as a medicinal plant against inflammation because of its pheophorbide a and flavonoid content.

The Synergistic Beneficial Effects of Ginkgo Flavonoid and Coriolus versicolor Polysaccharide for Memory Improvements in a Mouse Model of Dementia.

This study reports the combination of Ginkgo flavonoid (GF) and Coriolus versicolor polysaccharide (CVP) in the prevention and treatment of a mouse model of Alzheimer's disease (AD). GF is a traditional health product, and CVP is the main active ingredient of the medicinal fungus Coriolus versicolor. The Morris water maze test, the Y maze, and the step-through test showed that the combinational use of CVP and GF synergistically improved memory in a mouse model of AD. Based on H&E staining analysis, the combination of CVP and GF decreased the severity of the pathological findings in the brain. Given that the expression of IL-1ß, IL-6, and TNF-α was downregulated, the inflammation response in AD mice was considered to be inhibited. The downregulation of GFAP further demonstrated that inflammation was reduced in the brain of AD mice following treatment. Moreover, the expression levels of superoxide dismutase (SOD) and catalase (CAT) were elevated in the brains of treated mice, indicating that oxidation levels were reduced upon the combination treatment. Our results provide new insights into the efficient utilization of traditional medicine for preventing dementia.

Combined Molecular Docking, 3D-QSAR, and Pharmacophore Model: Design of Novel Tubulin Polymerization Inhibitors by Binding to Colchicine-binding Site.

Interference with dynamic equilibrium of microtubule-tubulin has proven to be a useful tactics in the clinic. Based on investigation into the structure-activity relationship (SAR) studies of tubulin polymerization inhibitors obtained from several worldwide groups, we attempted to design 691 compounds covering several main heterocyclic scaffolds as novel colchicine-site inhibitors (CSIs). Evaluated by a series of combination of commonly used computer methods such as molecular docking, 3D-QSAR, and pharmacophore model, we can obtain the ultimate 16 target compounds derived from five important basic scaffolds in the field of medicinal chemistry. Among these compounds, compound A-132 with in silico moderate activity was synthesized, and subsequently validated for preliminary inhibition of tubulin polymerization by immunofluorescence assay. In additional, the work of synthesis and validation of biological activity for other 15 various structure compounds will be completed in our laboratory. This study not only developed a hierarchical strategy to screen novel tubulin inhibitors effectively, but also widened the spectrum of chemical structures of canonical CSIs.

[Preparation of baicalein using thermophilic and sugar-tolerant beta-glucosidase].

The reaction conditions of baicalin hydrolyzed into baicalein by a kind of thermophilic and sugar-tolerant beta-glucosidase were studied in this paper. The beta-glucosidase could catalyze baicalin into baicalein well in the acetic acid-sodium acetate buffer. The optimal enzyme activity was at 85 degrees C and pH 5.5. The enzyme was stable at the temperature less than 85 degrees C and pH range of 5-7.5. The maximum reaction rate V. and michaelis constant K. were 0.41 mmol x L(-1) x min(-1) and 3.31 mmol x L(-1) respectively. Different metal ions had different effects on the activity of enzyme. Na+ existing in acetic acid-sodium acetate buffer had an activation effect on enzyme. The enzyme activity was enhanced by the concentrations of glucose below 0.6 mol x L(-1), and was gradually inhibited when monosaccharide concentration was over 0.6 mol x L(-1). When the monosaccharide concentration reached 1.2 mol x L(-1), the inhibition rate of enzyme activity was about 50%, which showed good glucose tolerance. The good reaction conditions through the experiment have been determined as follows, the substrate: enzyme dose was 1 g: 0.2 mL, acetic acid-sodium acetate buffer pH 5.5, reaction temperature 85 degrees C, reaction time 10 h, and the enzymatic hydrolyzation ratio could reach 97%.

Improving flavonoid extraction from Ginkgo biloba leaves by prefermentation processing.

This paper presents a prefermentation treatment method involving fungi to improve flavonoid extraction from the leaves of Ginkgo biloba . The fungi employed for this treatment were screened from the soil present under an ancient ginkgo tree. Seventy-six strains belonging to 23 genera were isolated and identified by a molecular identification method employing 18S rDNA sequences. Thirty-three strains grew well using ginkgo leaves as the growth medium. One strain, Gyx086, with higher extracted yield of flavonoids and more similar to the control, was finally selected for prefermentation processing. The major fermentation factors were optimized by response surface methodology. The optimal conditions for the highest total falvonoid yield were 27.8 °C for temperature, 64.2% for moisture content, and 61 h for fermentation time. Under the optimal condition, a actual total flavonoid yield of 27.59 ± 0.52 mg/g dry weight culture sample was obtained, which was about 70% higher than that of unfermented gingko leaf samples.