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Prenyl groups increase the lipophilicity of flavonoids, endowing them with a special activity, selectivity, and pharmacological properties by prenylation. Herein, a novel prenyltransferase (ShFPT) gene from Streptomyces sp. NT11 was expressed in Escherichia coli, and its biochemical characteristics were determined. ShFPT exhibited high selectivity to prenylate naringenin at C-6 to generate 6-prenylnaringenin. The optimal activity was observed at pH 6.0 and 55 °C. The Kcat and Km for naringenin were 0.0095 s-1 and 0.20 mM, respectively. Several promiscuous kinase and isopentenyl phosphate kinase genes were screened to develop the most efficient dimethylallyl diphosphate (DMAPP) synthesis pathway for 6-prenylnaringenin synthesis in E. coli. The 6-prenylnaringenin production was improved by changing the induction strategies and optimizing the bioconversion conditions. Finally, 6-prenylnaringenin production reached the highest yield of 69.9 mg/L with average productivity of 4.0 mg/L/h after 16 h incubation, which is the highest yield for any prenylated flavonoid reported to date in E. coli. Therefore, this study provides an efficient method for 6-prenylnaringenin production and reveals the DMAPP synthesis pathway.

Introduction Berberine (BBR), as one of the outstanding representatives of isoquinoline alkaloids, has been used as an antibacterial drug for a long time in China since ancient times. Currently, a large number of studies have been reported that berberine has a wide spectrum of pharmacological activities, such as anti-tumor, anti-inflammatory, hypoglycemic, hypolipidemic, anti-obesity, and the like. Areas covered This review systematically discussed important patents on berberine and berberine derivatives in terms of pharmacological activity between 2016 and 2020. These patents were mainly searched through the European Patent Office database and Web of Science. These berberine patents (~41) cover a wide range of applications, mainly including antitumor, anti-inflammatory, antibacterial, anti-metabolic disorder, and other newly reported pharmacological activities. Expert opinion Berberine is an important lead compound with great potential for optimization in drug development. However, there is a lack of research related to the biomolecular targets of BBR, which directly restricts the development of berberine in the pharmaceutical field. The problems involved with poor bioavailability and cytotoxicity are also worth considering in the development of berberine-based drugs. Accordingly, the increasing number of patents involving biomolecular targets in BBR's patent applications will be published as its related pharmacological mechanisms are further deciphered.

In recent years, with the global environmental deterioration and air pollution, the incidence of bronchitis has increased year by year, and the sales of anti-bronchitis drugs are growing rapidly, mainly due to the long treatment cycle and the difficulty of curing. Developing available traditional Chinese medicines with significant curative effect against bronchitis would be a promising strategy; for instance, Ginkgo seeds, as the fruit of natural plant ginkgo, has been used in ancient times to cure coughs. However, the detailed mechanism of curing cough has not been shown yet. Investigate the mechanism of Ginkgo seeds in the treatment of bronchitis by establishing a series of molecular networks including active ingredients-targets, proteins interactions, biological functions, pathway, and biological processes of targets. In this study, the main active ingredients of Ginkgo seeds and the potential targets related to bronchitis could be obtained by retrieving corresponding database. The molecular docking study between active molecules and protein targets was performed by Glide 6.6. Subsequently, a total of forty potential targets were manually selected. Based on this, the ingredients-target network was constructed using Cytoscape software, as well as proteins interactions network combing with the String database. Finally, the molecular biological function, metabolic pathway, and biological processes of these forty targets were analyzed by Clue GO plug-in. The results indicated that these protein targets were closely related to lipid transport, positive regulation of DNA replication, cAMP metabolic pathway, and other processes, which played a vital role in the treatment of bronchitis by mediating interleukin 17, fluid shear stress and atherosclerosis, asthma, renin secretion, p53, and other signaling pathways. Among these targets, the two protein ALB (Albumin) and DHRS2 (Dehydrogenase 2) can interact with compounds more frequently, and the top three compounds ranked by the docking scores were amentoflavone, (+)-catechin-5-O-glucoside, and liquiritin, implying that these compounds might be used for the treatment of bronchitis. It is obvious that the pharmacological effect of Ginkgo seeds on bronchitis displayed a characteristic of multi-components, multi-targets, and multi-pathways. Nevertheless, the two protein targets and three compounds derived from Ginkgo seeds could be further used for the explanation for Ginkgo seeds in curing bronchitis. This research can provide a scientific basis for studying on the anti-bronchitis mechanism of Ginkgo seeds.

Berberine (BBR), a well-known alkaloid, exhibits various pharmacological activities, especially hypolipidemic activity, which has attracted much interest from medicinal chemists in the past decade. However, little progress was made on the structural modification of BBR for improving lipid-lowering activity, mainly due to its unclear biological target and low safety. In this study, a new scaffold of 7,9-disulfatetrahydroberberine was discovered unexpectedly, provided with extremely low cytotoxicity. Hence, a novel series of highly safe 7,9-disulfatetrahydroberberines were designed, synthesized, and evaluated for their hypolipidemic activities. In order to investigate the significance of the 9-position substituent, another new series of 7-sulfatetrahydroberberines were designed and synthesized. Lipid-lowering experiments showed that among these compounds, 5f exhibited the best lipid-lowering activity based on two cell models, 3T3-L1 cells and HepG2 cells. Compared with the blank control, the inhibition rate of compound 5f against total cholesterol was over 60%, the inhibition rate against triglyceride was over 70%, the inhibition rate against low-density lipoprotein cholesterol was approximately 75%, and the inhibition rate against high-density lipoprotein cholesterol was close to 50%, which were far superior to the positive control BBR. This result also verified the feasibility of the development of BBR as a lipid-lowering drug via disubstituted modification at the 7- and 9-position.

Hyperoside is used as the standard to assess the quality of Hyperin perforatum L and exhibits many biological properties. To improve the yield of hyperoside in Escherichia coli, four UDP-galactose synthesis pathways were screened and a recombinant strain was reconstructed by using an efficient UDP-galactose generation system coupled with Petunia hybrida glycosyltransferase to biosynthesis hyperoside from quercetin. By adopting the resting cell transformation, maximal hyperoside production of 4574 mg/L and average productivity of 63.5 mg/L/h were achieved after 72 h of biotranformation. Subsequently through recycling of resting cells, average productivity within 72 h reached 126 mg/L/h, which was 100% higher than that in the batch fermentation. Hyperoside production reached equivalent to 18,000 mg/L by recycling seven times of resting cell in 168 h, which was 393% of that in the batch fermentation and the first to reach 10 g per liter scale in E. coli. Therefore, this study provides an efficient method for construction of UDP-galactose synthesis pathway and hyperoside production in E. coli.

UDP-rhamnose is the main type of sugar donor and endows flavonoids with special activity, selectivity, and pharmacological properties by glycosylation. In this study, several UDP-glucose synthesis pathways and UDP-rhamnose synthases were screened to develop an efficient UDP-rhamnose biosynthesis pathway in Escherichia coli. Maximal UDP-rhamnose production reached 82.2 mg/L in the recombinant strain by introducing the cellobiose phosphorolysis pathway and Arabidopsis thaliana UDP-rhamnose synthase (AtRHM). Quercitrin production of 3522 mg/L was achieved in the recombinant strain by coupling the UDP-rhamnose generation system with A. thaliana rhamnosyltransferase (AtUGT78D1) to recycle UDP-rhamnose. To further increase UDP-rhamnose supply, an NADPH-independent fusion enzyme was constructed, the UTP supply was improved, and NADPH regenerators were overexpressed in vivo. Finally, by optimizing the bioconversion conditions, the highest quercitrin production reached 7627 mg/L with the average productivity of 141 mg/(L h), which is the highest yield of quercitrin and efficiency of UDP-rhamnose supply reported to date in E. coli. Therefore, the method described herein for the regeneration of UDP-rhamnose from cellobiose may be widely used for the rhamnosylation of flavonoids and other bioactive substances.

The development of an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) is an ongoing and challenging research field. However, the dynamic motion of the binding site of EGFR has not been accurately depicted, hindering the improvement of EGFR TKI. For this reason, about 33 protein complexes (32 EGFR proteins plus 1 ErbB4 protein) were carefully curated and subsequently studied for dynamic movements of their binding sites by molecular dynamics simulations in this study. The analysis of root mean square deviation (RMSD) revealed that T790M mutation can make an impact on dynamic motion of binding sites; the RMSD value of the EGFR binding site was unrelated to inhibitory activity. The analysis of the radius of gyration (Rg) revealed that T790M can slightly shrink the value of Rg, thereby influencing the shape of the EGFR binding site. More interestingly, the Rg value can exhibit weak correlation with inhibitory activity of most inhibitors. The relationship between Rg and biological activity deserve our serious interest since the best scoring function, Xscore, cannot distinguish highly active EGFR inhibitors. The root mean square fluctuation (RMSF) analysis of key residues derived from binding sites indicated that the most flexible residue was ASP800 with a large RMSF value against the steady residue ALA743 with a small RMSF value, and two other residues (MET793 and LEU844) were supposed to be involved with molecular recognition. In short, the obtained results would be more effective for guiding the development of a novel EGFR kinase inhibitor.

Synthesis of β-ionone in microbial cell factories is limited by the efficiency of carotenoid cleavage dioxygenases (CCDs). To obtain genes responsible for specific cleavage of carotenoids generating β-ionone, a novel carotenoid cleavage dioxygenase 1 from Morus notabilis was cloned and overexpressed in Escherichia coli . The MnCCD1 protein was able to cleave a variety of carotenoids at the positions 9, 10 (9', 10') to produce β-ionone, 3-hydroxy-4-oxo-β-ionone, 3-hydroxy-β-ionone, and 3-hydroxy-α-ionone in vitro . MnCCD1 could also cleave lycopene and β-carotene at the 9, 10 (9', 10') bind bond to produce pseudoionone and β-ionone respectively in E.coli accumulating carotenoids. The enzyme activity of MnCCD1 was reached 2.98 U/mL at optimized conditions (temperature 28 ℃, IPTG 0.1 mM, induction time 24 h). The biochemical characterization of MnCCD1 revealed the optimal activities were at pH 8.4 and 35 ℃. The addition of 10% ethanol could increase enzyme activity at above 15%. However, an obvious decline was observed on enzyme activity as the concentration of Fe 2+ increased (0-1 mM). The V max for β-apo-8'-carotenal was 72.5 U/mg, while the K m was 0.83 mM. The results provide a foundation for developing the application of carotenoid cleavage dioxygenases as biocatalysis and synthetic biology platforms to produce volatile aroma components from carotenoids.

Natural β-ionone, a high-value flavoring agent, has been widely applied in the food, cosmetics, and perfume industry. However, attempts to overproduce β-ionone in microorganisms have been limited by the efficiency of carotenoid cleavage dioxygenases (CCDs), which catalyzes β-carotene in the biosynthesis pathway. In order to obtain CCD genes responsible for the specific cleavage of carotenoids generating β-ionone, a novel carotenoid cleavage dioxygenase 1 from Helianthus annuus was cloned and overexpressed in Escherichia coli BL21(DE3). The recombinant CCD was able to cleave a variety of carotenoids at the 9, 10 (9', 10') sites to produce C13 products in vitro , including β-ionone, pseudoionone, 3-hydroxy-4-oxo-β-ionone, 3-hydroxy-β-ionone, and 3-hydroxy-α-ionone, which vary depending on the carotenoid substrates. In comparison with lycopene and zeaxanthin, HaCCD1 also showed the high specificity for β-carotene to cleave the 9, 10 (9', 10') double bond to produce β-ionone in E.coli accumulating carotenoids. Finally, the expression of HaCCD1 in E.coli was optimized, and biochemical characterizations were further clarified. The optimal activity of HaCCD1 was at pH 8.8 and 50 °C. The V max for β-apo-8'-carotenal was 10.14 U/mg, while the K m was 0.32 mM. Collectively, our study provides a valuable enzyme for the synthesis of natural β-ionone by biotransformation and synthetic biology platform.

As a fast-growing tree, poplar is widely planted and typically used for wood processing in China. During poplar wood processing, a large amount of poplar sawdust (PS) and poplar leaves (PL) are produced and abandoned. To make full use of poplar resources and clarify the use of poplar as a feed additive, the active ingredients in PS and PL were extracted and isolated, and the anti-inflammatory effects of the extracts on mice with dextran sulfate sodium (DSS)-induced colitis were investigated. In vitro anti-inflammatory experiments showed that the ethyl acetate extract of PS and PL (PSE and PLE, respectively) could significantly inhibit the proliferation of concanavalin A (Con A)-activated lymphocytes. Salicortin, tremulacin and salireposide were identified in both PSE and PLE. Oral administration of PSE and PLE rescued DSS-induced colonic shortening, repaired tissue damage, and decreased the disease activity index (DAI). The antioxidant capacity, including the increased activities of glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD and catalase (CAT) and decreased activity of myeloperoxidase (MPO), in the colons of mice with colitis was enhanced through the activation of ERK after treatment with PSE and PLE. The ratio of Th1 to Th17 cells, which can lead to inflammation in the spleen, was significantly decreased by the administration of PSE and PLE, while the phosphorylation of related transcription factors (p65, STAT1, and STAT3) was inhibited. Furthermore, PSE and PLE could induce apoptosis in Con A-activated lymphocytes, which may be associated with the increase in p-TBK1, as the molecular docking results also indicated that salireposide in PSE and PLE could interact with the TBK1 protein. Overall, our study provides a promising feed additive for improving intestinal inflammation in animals and a method for the full utilization of poplar resources.

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.

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.

A thermostable β-xylosidase gene Tpexyl3 from Thermotoga petrophila DSM 13,995 was cloned and overexpressed by Escherichia coli. Recombinant Tpexyl3 was purified, and its molecular weight was approximately 86.7 kDa. Its optimal activity was exhibited at pH 6.0 and 90 °C. It had broad specificity to xylopyranosyl, arabinopyranosyl, arabinofuranosyl and glucopyranosyl moieties. The β-xylosidase activity of the recombinant Tpexyl3 was 6.81 U/mL in the LB medium and 151.4 U/mL in a 7.5 L bio-reactor. It was applied to transform ginsenoside extract into the pharmacologically active minor ginsenoside 20(S)-Rg3, which was combined with thermostable β-glucosidase Tpebgl3. After transforming under optimal condition, the 20 g/L of ginsenoside extract was transformed into 6.28 g/L of Rg3 within 90 min, with a corresponding molar conversion of 95.0% and Rg3 productivity of 1793.49 mg/L/h, respectively. This study is the highest report of a GH3 family glycosidase with arabinopyranosidase activity and also the first report on the high substrate concentration bioconversion of ginsenoside extract to ginsenoside 20(S)-Rg3 by using two thermostable glycosidases.

Kaempferol and astragalin are used as standards to assess the quality of Ginkgo biloba extract and Radix astragali, respectively, and possess numerous biological properties. In this study, we constructed a recombinant strain with a highly efficient biosynthetic pathway of kaempferol by screening key enzyme genes, designing a synthetic fusion enzyme and increasing the gene copy number. By optimizing conversion and fed-batch fermentation conditions, maximal kaempferol production reached 1184.2 ± 16.5 mg/L, which represents the highest yield of kaempferol from naringenin reported to date. Based on this result, glycosyltransferase (AtUGT78D2) and an efficient UDP-glucose synthesis pathway were introduced into the recombinant strain to produce astragalin, resulting in maximal astragalin production at 1738.5 ± 24.8 mg/L without kaempferol accumulation. The efficient synthesis pathway described in this study for kaempferol and astragalin biosynthesis can be widely used for flavonoid biosynthesis in Escherichia coli.

In this study, a combined optimization strategy, based on co-expression of molecular chaperones and supplementation of osmolytes, was used to reduce the formation of inclusion bodies and enhance the expression of the soluble form of 4-α-glucanotransferase. The 4-α-glucanotransferase yield was enhanced by co-expression with pGro7 and supplementation of trimetlylamine oxide. Subsequently, the effects of process conditions (temperature, inducer concentration, and arabinose concentration) on cell growth and 4-α-glucanotransferase production were also investigated in shake flasks. In addition, a modified high-cell-density fermentation approach was proposed and applied in 3-L fermentor supplied with l -arabinose and trimetlylamine oxide, which achieved a dry cell weight of 65.92 g·L−1. Through this cultivation approach at 28 °C, the activity of 4-α-glucanotransferase reached 332.5 U·g−1 dry cell weight, which was 24.6-fold greater than the initial activity in shake flask cultivation. This combined strategy is expected to provide an efficient and economical approach to overproduction of aggregation prone proteins on a large scale.

It has been reported that Celtis sinensis Pers. is employed as a folk medicine for the treatment of inflammation diseases. But the mechanism supporting its use as anti-inflammatory remain unclear. To investigate the anti-inflammatory of Celtis sinensis Pers. ICR mice were provided Celtis sinensis Pers. leaf extract (CLE) at 100, 200mg/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, 20mg/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 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.

Ginkgo acids (GAs) in ginkgo products usually lead to allergies or liver toxicity. In this study, the GA-induced toxicity was attenuated and Con A-induced T-lymphocyte proliferation was inhibited by extracts of Celtis sinensis leaves (ECSL). So, the active ingredients in ECSL were studied to solve the problems caused by GAs. First, the eight components of MeOH extracts were determined by HPLC-DAD/LC-MS. Then, the 12 active ingredients were separated based on the anti-inflammatory activity. Lymphocyte conversion showed that the inhibition rates of apigenin, quercetin, and isovitexin at 100 μM on Con A-activated proliferation of T cells were up to 82.46%, 62.86% and 42.76%, respectively. The inhibition rate on the LPS-induced NO release in RAW 264.7 cells of quercetin, apigenin, isovitexin, and vitexin were exceeding 80% at 100 μM. Taken together, the material foundation for the screen of GAs toxicity-attenuated ingredients were provided here. PRACTICAL APPLICATIONS: Ginkgo biloba extracts (EGBs) have been conducted to develop functional food which could increase blood circulation and enhance memory. Nevertheless, people in consumption of ginkgo products, often caused severe allergic reactions due to the potential allergens identified ginkgolic acids (GAs) of ginkgo products. We first find that the extracts of Celtis sinensis leaves can reduce GAs-induced damage on HepG2 liver cells. Then, the bioactive compounds in C. sinensis leaves were separated and purified based on anti-inflammatory activities against T cells. Quercetin, apigenin, and isovitexin showed well anti-inflammatory activities against Con A-activated T-lymphocytes and LPS activated RAW 264.7 macrophages. However, quercetin and apigenin are flavones O-glycosides which are rich in Ginkgo biloba. To solve the problems in Ginkgo biloba products caused by GAs, flavone C-glycoside (isovitexin) may be used for the further study in GAs toxicity-reduction.

Berberine (BBR), a kind of quaternary ammonium benzylisoquinoline alkaloids with multiple pharmacological activities, has been regarded as a promising lipid-lowering agent in the field of drug repurposing. Particularly, the chemical modification at the C-9 position of BBR can remarkably improve its lipid-lowering efficacy. In this study, thirteen novel BBR derivatives were rationally designed, synthesized, and evaluated by preliminary pharmacological tests. The results showed that most compounds exhibited more potent hypolipidemic activities when compared with BBR and simvastatin. Among these compounds, compound 2h-1 and 2h-2 exhibited better activity profiling in these four tests involving with inhibition of total cholesterol (TCHO), triglyceride (TG), and low-density lipoprotein cholesterol (LDLC) and the increase of high-density lipoprotein cholesterol (HDLC). Correspondingly, the BBR analogs with 9-O-cinnamic moiety probably exhibited potent lipid-lowering activity, and should be exploited as an important versatile template for the development of BBR-like lipid-lowering agents.

Ginkgo biloba extract (EGB) has many pharmacological activities. In the quality standard of EGB, the main quality control indexes are total flavone (content &ge, 24%) and total lactone (content &ge, 6%). There are no specific limits for nearly 70% of &ldquo, other components&rdquo, In recent years, in order to pursue the production of a high-ketone ester, some enterprises removed the unwanted components, including some organic acids. Protocatechuic acid (PCA), as an important organic acid, has been reported to have a variety of biological activities. It is necessary to explore whether it can promote the biological activities of the main functional components of EGB. In this study, PCA was selected to be combined with Ginkgolide B (GB) for the treatment of Parkinson&rsquo, s disease. In vitro, rotenone (rot) was used to induce PC12 cells. The survival rate was tested by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-dimethyltetrazolium bromide (MTT) assay. Reactive oxygen species (ROS) and antioxidase were detected to analyze the effects of drugs on oxidative stress. The apoptosis was tested via Western blot. The results show that the cell viability was increased, morphology was improved, the oxidative stress level decreased, and the apoptosis was inhibited after the combination treatment of GB and PCA, and the effect was better than GB or PCA alone. In vivo, MPTP (30 mg/kg) was used to induce Parkinson&rsquo, s disease (PD) in male C57BL/6 mice. The motor ability of the mice was measured by pole-climbing and the suspension. The injury of nerve cells was indicated by HE staining. Oxidative stress levels were tested via antioxidant enzyme activity. The number of dopaminergic neurons was reflected by TH staining. Results show that the combination treatment of GB and PCA could significantly restore the motor ability of PD mice, reduce the injury of nerve cells, improve the activity of the antioxidant enzyme in the brain tissue, and increase the expression of TH in the substantia nigra of midbrain. Our study shows that PCA increases the efficacy of GB (the main functional ingredient of EGB) in the treatment of Parkinson&rsquo, s disease, which provides a new idea for the treatment of nervous system diseases and a new concept for the efficient utilization of active components in Ginkgo biloba leaves.

Quercetin glycoside derivatives (QGDs) are a class of common compounds with a wide range of biological activities, such as antitumor activities. However, their molecular targets associated with biological activities have not been investigated. In this study, four common QGDs with mutual bioconversion were selected, and studied in the large-scale reverse docking experiments. Network pharmacology analysis showed that most of the four QGDs can bind several potential protein targets that were closely related to breast cancer disease. Among them, a druggable protein, transforming growth factor beta receptor I (TGFBR1/ALK5) was screened via high docking scores for the four QGDs. This protein has been proven to be an important target for the treatment of breast cancer by regulating the proliferation and migration of cancer cells in the past. Subsequently, the molecular dynamics (MD) simulation and MM/GBSA calculation demonstrated that all QGDs could thermodynamically bind with TGFBR1, indicating that TGFBR1 might be one of the potential protein targets of QGDs. Finally, the cytotoxicity test and wound-healing migration assay displayed that isoquercetin, which can perform best in MD experiment, might be a promising agent in the treatment of breast cancer metastasis.

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.

Isorhamnetin-3-O-rhamnoside was synthesized by a highly efficient three-enzyme (rhamnosyltransferase, glycine max sucrose synthase and uridine diphosphate (UDP)-rhamnose synthase) cascade using a UDP-rhamnose regeneration system. The rhamnosyltransferase gene (78D1) from Arabidopsis thaliana was cloned, expressed, and characterized in Escherichia coli. The optimal activity was at pH 7.0 and 45 &deg, C. The enzyme was stable over the pH range of 6.5 to 8.5 and had a 1.5-h half-life at 45 &deg, C. The Vmax and Km for isorhamnetin were 0.646 U/mg and 181 &mu, M, respectively. The optimal pH and temperature for synergistic catalysis were 7.5 and 25 &deg, C, and the optimal concentration of substrates were assayed, respectively. The highest titer of isorhamnetin-3-O-rhamnoside production reached 231 mg/L with a corresponding molar conversion of 100%. Isorhamnetin-3-O-rhamnoside was purified and the cytotoxicity against HepG2, MCF-7, and A549 cells were evaluated. Therefore, an efficient method for isorhamnetin-3-O-rhamnoside production described herein could be widely used for the rhamnosylation of flavonoids.

A novel β-xylosidase Dt-2286 from Dictyoglomus turgidum was cloned and overexpressed in Escherichia coli BL21 (DE3). Dt-2286 belonging to glycoside hydrolase (GH) family 3 encodes a polypeptide with 762 amino acid residues with a molecular weight of 85.1 kDa. By optimization of the growth and induction conditions, the activity of β-xylosidase reached 273 U/mL, which is the highest yield reported to date from E. coli in a shake-flask. The optimal activities of the purified Dt-2286 were found at pH 5.0 and 98 °C. It also shows excellent thermostable/haloduric/organic solvent-tolerance. Dt-2286 was revealed to be a multifunctional enzyme with β-xylosidase, α-arabinofuranoside, α-arabinopyranoside and β-glucosidase activities, and Kcat/Km was 5245.316 mM−1 s−1, 2077.353 mM−1 s−1, 1626.454 mM−1 s−1, and 470.432 mM−1 s−1 respectively. Dt-2286 showed significant synergistic effects on the degradation of xylans, releasing more reduced sugars (up to 15.08 fold) by simultaneous addition with endoxylanase. Moreover, this enzyme has good activity in the hydrolysis of epimedium B, demonstrating its versatility in practical applications.

Alcoholic liver disease (ALD) is one of the most common health problems for drinkers, especially in men. Echinacoside (ECH), a natural phenylethanoid glycoside welcomed by the market, has been shown to have a variety of biological activities, such as neuroprotective, anti-fatigue, anti-diabetes and so on. Here, the protective effect and the underlying mechanism of ECH on ethanol-induced liver injuries were studied. In vitro, the HepG2 cells were treated with ECH prior to ethanol. In vivo, C57BL/6 J mice were fed a Lieber-DeCarli ethanol liquid diet and gave with or without 100 mg/kg ECH for 10 days. Our experiments showed that ECH significantly enhanced the levels of antioxidants and reduced the level of ROS, thus attenuating ethanol-induced oxidative stress. Besides, ECH attenuated lipid accumulation caused by ethanol, as evidenced by oil-red O staining, histological examination and the quantification of TG and TC. Finally, ECH increased the level of PPAR-α, and reduced the levels of SREBP-1c and FASN. When PPAR-α inhibitor was introduced in the system, the effects of ECH on SREBP-1c and FASN were reversed. Taken together, our study suggest that ECH can protect against ethanol-induced liver injuries via alleviating oxidative stress and hepatic steatosis by affecting SREBP-1c/FASN pathway via PPAR-α.

In this study, a recombinant carotenoid cleavage dioxygenase 4 was produced from Osmanthus fragrans by E . coli under different bacterial growth conditions and used to develop a biotechnological method for preparation of natural β-ionone from β-carotene. β-ionone was analyzed by HPLC and OfCCD4 activity was measured based on concentration change of β-ionone. At pH 8.0 and 35 °C, the greatest activity of the purified recombinant protein was 14.3 U/mg and the maximum concentration of β-ionone was 71.186 mg/L within 1 h. Both the enzyme activity and the concentration of β-ionone could increase by nearly 6 times with addition of 9% Triton X-100 and 2% liquid paraffin.

Astragalin (kaempferol 3-O-glucoside) is used as a standard to assess the quality of Radix astragali and has exhibited a number of biological properties. In this work, we screened several UDP-dependent glycosyltransferases (UGT) for their potential as efficient biocatalysts for astragalin synthesis. The highest astragalin production with 285 mg/L was detected in the recombinant strain expressing UGT from Arabidopis thaliana (AtUGT78D2). To further improve astragalin production, an efficient UDP-glucose synthesis pathway was reconstructed in the recombinant strain by introducing sucrose permease, sucrose phosphorylase, and uridylyltransferase. On the basis of those results, a recombinant strain, BL21-II, was constructed to produce astragalin. By optimizing conversion conditions, astragalin production was increased from 570 to 1708 mg/L. The production was scaled up using a fed-batch fermentation, and maximal astragalin production was 3600 mg/L, with a specific productivity of 150 mg/L/h after 24 h incubation and a corresponding molar conversion of 91.9%, the highest yield reported to date.

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.

The thermostable β-glucosidase gene from Thermotoga petrophila DSM 13995 was cloned and overexpressed in Escherichia coli. The activity of the recombinant β-glucosidase was 21 U/mL in the LB medium. Recombinant β-glucosidase was purified, and its molecular weight was approximately 81 kDa. The optimal activity was at pH 5.0 and 90 °C, and the thermostability of the enzyme was improved by Ca2+. The β-glucosidase had high selectivity for cleaving the outer and inner glucopyranosyl moieties at the C-20 carbon of ginsenoside Rb1, which produced the pharmacologically active minor ginsenoside 20(S)-Rg3. In a reaction at 90 °C and pH 5.0, 10 g/L of ginsenoside Rb1 was transformed into 6.93 g/L of Rg3 within 90 min, with a corresponding molar conversion of 97.9 %, and Rg3 productivity of 4620 mg/L/h. This study is the first report of a GH3-family enzyme that used Ca2+ to improve its thermostability, and it is the first report on the high substrate concentration bioconversion of ginsenoside Rb1 to ginsenoside 20(S)-Rg3 by using thermostable β-glucosidase under high temperature.

The GH3 β-glucosidase gene from Thermotoga thermarum DSM 5069T was cloned and overexpressed in Escherichia coli. The activity of recombinant β-glucosidase was 26 U/mL in LB medium after IPTG induction. The recombinant β-glucosidase was purified by heat treatment followed by Ni-NTA affinity, and the protein's molecular weight was approximately 82 kDa. The optimal activity was at pH 5.0 and 90 °C. The enzyme was stable over the pH range of 4.5–7.5 and had a 1-h half-life at 90 °C. The Kcat and Km for p-nitrophenyl-β- d -glucopyranoside were 121 s−1 and 0.065 mM, respectively. The β-glucosidase exhibited high selectivity to cleave the outer and inner glucopyranosyl moieties at the C-20 carbon of ginsenoside Rb1 and catalyzed the conversion of ginsenoside Rb1 or Rd to the more pharmacologically active minor ginsenoside 20(S)-Rg3. At a concentration of 0.75 U/mL of the enzyme, a temperature of 85 °C and pH 5.0, 10 g/L ginsenoside Rb1 was transformed into 6.9 g/L Rg3 within 60 min with a corresponding molar conversion of 97.8%. BGL3T hydrolyzed ginsenoside Rb1 along the following pathway: Rb1 → Rd → Rg3. This manuscript provides the first report of the production of ginsenoside 20(S)-Rg3 by a highly thermostable β-glucosidase.

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 β-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.

In the fermentation progress, fermentation parameters including the feed rate, induction temperature, and induction pH evidently regulate the accumulation of acetic acid generated by recombinant E. coli in the medium. The production of thermostable β-glucosidase (Tpebgl3) was increased by optimizing the parameters mentioned step by step. The optimal conditions were obtained with the highest enzyme expression (560.4 U/mL) and the maximum DCW (65 g/L) at the pre-induction specific growth rate of 0.2 h−1 followed by a post-induction specific growth rate (0.18 h−1); induction temperature is 39 °C; the pH is 7.2; the concentration of acetic acid was maintained all along below 0.9 g/L. Results show it is necessary for the synthesis of Tpebgl3 to regulate the accumulation of acetic acid at the premise of feeding to meet the normal growth of E. coli. The production of Tpebgl3 by recombinant E. coli is the highest reported to date.

The β-glucosidase gene Tt-bgl from Thermotoga thermarum DSM 5069T was cloned and overexpressed in Escherichia coli. A simple strategy, induction at 37 °C with no IPTG, was explored to reduce the inclusion bodies, by which the activity of Tt-BGL was 13 U/mL in LB medium. Recombinant Tt-BGL was purified by heat treatment followed by Ni–NTA affinity. The optimal activity was at pH 4.8 and 90 °C. The activity of Tt-BGL was significantly enhanced by methanol and Al3+. The enzyme was stable over pH range of 4.4–8.0, and had a 2-h half life at 90 °C. The Vmax for p-nitrophenyl-β- d -glucopyranoside and ginsenoside Rb1 was 142 U/mg and 107 U/mg, while the Km was 0.59 mM and 0.15 mM, respectively. The activity of the enzyme was not inhibited by ginsenoside Rb1 (36 g/L). It was activated by glucose at concentrations lower that 400 mM. With glucose further increasing, the activity of Tt-BGL was gradually inhibited, but remained 50% of the original value in even as high as 1500 mM glucose. Under the optimal conditions, Tt-BGL transformed ginsenoside Rb1 (36 g/L) to Rd by 95% in 1 h.

Aspergillus niger-fermented-Ginkgo biloba leaves (FG) and the comparative effect with nonfermented (NF)-Ginkgo leaves were investigated on morphology, absorption, and immunity of small intestine after lipopolysaccharide (LPS)-challenge in chicks at an early age. Broiler chicks (180 d of age) were divided into 3 treatment groups and were fed 1 of 3 diets: basal diet or basal diet supplemented with 0.5% NF or FG (control, NF, and FG group, respectively). Half of the birds from each treatment group were injected intraperitoneally with LPS (500 μg/kg of BW) or 0.9% NaCl solution (the sham control groups) at 10, 12, 15, 17, 19, and 21 d of age. The results indicated that when LPS-challenged birds were pretreated with FG, the decrease of ADG, ADFI, duodenal and jejunal relative weights, villus height, crypt depth, alkaline phosphatase activity, and plasma d-xylose were dramatically attenuated (P < 0.05 or P < 0.01). Meanwhile, a significant decrease (P < 0.05 or P < 0.01) of duodenal and jejunal interferon-γ, interleukin (IL)-4, IL-13, IL-18, inducible nitric oxide synthase, and duodenal sodium glucose co-transporter 1 mRNA expressional levels were found in LPS-challenged birds pretreated with FG. In conclusion, FG-supplemented diets minimized the deleterious effects of LPS and improved intestinal development, absorption, and immunity in immune-stressed chickens.

The present study was conducted to investigate the influence of dietary supplementation with different levels of fermented Ginkgo biloba leaves (FGBL) on growth performance, slaughter performance, meat quality, antioxidant enzyme capacity, and free radical scavenging activities of muscles in broiler chickens. A total of 648 one-d-old broiler chickens were randomly allocated into six dietary treatments, including control group (CON group: basal diet), FGBL1, FGBL2, FGBL3, FGBL4, and FGBL5 groups (basal diet containing 1.5, 2.5, 3.5, 4.5, and 5.5 g/kg FGBL, respectively). Body weight gain and feed intake were recorded at 1, 21, and 42 d. At 42 d, 2 birds from each replicate were slaughtered. The results indicated that 3.5 g/kg FGBL diet significantly increased (P 0.05) ADFI and ADG in 1 to 42 d and ADFI in 22 to 42 d compared with the CON group. In 1 to 21 d, 4.5 g/kg FGBL diet improved (P 0.05) ADFI and ADG. With dietary FGBL increasing, the feed: gain ratio (F/G) in 1 to 21 d was significantly decreased (P 0.05). However, birds fed with 5.5 g/kg FGBL had a higher (P 0.05) F/G compared with other groups in 22 to 42 d and 1 to 42 d. In addition, FGBL3 and FGBL4 showed lower (P 0.05) L* value in breast muscle, cooking loss in thigh muscle and lower 24 h and 48 h drip loss in both breast and thigh muscles than those of other groups. Furthermore, birds in the FGBL3 and FGBL4 groups increased (P 0.05) the activity of total superoxide dismutase and total antioxidant capability in muscles, and the scavenging activities of 2,2΄-azino-bis (3-ethylbenzothiazoline-6-sulfonic) acid radical, OH•, and O2•- in thigh muscle, decreased (P 0.05) malondialdehyde concentration in thigh muscle, as compared to the CON group. In conclusion, FGBL had the potential to improve the growth performance, meat quality and antioxidant status of broiler chickens. The optimal dose in the present study of FGBL in broiler diets was from 3.5 to 4.5 g/kg.

Aspergillus niger-fermented Ginkgo biloba leaves (FR) and its comparative effect with non-fermented G. biloba leaves (NF) were investigated on growth performance, small intestinal structure and function of broiler chicks. A total of 300 d-old Arbor Acress broilers were randomly allocated into 5 dietary treatments, which were then denoted as the control group (basal diet), and the NF group (containing 0.35% and 0.7% NF in the starter and grower diets, respectively), FR1, FR2 and FR3 groups containing 0.2%, 0.35% and 0.5% FR in the starter diets and 0.4%, 0.7% and 1.0% FR in the grower diets, respectively. The results showed that, compared with the control and NF groups, reduced (P

Aspergillus niger-fermented Ginkgo biloba leaves (FR) and its comparative effect with vitamin E (VE) and nonfermented (NF) Ginkgo leaves on growth, lipid metabolism, antioxidant capacity, and meat quality of broiler chicks were investigated. In total, 360 one-day-old broiler chicks were randomly allocated into 6 dietary treatments, which were then denoted as control group (basal diet), VE group (containing respectively 15 and 30 IU/kg of all-rac-α-tocopherol acetate in the starter and grower phase), NF group (containing respectively 0.35% and 0.7% NF in the starter and grower phase), and FR1, FR2, and FR3 groups containing respectively 0.2, 0.35, and 0.5% FR in the starter and 0.4, 0.7, and 1.0% FR in the grower phase. The results on performance showed that a significant (P < 0.05) reduction of feed:gain ratio of birds in the FR2 group (22-42 d and 1-42 d) was observed when compared with that of the control and NF groups. With dietary FR increasing, the serum α-tocopherol concentration increased linearly (P = 0.001). Compared with the control, broilers had higher (P < 0.05) serum high-density lipoprotein concentration, total superoxide dismutase activities, and total antioxidant capacity when they were provided with the FR2 and FR3 diet. Whereas the low-density lipoprotein and triglyceride concentrations were lower (P < 0.05 or P < 0.01) in broilers from FR2 or FR3 groups. As the dietary FR increased, abdominal fat (P = 0.002) and muscle malondialdehyde (P = 0.001) concentrations decreased. Furthermore, 24-h pH, 24-h drip loss, and cooking loss were greatly improved (P < 0.05) as the levels of FR increased. Birds fed with FR had a lower (P < 0.05) C16:0 and C18:0 concentrations but a greater (P = 0.001) concentration of C18:2, C18:3, and C20:4 than that of the control. In conclusion, FR can improve the growth performance and lipid metabolism of broilers with decreased abdominal fat deposition. Also, the antioxidant capacity and meat quality improving effects observed in broilers fed FR products might result from the increased retention of α-tocopherol and reduction in lipid peroxidation, as evidenced by the decrease in malondialdehyde and the increase in total superoxide dismutase activities.

This study reports the combination of Ginkgo flavonoid (GF) andCoriolus versicolorpolysaccharide (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 fungusCoriolus 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-1β, IL-6, and TNF-αwas 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.

This study was conducted to investigate the effect of Candida utilis and Aspergillus niger, combined with fermented Ginkgo biloba leaves (FGB) on immunity, antioxidant capability, lipid metabolism, and disease resistance against Aeromonas hydrophila infection in juvenile blunt snout bream (Megalobrama amblycephala). Fish were fed basal diets supplemented with fermented Ginkgo biloba leaves FGB1(0.125%), FGB2(0.25%), FGB3(0.5%), FGB4(1.0%), or Control (0.00%) respectively. At the end of the 60-day feeding trial, fish were challenged by A. hydrophila and mortality rate was recorded for the next 7 days. Results showed that, compared with the control, serum myeloperoxidase (MPO) activity of groups FGB1 and FGB2, alkaline phosphatase (AKP) and lysozyme (LZM) activity of group FGB2 increased significantly (P0.05). After A. hydrophila challenge, the relative mortality of group FGB1 and FGB2 were significantly lower than the control (P

The fungal degradation of toxic alkaloids, papaverine and harmaline, which present in some wild grass, were firstly investigated by using the lignin-degrading basidiomycete Phanerochaete chrysosporium. The fungi could quickly degrade the papaverine and harmaline, especially in the case of P. chrysosporium in Mn-including medium. The nutrient nitrogen concentration had a great effect on the degradation rate of substrates. The papaverine was degraded more effectively under low concentration of nitrogen (LN) condition. But the harmaline could be greatly degraded under both low and high nitrogen concentration. These results strongly suggest that the lignin-degrading basidiomycete Phanerochaete chrysosporium have great potential in degradation of toxic alkaloids of wild grass.