UDP-glycosyltransferase UGT96C10 functions as a novel detoxification factor for conjugating the activated dinitrotoluene sulfonate in switchgrass.

Dinitrotoluene sulfonates (DNTSes) are highly toxic hazards regulated by the Resource Conservation and Recovery Act (RCRA) in the United States. The trinitrotoluene (TNT) red water formed during the TNT purification process consists mainly of DNTSes. Certain plants, including switchgrass, reed and alfalfa, can detoxify low concentrations of DNTS in TNT red water-contaminated soils. However, the precise mechanism by which these plants detoxify DNTS remains unknown. In order to aid in the development of phytoremediation resources with high DNTS removal rates, we identified and characterized 1-hydroxymethyl-2,4-dinitrobenzene sulfonic acid (HMDNBS) and its glycosylated product HMDNBS O-glucoside as the degradation products of 2,4-DNT-3-SO3Na, the major isoform of DNTS in TNT red water-contaminated soils, in switchgrass via LC-MS/MS- and NMR-based metabolite analyses. Transcriptomic analysis revealed that 15 UDP-glycosyltransferase genes were dramatically upregulated in switchgrass plants following 2,4-DNT-3-SO3Na treatment. We expressed, purified and assayed the activity of recombinant UGT proteins in vitro and identified PvUGT96C10 as the enzyme responsible for the glycosylation of HMDNBS in switchgrass. Overexpression of PvUGT96C10 in switchgrass significantly alleviated 2,4-DNT-3-SO3Na-induced plant growth inhibition. Notably, PvUGT96C10-overexpressing transgenic switchgrass plants removed 83.1% of 2,4-DNT-3-SO3Na in liquid medium after 28 days, representing a 3.2-fold higher removal rate than that of control plants. This work clarifies the DNTS detoxification mechanism in plants for the first time, suggesting that PvUGT96C10 is crucial for DNTS degradation. Our results indicate that PvUGT96C10-overexpressing plants may hold great potential for the phytoremediation of TNT red water-contaminated soils.

Chlorogenic Acid and its Isomers Attenuate NAFLD by Mitigating Lipid Accumulation in Oleic Acid-Induced HepG2 Cells and High-Fat Diet- Fed Zebrafish.

Chlorogenic acid (Chl), isochlorogenic acid A (Isochl A), and isochlorogenic acid B (Isochl B) are naturally occurring phenolic compounds, which have been shown to exert a regulatory effect on lipid metabolism. However, the mechanism underlying this effect remains unclear. Herein, we investigated the inhibitory effects and underlying mechanisms of these three phenolic compounds on oleic acid (OA)-induced HepG2 cells and high-fat diet (HFD)-fed zebrafish. Lipid accumulation and triacylglycerol levels increased in OA-induced cells, which was attenuated by Chl, Isochl A, and Isochl B. Moreover, the levels of malondialdehyde (MDA) and reactive oxygen species (ROS) decreased, while superoxide dismutase (SOD) levels increased by Chl, Isochl A and Isochl B treatment. Western blot analysis demonstrated that Chl, Isochl A and Isochl B reduced the expression of lipogenesis-related protein, including fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) and peroxisome proliferator-activated receptor gamma (PPARγ). Moreover, peroxisome proliferator-activated receptor alpha gamma (PPARα) was increased by Chl, Isochl A, and Isochl B treatment. In addition, our results indicated that Chl, Isochl A and Isochl B decreased lipid profiles and lipid accumulation in HFD-fed zebrafish. Thus, these findings highlight the potential of Chl, Isochl A, and Isochl B as effective agents for treating or/and ameliorating non-alcoholic fatty liver disease (NAFLD).

Kaempferol Derivatives from Hippophae rhamnoides Linn. Ameliorate H2O2-Induced Oxidative Stress in SH-SY5Y Cells by Upregulating Nrf2.

As a medicinal and edible resource, Hippophae rhamnoides Linn. subsp. sinensis Rousi is rich in bioactive secondary metabolites, including flavonoids and their derivatives, which offer protective effects against oxidative damage. This study reported the isolation of three new kaempferol derivatives from the seed residue of H. rhamnoides - Hippophandine A, B, and C (compounds 1-3). Their structures were elucidated by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), nuclear magnetic resonance (NMR), and chemical analyses. The compounds were evaluated for their ability to mitigate hydrogen peroxide (H2O2)-induced cell death in SH-SY5Y cells. The results elucidated that Hippophandine A-C at concentrations of 1, 5, and 10 µM reduced the levels of malondialdehyde (MDA) and increased the activity of antioxidative enzymes, such as superoxide dismutase (SOD), glutathione (GSH), and catalase (CAT). Furthermore, they significantly altered the protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream heme oxygenase-1 (HO-1), which is an indicator of redox detection in H2O2-induced SH-SY5Y.

Petanin Potentiated JNK Phosphorylation to Negatively Regulate the ERK/CREB/MITF Signaling Pathway for Anti-Melanogenesis in Zebrafish.

Petanin, an acylated anthocyanin from the Solanaceae family, shows potential in tyrosinase inhibitory activity and anti-melanogenic effects; however, its mechanism remains unclear. Therefore, to investigate the underlying mechanism of petanin's anti-melanogenic effects, the enzyme activity, protein expression and mRNA transcription of melanogenic and related signaling pathways in zebrafish using network pharmacology, molecular docking and molecular dynamics simulation were combined for analysis. The results showed that petanin could inhibit tyrosinase activity and melanogenesis, change the distribution and arrangement of melanocytes and the structure of melanosomes, reduce the activities of catalase (CAT) and peroxidase (POD) and enhance the activity of glutathione reductase (GR). It also up-regulated JNK phosphorylation, inhibited ERK/RSK phosphorylation and down-regulated CREB/MITF-related protein expression and mRNA transcription. These results were consistent with the predictions provided through network pharmacology and molecular docking. Thus, petanin could inhibit the activity of tyrosinase and the expression of tyrosinase by inhibiting and negatively regulating the tyrosinase-related signaling pathway ERK/CREB/MITF through p-JNK. In conclusion, petanin is a good tyrosinase inhibitor and anti-melanin natural compound with significant market prospects in melanogenesis-related diseases and skin whitening cosmetics.

Isolation, Purification and Tyrosinase Inhibitory Activity of Anthocyanins and Their Novel Degradation Compounds from Solanum tuberosum L.

To explore the composition of anthocyanins and expand their biological activities, anthocyanins were systematically isolated and purified from tubers of Solanum tuberosum L., and their tyrosinase inhibitory activity was investigated. In this study, two new anthocyanin degradation compounds, norpetanin (9) and 4-O-(p-coumaryl) rhamnose (10), along with 17 known anthocyanins and their derivatives, were isolated and purified from an acid-ethanolic extract of fresh purple potato tubers. Their structures were elucidated via 1D and 2D NMR and HR-ESI-MS and compared with those reported in the literature. The extracts were evaluated for anthocyanins and their derivatives using a tyrosinase inhibitor screening kit and molecular docking technology, and the results showed that petanin, norpetanin, 4-O-(p-coumaryl) rhamnose, and lyciruthephenylpropanoid D/E possessed tyrosinase inhibitory activity, with 50% inhibiting concentration (IC50) values of 122.37 ± 8.03, 115.53 ± 7.51, 335.03 ± 12.99, and 156.27 ± 11.22 µM (Mean ± SEM, n = 3), respectively. Furthermore, petanin was validated against melanogenesis in zebrafish; it was found that it could significantly inhibit melanin pigmentation (p < 0.001), and the inhibition rate of melanin was 17% compared with the normal group. This finding may provide potential treatments for diseases with abnormal melanin production, and high-quality raw materials for whitening cosmetics.

Rapid screening of α-glucosidase inhibitors in Hypericum perforatum L. using bio-affinity chromatography coupled with UPLC/MS.

α-glucosidase inhibitors (AGIs) are widely used for the treatment of type 2 diabetes, but their side effects have made it to develop novel and alternative AGIs immediately. In this study, the extract of Hypericum perforatum L. (HPE) has been confirmed to have α-glucosidase inhibitory activity in vitro and in vivo. Seven active compounds, rutin, hyperoside, isoquercitrin, avicularin, quercitrin, quercetin, and biapigenin, were screened based on a bio-affinity chromatography column with α-glucosidase enzyme-conjugated solid phase and UPLC/MS, which exhibited excellent α-glycosidase inhibitory effects by the determined IC50 values. The mechanism of α-glycosidase inhibitory activity of biapigenin was studied for the first time. The results showed that biapigenin was a high-potential, reversible, and mixed enzyme inhibitor. Analysis by molecular docking further revealed that hydrophobic interactions were generated by interactions between biapigenin and amino acid residues LYS156, PHE303, PHE314, and LEU313. In addition, hydrogen bonding occurred between biapigenin and α-glucosidase amino acid residues ASP307, SER241, and LYS156. This research identified that biapigenin could be a novel AGI and further applied to the development of potential anti-diabetic drugs. Furthermore, our studies established a rapid in vitro screening method for AGIs from plants.

Flavonoids from Hippophae rhamnoides Linn. Revert Doxorubicin-Induced Cardiotoxicity through Inhibition of Mitochondrial Dysfunction in H9c2 Cardiomyoblasts In Vitro.

Doxorubicin (Dox) is one of the most frequently prescribed anti-cancer drugs. However, treatment with Dox is limited due to cumulative cardiotoxicity. 3-O-ß-d-Sophorosylkaempferol-7-O-{3-O-[2(E)-2,6-dimethyl-6-hydroxyocta-2,7-dienoyl]}-α-L-rhamnoside (F-A), kaempferol 3-sophoroside 7-rhamnoside (F-B), and hippophanone (F-C) were successfully obtained by purification and separation of seabuckthorn seed residue in our previous research. This study was undertaken to investigate the protective effect of three flavonoids against Dox-induced H9c2 cell apoptosis. Cell proliferation was detected by MTT assay. 2',7'-Dichlorofluorescein diacetate (DCFH-DA) was used to determine the production of intracellular reactive oxygen species (ROS). ATP content was measured using an assay kit. Transmission electron microscopy (TEM) was used to observe changes in mitochondrial ultrastructure. The expression levels of proteins (p-JNK, JNK, p-Akt, Akt, p-P38, P38, p-ERK, ERK, p-Src, Src, Sab, IRE1α, Mfn1, Mfn2, and cleaved caspase-3) were evaluated by Western blot. Molecular docking was performed using AutoDock Vina. The three flavonoids could significantly relieve Dox-induced cardiac injury and inhibit cardiomyocyte apoptosis. The mechanisms were mainly related to the stability of mitochondrial structure and function maintained by suppressing the production of intracellular ROS, p-JNK and cleaved caspase-3, and increasing ATP contents and protein expression of mitochondrial mitofusin (Mfn1, Mfn2), Sab and p-Src. Pretreatment with flavonoids from Hippophae rhamnoides Linn. can reduce Dox-induced H9c2 cell apoptosis based on the 'JNK-Sab-Ros' signal pathway.

Functional R2R3-MYB transcription factor NsMYB1, regulating anthocyanin biosynthesis, was relative to the fruit color differentiation in Nitraria sibirica Pall.

BACKGROUND: Nitraria sibirica Pall. is an economic plant with two kinds of fruit color, widely spreads in the Qinghai Tibet Plateau. The chemical analysis and pharmacological evaluation had been carried out for several tens of years, the mechanism behind the fruit color differentiation is still unclear. RESULTS: In this manuscript, the chemical analysis of the extractions showed that the chemical composition of fruit color was anthocyanin, and two kind of Nitraria sibirica Pall. were caused by the content differentiation with the same anthocyanin kinds. Cyanidin-3-[2"-(6'"-coumaroyl)-glucosyl]-glucoside (C3G) was the major anthocyanin. Transcriptome analysis and the qRT-PCR revealed that the structural genes relative to anthocyanin biosynthesis except CHS, F3'5'H and ANS were up-regulated in the peels of BF (Black fruit) compared with the peels of RF (Red fruit), which indicated that transcript factor should be the reason for the expression difference of the structure genes. In the unigenes of the transcript factor MYB and bHLH, relative to anthocyanin, only NsMYB1 (Cluster 8422.10600), was high-expression and up-expression in the peels of BF. NsMYB1 encoded the same length protein with four amino acid differences in the RF and BF, and both contained the intact DNA, HTH-MYB and SANT domains. NsMYB1 was close to the AtMYB114, AtMYB113 and AtPAP1, regulating anthocyanin biosynthesis, in phylogenetic relationship. Both NsMYB1r and NsMYB1b could promote the transcript of the structural genes, and induced the anthocyanin accumulation in all tissues of transgenic tobacco. The insertion of 'TATA' in the promoter of NsMYB1r gave one more promoter region, and was the reason for higher transcripts in black fruit possibly. CONCLUSIONS: Cyanidin-3-[2''-(6'"-coumaroyl)-glucosyl]-glucoside was the major anthocyanin in black fruit of Nitraria sibirica Pall.. NsMYB1 was a functional R2R3-MYB transcription factor, regulated the anthocyanin biosynthesis, and led to the fruit color differentiation in Nitraria sibirica Pall.

LATERAL BRANCHING OXIDOREDUCTASE, one novel target gene of Squamosa Promoter Binding Protein-like 2, regulates tillering in switchgrass.

Strigolactones (SLs) play a critical role in regulating plant tiller number. LATERAL BRANCHING OXIDOREDUCTASE (LBO) encodes an important late-acting enzyme for SL biosynthesis and regulates shoot branching in Arabidopsis. However, little is known about the function of LBO in monocots including switchgrass (Panicum virgatum L.), a dual-purpose fodder and biofuel crop. We studied the function of PvLBO via the genetic manipulation of its expression levels in both the wild-type and miR156 overexpressing (miR156OE ) switchgrass. Co-expression analysis, quantitative real-time polymerase chain reaction (qRT-PCR), transient dual luciferase assay, and chromatin immunoprecipitation-qPCR were all used to determine the activation of PvLBO by miR156-targeted Squamosa Promoter Binding Protein-like 2 (PvSPL2) in regulating tillering of switchgrass. PvLBOtranscripts dramatically declined in miR156OE transgenic switchgrass, and the overexpression of PvLBO in the miR156OE transgenic line produce fewer tillers than the control. Furthermore, we found that PvSPL2 can directly bind to the promoter of PvLBO and activate its transcription, suggesting that PvLBO is a novel downstream gene of PvSPL2. We propose that PvLBO functions as an SL biosynthetic gene to mediate tillering and acts as an important downstream factor in the crosstalk between the SL biosynthetic pathway and the miR156-SPL module in switchgrass.

Down-regulation of PvSAMS impairs S-adenosyl-L-methionine and lignin biosynthesis, and improves cell wall digestibility in switchgrass.

S-adenosyl- l-methionine (SAM) is the methyl donor involved in the biosynthesis of guaiacyl (G) and syringyl (S) lignins in vascular plants. SAM is synthesized from methionine through the catalysis of the enzyme S-adenosylmethionine synthase (SAMS). However, the detailed function of SAMS in lignin biosynthesis has not been widely investigated in plants, particularly in monocot species. In this study, we identified PvSAMS genes from switchgrass (Panicum virgatum L.), an important dual-purpose fodder and biofuel crop, and generated numerous transgenic switchgrass lines through PvSAMS RNA interference technology. Down-regulation of PvSAMS reduced the contents of SAM, G-lignins, and S-lignins in the transgenic switchgrass. The methionine and glucoside derivatives of caffeoyl alcohol were found to accumulate in the transgenic plants. Moreover, down-regulation of PvSAMS in switchgrass resulted in brownish stems associated with reduced lignin content and improved cell wall digestibility. Furthermore, transcriptomic analysis revealed that most sulfur deficiency-responsive genes were differentially expressed in the transgenic switchgrass, leading to a significant increase in total sulfur content; thus implying an important role of SAMS in the methionine cycle, lignin biosynthesis, and sulfur assimilation. Taken together, our results suggest that SAMS is a valuable target in lignin manipulation, and that manipulation of PvSAMS can simultaneously regulate the biosynthesis of SAM and methylated monolignols in switchgrass.

Genome-Wide Identification of Switchgrass Laccases Involved in Lignin Biosynthesis and Heavy-Metal Responses.

Plant laccase genes belong to a multigene family, play key roles in lignin polymerization, and participate in the resistance of plants to biotic and abiotic stresses. Switchgrass is an important resource for forage and bioenergy production, yet information about the switchgrass laccase gene family is scarce. Using bioinformatic approaches, a genome-wide analysis of the laccase multigene family in switchgrass was carried out in this study. In total, 49 laccase genes (PvLac1 to PvLac49) were identified; these can be divided into five subclades, and 20 of them were identified as targets of miR397. The tandem and segmental duplication of laccase genes on Chr05 and Chr08 contributed to the expansion of the laccase family. The laccase proteins shared conserved signature sequences but displayed relatively low sequence similarity, indicating the potential functional diversity of switchgrass laccases. Switchgrass laccases exhibited distinct tissue/organ expression patterns, revealing that some laccases might be involved in the lignification process during stem development. All five of the laccase isoforms selected from different subclades responded to heavy metal. The immediate response of lignin-related laccases, as well as the delayed response of low-abundance laccases, to heavy-metal treatment shed light on the multiple roles of laccase isoforms in response to heavy-metal stress.

Improved Stability of Blue Colour of Anthocyanins from Lycium ruthenicum Murr. Based on Copigmentation.

Natural blue food colourant is rare. The aim of this work was to screen compounds from the common copigments that could improve the blue tones of anthocyanins (ACNs) and to investigate the effect of different copigments on the colour stability of anthocyanins in neutral species. International Commission on Illumination (CIE) colour space, UV, IR, NMR, atomic force microscopy (AFM) and computational chemistry methods were utilised to evaluate ACNs from Lycium ruthenicum Murr. (LR), which is complexed with food additives and biological agents. The results indicate that Pro-Xylane (PX), Ectoin (ECT) and dipotassium glycyrrhizinate (DG) enhance the blue colour of the ACNs. ACNs-PX presents a colour close to Oxford Blue and has a surface height of 2.13 ± 0.14 nm and slightly improved stability. The half-life of ACNs-DG is improved 24.5-fold and had the highest complexation energy (-50.63/49.15) kcal/mol, indicating hydrogen bonds and π-π stacking forces enhance stability. These findings offer a new perspective for anthocyanin utilisation as a blue colourant and contribute to the large-scale application of LR.

[Preparation and characterization of fenugreek leaf flavonoids and their protective effects against oxidative damage to hepatocytes].

The present study investigated the main components of fenugreek(Trigonella foenum-graecum L.) leaf flavonoids(FLFs) and their antioxidant activity. FLFs were prepared and enriched by solvent extraction, and the flavonoids were characterized by high-performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry(HPLC-Q-TOF-MS/MS). The protective effect of FLFs against H_2O_2-induced stress damage to L02 hepatocytes was also investigated. Firstly, the cell viability was measured by MTT assay. The oxidative stress injury model was induced by H_2O_2 in L02 cells. The release of lactate dehydrogenase(LDH), the content of reduced glutathione(GSH) and malondialdehyde(MDA), and the activities of superoxide dismutase(SOD) and catalase(CAT) were measured by assay kits. Hoechst fluorescence staining was performed to observe the cell apoptosis. The expression levels of c-Jun N-terminal kinase(JNK), extracellular signal-regulated kinase 1/2(ERK1/2), nuclear factor erythroid-2 related factor 2(Nrf2), heme oxygenase 1(HO-1), and their phosphorylated proteins were detected by Western blot. Based on the MS fragment ion information and data in databases, FLFs contained eight flavonoids with quercetin and kaempferol as the main aglycons. The cell viabi-lity assay revealed that as compared with the conditions in the H_2O_2 treatment group, 3.125-25 µg·mL~(-1) FLFs could increase the viability of L02 cells, reduce LDH release and MDA content in a dose-dependent manner, potentiate the activities of SOD, CAT, and GSH, decrease the phosphorylation of JNK and ERK1/2 proteins, and up-regulate the expression of Nrf2 and HO-1. The results of fluorescence staining showed that the nucleus of the H_2O_2 treatment group showed concentrated and dense strong blue fluorescence, while the blue fluorescence intensity of the FLFs group decreased significantly. FLFs showed a protective effect against H_2O_2-induced oxidative damage in L02 cells, and the underlying mechanism is associated with the enhancement of cell capability in clearing oxygen free radicals and the inhibition of apoptosis by the activation of the MAPKs/Nrf2/HO-1 signaling pathway. The antioxidant effect of fenugreek leaf is related to its rich flavonoids.

Kaempferol and Kaempferide Attenuate Oleic Acid-Induced Lipid Accumulation and Oxidative Stress in HepG2 Cells.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases which lacks ideal treatment options. Kaempferol and kaempferide, two natural flavonol compounds isolated from Hippophae rhamnoides L., were reported to exhibit a strong regulatory effect on lipid metabolism, for which the mechanism is largely unknown. In the present study, we investigated the effects of kaempferol and kaempferide on oleic acid (OA)-treated HepG2 cells, a widely used in vitro model of NAFLD. The results indicated an increased accumulation of lipid droplets and triacylglycerol (TG) by OA, which was attenuated by kaempferol and kaempferide (5, 10 and 20 µM). Western blot analysis demonstrated that kaempferol and kaempferide reduced expression of lipogenesis-related proteins, including sterol regulatory element-binding protein 1 (SREBP1), fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD-1). Expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT enhancer binding proteins ß (C/EBPß), two adipogenic transcription factors, was also decreased by kaempferol and kaempferide treatment. In addition, western blot analysis also demonstrated that kaempferol and kaempferide reduced expression of heme oxygenase-1 (HO-1) and nuclear transcription factor-erythroid 2-related factor 2 (Nrf2). Molecular docking was performed to identify the direct molecular targets of kaempferol and kaempferide, and their binding to SCD-1, a critical regulator in lipid metabolism, was revealed. Taken together, our findings demonstrate that kaempferol and kaempferide could attenuate OA-induced lipid accumulation and oxidative stress in HepG2 cells, which might benefit the treatment of NAFLD.

Anti-Inflammatory Activity of Three Triterpene from Hippophae rhamnoides L. in Lipopolysaccharide-Stimulated RAW264.7 Cells.

Oleanolic acid (OA), asiatic acid (AA), and maslinic acid (MA) are ubiquitous isomeric triterpene phytochemicals with many pharmacological effects. To improve their application value, we used lipopolysaccharide (LPS) to induce RAW264.7 cells and studied the differences in the anti-inflammatory effects of the triterpenes according to their structural differences. MTT, Griess, and immunofluorescence assays, ELISA, flow cytometry, and Western blotting, were performed. The release of LPS-induced pro-inflammatory mediators, such as nitric oxide (NO), inducible nitric oxide synthase (iNOS), and interleukin (IL-6), was significantly inhibited by OA, AA, and MA at the same concentration, and AA and MA promoted the production of anti-inflammatory factor IL-10. OA, AA, and MA inhibited LPS-induced NF-κB nuclear translocation in RAW264.7 cells. OA and AA inhibited the phosphorylation of ERK1/2, P38, and JNK1/2 in LPS-stimulated RAW264.7 cells. Moreover, OA increased LPS-induced Nrf2 expression and decreased Keap1 expression in RAW264.7 cells. OA, AA, and MA inhibited LPS-stimulated intracellular reactive oxygen species (ROS) production and alleviated mitochondrial membrane potential depletion. Overall, our data suggested that OA, AA, and MA exhibited significant anti-inflammatory effects in vitro. In particular, OA and AA take effects through the MAPKs, NF-κB, and Nrf2 signaling pathways.

Inhibitory Activity and Mechanism Investigation of Hypericin as a Novel α-Glucosidase Inhibitor.

α-glucosidase is a major enzyme that is involved in starch digestion and type 2 diabetes mellitus. In this study, the inhibition of hypericin by α-glucosidase and its mechanism were firstly investigated using enzyme kinetics analysis, real-time interaction analysis between hypericin and α-glucosidase by surface plasmon resonance (SPR), and molecular docking simulation. The results showed that hypericin was a high potential reversible and competitive α-glucosidase inhibitor, with a maximum half inhibitory concentration (IC50) of 4.66 ± 0.27 mg/L. The binding affinities of hypericin with α-glucosidase were assessed using an SPR detection system, which indicated that these were strong and fast, with balances dissociation constant (KD) values of 6.56 × 10-5 M and exhibited a slow dissociation reaction. Analysis by molecular docking further revealed that hydrophobic forces are generated by interactions between hypericin and amino acid residues Arg-315 and Tyr-316. In addition, hydrogen bonding occurred between hypericin and α-glucosidase amino acid residues Lys-156, Ser-157, Gly-160, Ser-240, His-280, Asp-242, and Asp-307. The structure and micro-environment of α-glucosidase enzymes were altered, which led to a decrease in α-glucosidase activity. This research identified that hypericin, an anthracene ketone compound, could be a novel α-glucosidase inhibitor and further applied to the development of potential anti-diabetic drugs.

Protective Effect of Two Alkaloids from Hippophae rhamnoides Linn. against Doxorubicin-Induced Toxicity in H9c2 Cardiomyoblasts.

BACKGROUND: Doxorubicin (Dox) is one of the most frequently prescribed anti-cancer drugs. However, clinical application with Dox is limited due to its potentially fatal cumulative cardiotoxicity. N-p-coumaroyl-4-aminobutan-1-ol (alk-A), an organic amide alkaloid and hippophamide (alk-B), a rare pyridoindole alkaloid were successfully obtained by purification and separation of seabuckthorn seed residue in our previous research. This study was undertaken to investigate the protective effect of alk-A and alk-B against Dox-induced embryonic rat cardiac cells (H9c2 cells) apoptosis. METHODS: H9c2 cells were treated with Dox (2.5 µM) in the presence of alk-A and alk-B (10, 20, and 40 µM) and incubated for 24 h. RESULTS: It was shown that pretreatment of the H9c2 cells with alk-A and alk-B significantly reduced Dox-induced apoptosis. Alk-A and alk-B both inhibited reactive oxygen species (ROS) production and suppressed cleaved-caspase-3 protein expression and the activation of JNK (Jun N-terminal kinases), as well as increasing ATP levels, favoring mitochondrial mitofusin protein expression, and relieving damage to mitochondrial DNA. CONCLUSIONS: These results suggest that alk-A and alk-B can inhibit Dox-induced apoptosis in H9C2 cardiac muscle cells via inhibition of cell apoptosis and improvement of mitochondrial function, while alk-B showed more protection. Alk-B could be a potential candidate agent for protecting against cardiotoxicity in Dox-exposed patients.

Methylated mud snail protein as a bio-flocculant for high turbidity wastewater treatment.

The authors reported a potential candidate methylated mud snail protein (MeMsp) as an effective and eco-friendly flocculant to treat the high turbidity wastewater. MeMsp was obtained by extraction of mud snail protein (Msp) through isoelectric precipitation (PSC-IP) and then methylated via the esterification with side-chain carboxyl. Structural characterization of FT-IR, zeta potential and elemental analysis were carried out and further confirmed the successful of the methylation. Flocculation experiments with kaolin suspension simulated wastewater indicated that MeMsp-24 displayed more excellent flocculation efficiency at a low dosage. At the optimum dosage 27 mg/L, the maximum clarification efficiency of MeMsp-24 was 97.46% under pH 7.0. Furthermore, MeMsp-24 exhibited a wide flocculation window in the pH range 1.0-9.0, and faster sedimentation velocity and larger flocs size. In addition, MeMsp-24 exhibited 92.12% clarification efficiency in treating railway tunnel construction effluent. The flocculation kinetic and mechanism analysis revealed that the most effective particle collision occurred at the optimal dosage, with charge neutralization and adhesion playing irreplaceable roles in different environments, respectively. Therefore, through extraction and methylation modification, MeMsp could be a promising eco-friendly flocculant for high turbidity wastewater treatment.

[Research progress on chemical constituents and pharmacological activities of Trigonella foenum-graecum].

Trigonella foenum-graecum is an annual plant of the genus Trigonella in the Leguminosae family. It is widely distributed in China and has a long history of application. According to phytochemistry research, the seeds, stem, and leaves of this herb contain not only a variety of bioactive ingredients, including alkaloids, saponins, polysaccharides, flavonoids, and phenols, but also abundant nutrients such as unsaturated fatty acids and amino acids and various trace elements. Pharmacological studies have shown that both the extract of T. foenum-graecum and its chemical constituents exhibit hypoglycemic, hypolipidemic, antitumor, antioxidative, antimicro-bial, and hepatoprotective activities. This paper reviews the research progress on the chemical constituents and pharmacological effects of T. foenum-graecum, which may contribute to further development, application, and clinical research of this herb.

(20S)-Protopanaxadiol Ginsenosides Induced Cytotoxicity via Blockade of Autophagic Flux in HGC-27 Cells.

(20S)-Protopanaxadiol ginsenosides Rg3, Rh2 and PPD have been demonstrated for their anticancer activity. However, the underlying mechanism of their antitumor activity remains unclear. In the present study, we investigated the role of these three ginsenosides on cell proliferation and death of human gastric cancer cells (HGC-27 cells). The sulforhodamine B (SRB) assay, Western blot analysis, fluorescence microscopy, confocal microscopy, high performance liquid chromatography (HPLC) analysis, flow cytometry, and transmission electron microscopy (TEM) were used to evaluate cell proliferation, apoptosis, and autophagy. The results showed that both Rh2 and PPD were more effective than Rg3 in inhibiting HGC-27 cell proliferation and inducing cytoplasmic vacuolation, while no significant changes in apoptosis were observed. Interestingly, cytoplasmic vacuolation and blockade of autophagy flux were observed after treatment with Rh2 and PPD. Rh2 obviously up-regulated the expression of the LC3II and p62. Furthermore, the increase in lysosomal pH and membrane rupture was observed in Rh2-treated and PPD-treated cells. When HGC-27 cells were pretreated with bafilomycin A1, a specific inhibitor of endosomal acidification, cellular vacuolization was increased, and the cell viability was significantly decreased, which indicated that Rh2-induced lysosome-damage accelerated cell death. Furthermore, data derived from mitochondrial analysis showed that excessive mitochondrial reactive oxygen species (ROS) and dysregulation of mitochondrial energy metabolism were caused by Rh2 and PPD treatment in HGC-27 cells. Taken together, these phenomena indicated that Rh2 and PPD inhibited HCG-27 cells proliferation by inducing mitochondria damage, dysfunction of lysosomes, and blockade of autophagy flux. The number of glycosyl groups at C-3 position could have an important effect on the cytotoxicity of Rg3, Rh2 and PPD.