A series of myricetin derivatives containing amide, thioether, and 1,3,4-thiadiazole moieties were designed and synthesized, and their antiviral and antibacterial activities were assessed. The bioassays showed that all the title compounds exhibited potent in vitro antibacterial activities against Xanthomonas citri (Xac), Ralstonia solanacearum (Rs), and Xanthomonas oryzae pv. Oryzae (Xoo). In particular, the compounds 5a, 5f, 5g, 5h, 5i, and 5l, with EC50 values of 11.5⁻27.3 μg/mL, showed potent antibacterial activity against Xac that was better than the commercial bactericides Bismerthiazol (34.7 μg/mL) and Thiodiazole copper (41.1% μg/mL). Moreover, the in vivo antiviral activities against tobacco mosaic virus (TMV) of the target compounds were also tested. Among these compounds, the curative, protection, and inactivation activities of 5g were 49.9, 52.9, and 73.3%, respectively, which were better than that of the commercial antiviral Ribavirin (40.6, 51.1, and 71.1%, respectively). This study demonstrates that myricetin derivatives bearing amide, thioether, and 1,3,4-thiadiazole moieties can serve as potential alternative templates for the development of novel, highly efficient inhibitors against plant pathogenic bacteria and viruses.

Magnolia zenii is a critically endangered species known from only 18 trees that survive on Baohua Mountain in Jiangsu province, China. Little information is available regarding its molecular biology, with no genomic study performed on M. zenii until now. We determined the complete plastid genome of M. zenii and identified microsatellites. Whole sequence alignment and phylogenetic analysis using BI and ML methods were also conducted. The plastome of M. zenii was 160,048 bp long with 39.2% GC content and included a pair of inverted repeats (IRs) of 26,596 bp that separated a large single-copy (LSC) region of 88,098 bp and a small single-copy (SSC) region of 18,757 bp. One hundred thirty genes were identified, of which 79 were protein-coding genes, 37 were transfer RNAs, and eight were ribosomal RNAs. Thirty seven simple sequence repeats (SSRs) were also identified. Comparative analyses of genome structure and sequence data of closely-related species revealed five mutation hotspots, useful for future phylogenetic research. Magnolia zenii was placed as sister to M. biondii with strong support in all analyses. Overall, this study providing M. zenii genomic resources will be beneficial for the evolutionary study and phylogenetic reconstruction of Magnoliaceae.

Psidium guajava, a popular food and medicine dual purposes plant cultivated in tropical and subtropical regions, has been widely used as food crop and folk medicine, such as anti-diabetes agent, around the world. Triterpenoids have been considered as the major active ingredients of P. guajava. In the present study, a high-performance liquid chromatography coupled with diode array and evaporative light scattering detectors (HPLC-DAD-ELSD) method was developed for simultaneous determination of nine triterpenoids in P. guajava. Pressurized liquid extraction (PLE) was performed for sample preparation, and the analysis was achieved on a Cosmosil 5C18-MS-II (Nacalai Tesque, Kyoto, Japan) column eluted with gradient 0.1% aqueous formic acid-methanol system. The drift tube temperature of ELSD was set at 40 °C, and nitrogen flow-rate was at 1.6 L/min. All calibration curves for the analytes showed good linear regression (R2 > 0.9992) within test ranges. The established method was validated for intra-day and inter-day precisions (RSDs < 5%) and accuracy (recovery 94.23-106.87%). The validated method was successfully applied to determinate nine triterpenoids in 15 samples from the leave or fruit of P. guajava. In addition, the α-glucosidase inhibition assay showed good α-glucosidase inhibition activity in almost all the determined triterpenoids. The present study suggested that triterpenoids should be the quality control markers for P. guajava and HPLC-DAD-ELSD was an effective tool for the quality control of P. guajava.

A novel class of styryl sulfones were designed and synthesized as CAPE derivatives by our work team, which showed a multi-target neuroprotective effect, including antioxidative and anti-neuroinflammatory properties. However, the underlying mechanisms remain unclear. In the present study, the anti-Parkinson's disease (PD) activity of 10 novel styryl sulfone compounds was screened by the cell viability test and the NO inhibition test in vitro. It was found that 4d exhibited the highest activity against PD among them. In a MPTP-induced mouse model of PD, the biological activity of 4d was validated through suppressing dopamine neurotoxicity, microglial activation, and astrocytes activation. With compound 4d, we conducted the mechanistic studies about anti-inflammatory responses through inhibition of p38 phosphorylation to protect dopaminergic neurons, and antioxidant effects through promoting nuclear factor erythroid 2-related factor 2 (Nrf2). The results revealed that 4d could significantly inhibit 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/1-methyl-4-phenylpyridinium (MPTP/MPP+)-induced p38 mitogen-activated protein kinase (MAPK) activation in both in vitro and in vivo PD models, thus inhibiting the NF-κB-mediated neuroinflammation-related apoptosis pathway. Simultaneously, it could promote Nrf2 nuclear transfer, and upregulate the expression of antioxidant phase II detoxification enzymes HO-1 and GCLC, and then reduce oxidative damage.

Venom-induced thrombocytopenia (VIT) is one of the most important hemotoxic effects of a snakebite, which is often associated with venom-induced consumptive coagulopathy (VICC). Refractory thrombocytopenia without significant coagulation abnormalities has also been reported after envenomation by some viperid snakes; however, the mechanisms are not well understood and therapeutic strategies are lacking. Here, we found that patients injured by Daboia siamensis or Agkistrodon halys snakes, who were resistant to standard antivenom treatment, had developed coagulopathy-independent thrombocytopenia. Venoms from these viperid snakes, rather than from the elapid snake (Bungarus multicinctus), induced platelet surface expression of neuraminidase-1 (NEU-1), and significantly increased the desialylation of the glycoproteins on human platelets. The desialylated platelets caused by viperid snake venoms were further internalized by macrophages, which resulted in reduced platelet numbers in peripheral blood. Importantly, neuraminidase inhibitor significantly decreased viper venom-induced platelet desialylation, therefore inhibiting platelet phagocytosis by macrophages, and alleviating venom-induced thrombocytopenia. Collectively, these findings support an important role for desialylated platelet clearance in the progression of viper envenomation-induced, coagulopathy-independent thrombocytopenia. Our study demonstrates that the neuraminidase inhibitor may be a potential therapy or adjuvant therapy to treat snakebite-induced thrombocytopenia.

Bilobalide exhibits numerous beneficial bioactivities, including neuroprotective, anti-inflammatory, and antioxidant activity. Our previous study demonstrated that bilobalide inhibits adipogenesis and promotes lipolysis. The dose-dependent cytotoxicity was found to be specific to the mature adipocytes only, indicating the potential for regulating apoptosis in them. Herein, we aimed to investigate the apoptotic effects of bilobalide on 3T3-L1 mature adipocytes and elucidate the underlying mechanisms thereof. Flow cytometry analysis (FACS) revealed the pro-apoptotic effects of bilobalide on these cells. Bilobalide induced early apoptosis by reducing the mitochondrial membrane potential (MMP). DNA fragmentation was confirmed using TUNEL staining. Additionally, bilobalide increased the intracellular reactive oxygen species (ROS) levels and activities of Caspases 3/9. Pre-treatment with NAC (an ROS scavenger) confirmed the role of ROS in inducing apoptosis. Moreover, bilobalide up- and down-regulated the expression of Bax and Bcl-2, respectively, at the mRNA and protein expression levels; upregulated the Bax/Bcl-2 ratio; triggered the release of cytochrome c from the mitochondria; and increased the protein expression of cleaved Caspase 3, cleaved Caspase 9, and PARP cleavage. These results support the conclusion that bilobalide induces apoptosis in mature 3T3-L1 adipocytes through the ROS-mediated mitochondrial pathway, and offers potential novel treatment for obesity.

A series of novel phosphorylated penta-1,4-dien-3-one derivatives were designed and synthesized. The structures of all title compounds were determined by ¹H-NMR, 13C-NMR, 31P-NMR, and high-resolution mass spectrometry (HRMS). Bioassay results showed that several of the title compounds exhibited remarkable antibacterial and antiviral activities. Among these, compound 3g exhibited substantial antibacterial activity against Xanthomonas oryzae pv. Oryzae (Xoo), with a 50% effective concentration (EC50) value of 8.6 μg/mL, which was significantly superior to bismerthiazol (BT) (58.8 µg/mL) and thiodiazole-copper (TC) (78.7 μg/mL). In addition, compound 3h showed remarkable protective activity against tobacco mosaic virus (TMV), with an EC50 value of 104.2 μg/mL, which was superior to that of ningnanmycin (386.2 μg/mL). Furthermore, the microscale thermophoresis and molecular docking experiments on the interaction of compounds 3h and 3j with TMV coat protein (TMV CP) were also investigated. Compounds 3h and 3j bound to TMV CP with dissociation constants of 0.028 and 0.23 μmol/L, which were better than that of ningnanmycin (0.52 μmol/L). These results suggest that novel phosphorylated penta-1,4-dien-3-one derivatives may be considered as an activator for antibacterial and antiviral agents.

Uropathogenic Escherichia coli (UPEC) is the most common pathogenic bacteria associated with urinary tract infection (UTI). UPEC can cause UTI by adhering to and invading uroepithelial cells. Fimbriae is the most important virulence factor of UPEC, and a potentially promising target in developing novel antibacterial treatments. In this study, the antibacterial properties and effects of the compound dictamnine, extracted from the traditional Chinese medicine Cortex Dictamni, on the bacterial morphology, cell adhesion, and invasion of UPEC were studied. Dictamnine exhibited no obvious antibacterial activity against UPEC, but significantly impeded the ability of UPEC to adhere to and invade uroepithelial cells. RT-qPCR analysis showed that treatment downregulated the expression of type 1 fimbriae, P fimbriae, and curli fimbriae adhesion genes, and also downregulated adhesion-related receptor genes of uroepithelial cells. Transmission electron microscopy showed that dictamnine destroyed the structure of the fimbriae and the surface of the bacteria became smooth. These results suggest that dictamnine may help to prevent UTI by simultaneously targeting UPEC fimbriae and urothelial adhesin receptors, and may have a potential use as a new anti-UPEC drug.

Pulsatilla saponins (PS) extracts from Pulsatilla chinensis (Bge.) Regel, are a commonly used traditional Chinese medicine. In the previous study, we found Pulsatilla saponins displayed anti-tumor activity without side effects such as bone marrow suppression. However, the mechanism of the anti-tumor effect was not illustrated well. Since M2-like tumor-associated macrophages (TAMs) that required activation of the signal transducer and activator of transcription 6 (STAT6) for polarization are the important immune cells in the tumor microenvironment and play a key role in tumor progress and metastasis, this study aimed to confirm whether Pulsatilla saponins could inhibit the development and metastasis of tumors by inhibiting the polarization of M2 macrophages. We investigated the relevance of M2 macrophage polarization and the anti-tumor effects of Pulsatilla saponins in vitro and in vivo. In vitro, Pulsatilla saponins could decrease the mRNA level of M2 marker genes Arg1, Fizz1, Ym1, and CD206, and the down-regulation effect of phosphorylated STAT6 induced by IL-4; moreover, the conditioned medium (CM) from bone marrow-derived macrophages (BMDM) treated with Pulsatilla saponins could inhibit the proliferation and migration of B16-F0 cells. In vivo, Pulsatilla saponins could reduce the number of lung metastasis loci, down-regulate the expression of M2 marker genes, and suppress the expression of phosphorylated STAT6 in tumor tissues. Furthermore, we used AS1517499 (AS), a STAT6 inhibitor, to verify the role of PS on M2 macrophage polarization both in vitro and in vivo. We found that Pulsatilla saponins failed to further inhibit STAT6 activation; the mRNA level of Arg1, Fizz1, Ym1, and CD206; and the proliferation and migration of B16-F0 cells after AS1517499 intervention in vitro. Similar results were obtained in vivo. These results illustrated that Pulsatilla saponins could effectively suppress tumor progress by inhibiting the polarization of M2 macrophages via the STAT6 signaling pathway; this revealed a novel mechanism for its anti-tumor activity.

The use of nano-encapsulated resveratrol (RSV) in self-micro-emulsified drug delivery systems (SMEDDS) formulations was investigated. Self-emulsifying grading tests were used to establish the optimal ratio of oil, surfactant, and co-surfactant. The optimized system was further investigated for the droplet size and zeta potential at the different medium pH values by a Malvern Zetasizer and transmission electron microscopy (TEM). The antioxidant capacity and cytotoxicity of the formulation were detected by DCFH-DA and a CCK-8 assays. The results showed that the nano-emulsion based on ethyl oleate, Tween-80, and PEG-400 (35:40:25, w/w/w) was the most stable formulation due to the small droplet size (approximately 50 nm) and high zeta potential in a neutral environment. Furthermore, this formulation also exhibited a greater antioxidant capacity with less toxicity than free RSV. Taken together, considering these results and the simple fabrication process, this formulation could be used to deliver nutritional food supplements in a stable, efficient, and safe manner.

Dihydroartemisinin (DHA) is a derivative of the herb Artemisia annua L. that has prominent immunomodulatory activity; however, its underlying mechanism remains elusive. Inflammatory bowel disease (IBD) is an idiopathic inflammatory condition characterized as an autoimmune disorder that includes dysfunctions in the T helper (Th)/T regulatory cell (Treg) balance, which normally plays pivotal roles in immune homeostasis. The aim of this study was to explore the potential of DHA to ameliorate IBD by restoring the Th/Treg cell balance. To this end, we established mouse models of colitis induced by oxazolone (OXA) and 2,4,6-trinitro-benzene sulfonic acid (TNBS). We then treated mice with DHA at 4, 8, or 16 mg/kg/day. DHA treatment ameliorated colitis signs and reduced lymphocyte infiltration and tissue fibrosis. Moreover, DHA decreased the numbers of Th1 and Th17 cells and Th9 and Th22 cells in TNBS- or OXA-induced colitis, respectively, and increased Tregs in both models. DHA (0.8 mg/mL) also inhibited activated CD4+ T lymphocytes, which was accompanied by apoptosis induction. Moreover, it promoted heme oxygenase-1 (HO-1) production in vitro and in vivo, concomitant with CD4+ T cell apoptosis and restoration of the Th/Treg balance, and these effects were blocked by treatment with the HO-1 inhibitor Sn-protoporphyrin IX. Overall, these results suggest that DHA is a novel and valuable candidate for IBD therapy or Th/Treg immunoregulation.

Bilobalide, the only sesquiterpene compound from Ginkgo biloba leaf, exhibits various beneficial pharmaceutical activities, such as antioxidant, anti-inflammation, and protective effects for the central nervous system. Several bioactive components extracted from Ginkgo biloba extract reportedly have the potential to attenuate lipid metabolism. However, the effect of bilobalide on lipid metabolism remains unclear. In this study, we used 3T3-L1 cells as the cell model to investigate the effect of bilobalide on adipogenesis. The results showed that bilobalide inhibited 3T3-L1 preadipocyte differentiation and intracellular lipid accumulation. Quantitative real-time PCR and western blotting results indicated that several specific adipogenic transcription factors and a few important adipogenesis-related genes were significantly down regulated on both mRNA and protein levels in bilobalide treatment groups. By contrast, the expression of some lipolytic genes, such as adipose triglyceride lipase, hormone-sensitive lipase (HSL), and carnitine palmitoyltransferase-1α, were all up-regulated by bilobalide treatment, and the phosphorylation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase 1, and HSL were stimulated. Furthermore, bilobalide treatment partially restored AMPK activity following its blockade by compound C (dorsomorphin). These results suggested that bilobalide inhibited adipogenesis and promoted lipolysis in 3T3-L1 cells by activating the AMPK signaling pathway.

Alismatis rhizoma (AR), the dried rhizoma of Alisma orientale Juzepzuk (Alismataceae), is a traditional Chinese medicine. AR is an important part of many prescriptions and is commonly used as a diuretic agent in Asia. This study aimed to evaluate the diuretic effects of total triterpene extract (TTE) and triterpene component compatibility (TCC, the mixture of alisol B 23-acetate, alisol B, alisol A 24-acetate, alisol A, and alisol C 23-acetate) of AR in saline-loaded rats. The optimal diuretic TCC of AR was optimized using a uniform design. Different doses (5, 20, and 40 mg/kg) of TTE and TCC groups (N1-N8) were orally administered to rats. Urinary excretion rate, pH, and electrolyte excretion were measured in the urine of saline-loaded rats. Results showed that TTE doses increased urine volume and electrolyte excretion compared with the control group. All uniformly designed groups of TCC also increased urine excretion. In addition, optimal diuretic TCC was calculated (alisol B 23-acetate: alisol B: alisol A 24-acetate: alisol A: alisol C 23-acetate 7.2:0.6:2.8:3.0:6.4) and further validated by saline-loaded rats. This study demonstrated that TTE presented a notable diuretic effect by increasing Na⁺, K⁺, and Cl − displacements. The most suitable TTC compatible proportion of alisol B 23-acetate: alisol B: alisol A 24-acetate: alisol A: alisol C 23-acetate for diuretic activity was validated, and triterpenes were the material basis for the diuretic activity of AR.

The crystal structure of the sorafenib and B-RAF complex indicates that the binding cavity occupied by the pyridine-2-carboxamide in sorafenib has a large variable space, making it a reasonable modification site. In order to identify novel compounds with anti-cancer activity, better safety and polar groups for further application, five sorafenib analogs with new pyridine-2-amide side chains were designed and synthesized. Preliminary pharmacologic studies showed that these compounds displayed much lower toxicities than that of sorafenib. Among them, compound 10b bearing mercaptoethyl group kept relevant antiproliferation potency compared to sorafenib in Huh7 and Hela cell lines with values of IC50 58.79 and 63.67 M, respectively. As a small molecule inhibitor targeting protein tyrosine kinases, thiol in compound 10b would be an active group to react with maleimide in a mild condition for forming nanoparticles Sorafenib-PEG-DGL, which could be developed as a delivery vehicle to improve the concentration of anti-tumor therapeutic agents in the target cancer tissue and reduce side effects in the next study.

Pseudostellaria heterophylla (Miq.) Pax is a popular clinical herb and nutritious health food. However, leaf spot disease caused by fungal pathogens frequently occurs and seriously influences the growth, quality, and yield of P. heterophylla. In this work, the field control roles of difenoconazole, chitosan, and their combination in the leaf spot disease in P. heterophylla and their effects on the disease resistance, photosynthetic capacity, medicinal quality, and root yield of P. heterophylla are investigated. The results manifest that 37% difenoconazole water-dispersible granule (WDG) with 5000-time + chitosan 500-time dilution liquid had a superior control capacity on leaf spot disease with the control effects of 91.17%~88.19% at 15~30 days after the last spraying, which significantly (p < 0.05) exceeded that of 37% difenoconazole WDG 3000-time dilution liquid and was significantly (p < 0.01) higher than that of 37% difenoconazole WDG 5000-time dilution liquid, chitosan 500-time dilution liquid, or chitosan 1000-time dilution liquid. Simultaneously, this combination could more effectively enhance the disease resistance, photosynthetic capacity, medicinal quality, and tuberous root yield of P. heterophylla compared to when these elements were applied alone, as well as effectively reduce difenoconazole application. This study emphasizes that chitosan combined with a low dosage of difenoconazole can be proposed as a green, efficient, and alternative formula for controlling leaf spot disease in P. heterophylla and enhancing its resistance, photosynthesis, quality, and yield.

The widespread and excessive use of ivermectin (IVM) will not only cause serious environmental pollution, but will also affect metabolism of humans and other mammals that are exposed. IVM has the characteristics of being widely distributed and slowly metabolized, which will cause potential toxicity to the body. We focused on the metabolic pathway and mechanism of toxicity of IVM on RAW264.7 cells. Colony formation and LDH detection assay showed that IVM significantly inhibited the proliferation of and induced cytotoxicity in RAW264.7 cells. Intracellular biochemical analysis using Western blotting assay showed that LC3-B and Beclin-1 were upregulated and p62 was down-regulated. The combination of confocal fluorescence, calcein-AM/CoCl2, and fluorescence probe results showed that IVM could induce the opening of the mitochondrial membrane permeability transition pore, reduce mitochondrial content, and increase lysosome content. In addition, we focused on induction of IVM in the autophagy signal pathway. The Western blotting results showed that IVM increased expression of p-AMPK and decreased p-mTOR and p-S6K expression in protein levels, indicating that IVM activated the AMPK/mTOR signaling pathway. Therefore, IVM may inhibit cell proliferation by inducing cell cycle arrest and autophagy.

Novel furoxan/coumarin hybrids were synthesized, and pharmacologic studies showed that the compounds displayed potent antiproliferation activities via downregulating both the phosphatidylinositide 3-kinase (PI3K) pathway and the mitogen-activated protein kinase (MAPK) pathway. To investigate the preclinical pharmacokinetic (PK) properties of three candidate compounds (CY-14S-4A83, CY-16S-4A43, and CY-16S-4A93), liquid chromatography, in tandem with the mass spectrometry LC-MS/MS method, was developed and validated for the simultaneous determination of these compounds. The absorption, distribution, metabolism, and excretion (ADME) properties were investigated in in vitro studies and in rats. Meanwhile, physiologically based pharmacokinetic (PBPK) models were constructed using only in vitro data to obtain detailed PK information. Good linearity was observed over the concentration range of 0.01−1.0 μg/mL. The free drug fraction (fu) values of the compounds were less than 3%, and the clearance (CL) values were 414.5 ± 145.7 mL/h/kg, 2624.6 ± 648.4 mL/h/kg, and 500.6 ± 195.2 mL/h/kg, respectively. The predicted peak plasma concentration (Cmax) and the area under the concentration-time curve (AUC) were overestimated for the CY-16S-4A43 PBPK model compared with the experimental ones (fold error > 2), suggesting that tissue accumulation and additional elimination pathways may exist. In conclusion, the LC-MS/MS method was successively applied in the preclinical PK studies, and the detailed information from PBPK modeling may improve decision-making in subsequent new drug development.

Metastasis is an important cause of cancer-related death. Previous studies in our laboratory found that pregnane alkaloids from Pachysandra terminalis had antimetastatic activity against breast cancer cells. In the current study, we demonstrated that treatment with one of the alkaloid derivatives, (Z)-3β-ethylamino-pregn-17(20)-en (1), led to the downregulation of the HIF-1α/VEGF/VEGFR2 pathway, suppressed the phosphorylation of downstream molecules Akt, mTOR, FAK, and inhibited breast cancer metastasis and angiogenesis both in vitro and in vivo. Furthermore, the antimetastasis and antiangiogenesis effects of 1 treatment (40 mg/kg) were more effective than that of Sorafenib (50 mg/kg). Surface plasmon resonance (SPR) analysis was performed and the result suggested that HSP90α was a direct target of 1. Taken together, our results suggested that compound 1 might represent a candidate antitumor agent for metastatic breast cancer.

Currently, the authentication of camellia oil (CAO) has become very important due to the possible adulteration of CAO with cheaper vegetable oils such as rapeseed oil (RSO). Therefore, we report a Fourier transform infrared (FTIR) spectroscopic method for detecting the authenticity of CAO and quantifying the blended levels of RSO. In this study, two characteristic spectral bands (1119 cm-1 and 1096 cm-1) were selected and used for monitoring the purity of CAO. In combination with principal component analysis (PCA), linear discriminant analysis (LDA), and partial least squares regression (PLSR) analysis, qualitative and quantitative methods for the detection of camellia oil adulteration were proposed. The results showed that the calculated I1119/I1096 intensity ratio facilitated an initial check for pure CAO and six other edible oils. PCA was used on the optimized spectral region of 1800-650 cm-1. We observed the classification of CAO and RSO as well as discrimination of CAO with RSO adulterants. LDA was utilized to classify CAO from RSO. We could differentiate and classify RSO adulterants up to 1% v/v. In the quantitative PLSR models, the plots of actual values versus predicted values exhibited high linearity. Root mean square error of calibration (RMSEC) and root mean square error of cross validation (RMSECV) values of the PLSR models were 1.4518%-3.3164% v/v and 1.7196%-3.8136% v/v, respectively. This method was successfully applied in the classification and quantification of CAO adulteration with RSO.

Four new sesterterpenes, arthproliferins A-D (1-4), together with four known derivatives, were isolated and characterized from the mangrove-sediment-derived fungus Arthrinium sp. SCSIO41221. Their structures were determined using detailed nuclear magnetic resonance (NMR) and mass spectroscopic (MS) analyses. Some of the isolated compounds were evaluated for their cytotoxicity in vitro. The results revealed that terpestacin (6) exhibited significant activity with an IC50 value of 20.3 μM, and compounds 2 and 5 were found to show weak inhibitory effects against U87MG-derived GSCs.