Evaluation of the in vitro and in vivo antimicrobial activity of alkaloids prepared from Chelidonium majus L. using MRSA- infected C. elegans as a model host.

Chelidonium majus L. contained alkaloids as its main component, exhibiting various biological activities, particularly antibacterial activity. This study aimed to extract alkaloids from C. majus L. (total alkaloids) and evaluate their antibacterial activity both in vitro and in vivo. Reflux extraction was carried out on C. majus L., and the extract was purified with HPD-600 macroporous resin and 732 cation exchange resin columns. Infection modeling of Caenorhabditis elegans (C. elegans) was established to investigate the impact of Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-sensitive Staphylococcus aureus (MSSA) on the motility, longevity, and reactive oxygen species (ROS) levels of wild-type worms (N2 strain). The effects of total alkaloids on longevity and ROS were further evaluated in infected N2 worms. Additionally, the effect of total alkaloids on the stress resistance of C. elegans and the mechanism of action were investigated. By utilizing CB1370, DR26 and CF1038 transgenic strains of C. elegans to identify whether the antibacterial activity of total alkaloids was dependent on DAF-2/DAF-16 pathway. The results showed that total alkaloids exhibited a significant antibacterial activity against both MRSA and MSSA (MIC 31.25 µg/mL). Compared with MSSA, the MRSA exhibited a stronger inhibitory effect on the movement behavior and development of worms, along with faster pathogenicity and unique virulence factors. Total alkaloids also displayed the ability to extend the lifespan of C. elegans under oxidative stress and heat stress, and reduce the expression of ROS. The antibacterial activity of total alkaloids was primarily dependent on the DAF-2/DAF-16 pathway, and the presence of functional DAF-2 was deemed essential in total alkaloids mediated immune response against MRSA. Moreover, the antibacterial and anti-infection effects of total alkaloids were found to be associated with the daf-16 gene fragment.

[Explore antioxidant quality markers of Hippophae tibetana based on "dry-method + wet-method" technology].

The cross combination of dry-method(network pharmacology analysis) and wet-method(high-resolution mass spectro-metry with antioxidation experiment) was used to predict antioxidant quality markers(Q-markers) of Hippophae tibetana. Ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap mass spectrometry(UPLC-Q-Exactive Orbitrap-MS) was developed to rapidly separate and identify the chemical constituents in H. tibetana. Then in DPPH free radicals and superoxide anion scavenging experiment, the antioxidant activity of the four different polar parts with extracts of petroleumether, ethyl acetate, n-butanol and water was evaluated. Network pharmacology method was used for functional enrichment and pathway analysis to screen antioxidant-related components and preliminarily explain the mechanism of action. On this basis, multi-source information was integrated to predict the antioxidant Q-markers. The results showed that 51 components in H. tibetana were identified, including 18 flavonoids, 14 terpenoids, 6 alkaloids, 4 coumarins and phenylpropanoids, 3 volatile components and 2 polyphenols. The antioxidant capacity of different fractions: ethyl acetate > n-butanol > water > petroleum ether. The medicine mainly acted on PI3 K-Akt and FoxO signaling pathways to perform antioxidant effects through flavonoids such as quercetin, luteolin and kaempferol. According to the results of dry-method and wet-method, quercetin, luteolin and kaempferol, the representatives of poly-hydroxy flavone, may be the antioxidant Q-markers of H. tibetana. In this study, with the antioxidant Q-markers of H. tibetana as an example, an investigation model of predicting Q-marker was discussed based on the ternary system of composition, function and informatics, providing a scientific basis for the establishment of quality evaluation standards for H. tibetana.

Synergistic Effect of Berberine-Based Chinese Medicine Assembled Nanostructures on Diarrhea-Predominant Irritable Bowel Syndrome In Vivo.

Diarrhea-predominant irritable bowel syndrome (IBS-D) is one common chronic functional disease of the digestive system with limited treatments. The microbiota-gut-brain axis (MGBA) has a central function in the pathogeny of IBS-D, which includes the participation of many various factors, such as brain-gut peptides (BGPs), immune inflammation, and intestinal flora. Inspired by the drug combination in traditional Chinese medicine (TCM), our previous study discovered that berberine (BBR) and baicalin (BA) could form natural self-assemblies as BA-BBR nanoparticles (BA-BBR NPs) and showed synergistic effects against IBS-D. Here, we investigated the synergistic effects of BA-BBR NPs on IBS-D model mice induced by chronic restraint stress plus Senna alexandrina Mill decoction with the influence on MGBA. BA-BBR NPs showed the best therapeutic effect on improving visceral hypersensitivity and diarrhea on IBS-D model mice, compared with BBR, BA, and BA/BBR mixture. Furthermore, BA-BBR NPs significantly (P<0.05) reduced the levels of 5-hydroxytryptamine (5-HT), vasoactive intestinal polypeptide (VIP) and choline acety transferase (CHAT) in colon tissues or of serum from BGPs; it lowered the expressions of the nuclear factor kappa-B (NF-κB) in colon tissues and changed the levels of basophil granulocyte (BASO) and leukomonocyte (LYMPH) in whole blood from immune inflammation; it altered the intestinal flora of Bacteroidia, Deferribacteres, Verrucomicrobia, Candidatus_Saccharibacteria, and Cyanobacteria from intestinal flora. In conclusion, BA-BBR NPs, after forming the natural self-assembly between BBR and BA, promoted the synergistic effect on IBS-D mice than the sum of BBR and BA effects, based to the formation of self-assemblies rather than the simple mixing. It further proved that synergistic effect of BA-BBR NPs on IBS-D mice might be related to BGPs, immune inflammation, and intestinal flora from three important interrelated components of MGBA. This study will provide a novel idea for the interpretation of TCM compatibility theory and provide the basis for BA-BBR NPs as a medicinal plant-derived natural and efficient nanomaterial for clinical use.

Betulinic Acid-Nitrogen Heterocyclic Derivatives: Design, Synthesis, and Antitumor Evaluation in Vitro.

Betulinic acid (BA) is a star member of the pentacyclic triterpenoid family, which exhibits great prospects for antitumor drug development. In an attempt to develop novel antitumor candidates, 21 BA-nitrogen heterocyclic derivatives were synthetized, in addition to four intermediates, 23 of which were first reported. Moreover, they were screened for in-vitro cytotoxicity against four tumor cell lines (Hela, HepG-2, BGC-823 and SK-SY5Y) by a standard methylthiazol tetrazolium (MTT) assay. The majority of these derivatives showed much stronger cytotoxic activity than BA. Remarkably, the most potent compound 7e (the half maximal inhibitory concentration (IC50) of which was 2.05 ± 0.66 µM) was 12-fold more toxic in vitro than BA-treated Hela. Furthermore, multiple fluorescent staining techniques and flow cytometry collectively revealed that compound 7e could induce the early apoptosis of Hela cells. Structure-activity relationships were also briefly discussed. The present study highlighted the importance of introducing nitrogen heterocyclic rings into betulinic acid in the discovery and development of novel antitumor agents.

Design, synthesis, and biological evaluation of ligustrazine - betulin amino-acid/dipeptide derivatives as anti-tumor agents.

The ligustrazine - betulin derivative (TB), TB amino acids derivatives (TB-01 - TB-09) and TB dipeptide derivatives (TB-10 - TB-18) were designed and synthesized. And their in vitro cytotoxic activities were evaluated against four cancer cell lines (Hela, HepG2, BGC-823 and HT-29) and normal cells MDCK by standard methylthiazol tetrazolium (MTT) assay. Most of them demonstrated better antitumor activity than the relevant material betulin. Among them, compound TB-01 showed the best anti-tumor effect on the cancer cells and the lowest toxicity on the normal cells. For example, the cytotoxicity of TB-01 against the cancer cells (mean IC50 = 4.86 ±â€¯1.16 µM) was 3-fold higher than that against the normal cells MDCK (IC50 = 16.11 ±â€¯2.29 µM). Moreover, TB-01 showed better cytotoxic than positive drug cisplatin (DDP) on tumor cells. Besides, the Zebrafish toxicity evaluation test showed that TB-01 demonstrated high biosafety. Subsequently, fluorescent staining, apoptosis detection and cell cycle analysis indicated that TB-01 induced early apoptosis in HepG2 cells and blocked the cell cycle in the G1 phase. In addition, the structure-activity relationships of these derivatives were briefly discussed.

Design, Synthesis and Biological Evaluation of Diosgenin-Amino Acid Derivatives with Dual Functions of Neuroprotection and Angiogenesis.

Diosgenin, a natural product with steroidal structure, has a wide range of clinical applications in China. It also shows great potential in the treatment of blood clots and nerve damage. To enhance the bioavailability as well as efficacy of diosgenin, eighteen diosgenin-amino acid derivatives were designed and synthesized. The neuroprotective effects of these compounds were evaluated by SH-SY5Y cell line and the biosafety was evaluated by H9c2 cell line. The results displayed that part of the derivatives' activities (EC50 < 20 µM) were higher than positive control edaravone (EC50 = 21.60 ± 3.04 µM), among which, DG-15 (EC50 = 6.86 ± 0.69 µM) exhibited the best neuroprotection. Meanwhile, biosafety evaluation showed that DG-15 had no cytotoxicity on H9c2 cell lines. Interestingly, combined neuroprotective and cytotoxic results, part of the derivatives without their protecting group were superior to compounds with protecting group. Subsequently, Giemsa staining and DAPI (4',6-diamidino-2-phenylindole) staining indicated that DG-15 had a protective effect on damaged SH-SY5Y cells by reducing apoptosis. Moreover, DG-15 showed a higher role in promoting angiogenesis at high concentrations (4 mg/mL) on the chorioallantoic membrane model. This finding displayed that DG-15 had dual functions of neuroprotection and angiogenesis, which provided further insight into designing agent for the application in treatment of ischemic stroke.

Synthesis of Novel Baicalein Amino Acid Derivatives and Biological Evaluation as Neuroprotective Agents.

Baicalein, a famously effective component of the traditional Chinese medicine Rhizoma Huang Qin (Scutellaria altissima L.), has been proved to have potent neuroprotection and anti-platelet aggregation effects with few side effects. Meanwhile, recent studies have revealed that the introduction of amino acid to baicalein could improve its neuroprotective activity. In the present study, a series of novel baicalein amino acid derivatives were designed, synthesized, and screened for their neuroprotective effect against tert-butyl, hydroperoxide-induced, SH-SY5Y neurotoxicity cells and toxicity on the normal H9C2 cell line by standard methylthiazol tetrazolium (MTT) assay. In addition, all of the newly synthesized compounds were characterized by 1H-NMR, 13C-NMR, and high resolution mass spectrometry (HR-MS). The results showed that most of the compounds provided more potent neuroprotection than baicalein, and were equivalent to the positive drug edaravin. They showed no obvious cytotoxicity on normal H9C2 cells. Notably, the most active compound 8 displayed the highest protective effect (50% effective concentration (EC50) = 4.31 µM) against tert-butyl, hydroperoxide-induced, SH-SY5Y neurotoxicity cells, which was much better than the baicalein (EC50 = 24.77 µM) and edaravin (EC50 = 5.62 µM). Further research on the chick chorioallantoic membrane (CAM) model indicated that compound 8 could significantly increase angiogenesis, which might promote neurovascular proliferation. The detection of apoptosis analysis showed that compound 8 could dramatically alleviate morphological manifestations of cell damage. Moreover, the benzyloxycarbonyl (cbz)-protected baicalein amino acid derivatives showed better neuroprotective activity than the t-Butyloxy carbonyl (boc)-protected derivatives.

Design, synthesis and biological evaluation of cinnamic acid derivatives with synergetic neuroprotection and angiogenesis effect.

As for complex brain diseases involved with multiple pathogenic factors, it is extremely difficult to achieve curative effect by acting on a single target. Multi-approach drugs provide a promising prospect in the treatment of complex brain diseases and have been attracting more and more interest. Enlightened by synergetic effect of combination in traditional herb medicines, forty-two novel cinnamic acid derivatives were designed and synthesized by introducing capsaicin and/or ligustrazine moieties to enhance biological activities in both neurological function and neurovascular protection. Elevated levels of cell viability on human brain microvascular endothelium cell line (HBMEC-2) and human neuroblastoma cell line (SH-SY5Y) against free radical injury were observed in most of compounds. Among them, compound 14a exhibited the most potent activities with a significant EC50 value of 3.26 ±â€¯0.16 µM (HBMEC-2) and 2.41 ±â€¯0.10 µM (SH-SY5Y). Subsequently, the results of morphological staining and flow cytometry analysis experiments on both cell lines showed that 14a had the potential to block apoptosis, maintain cell morphological integrity and protect physiological function of mitochondria. Moreover, 14a displayed specific angiogenesis effect in the chick chorioallantoic membrane (CAM) assay; and the results of RT-PCR suggested that the mechanism for angiogenesis effect was associated with the enhancement of the expressions of VEGFR2 mRNA in chick embryo. Preliminary structure-activity relationship was analyzed. The above evidences suggested that conjunctures gained by combining active ingredients in traditional herb medicines deserved further study and might provide references in discovering dual-effective lead compounds for brain diseases.

Identification of a Quality Marker of Vinegar-Processed Curcuma Zedoaria on Oxidative Liver Injury.

Curcuma zedoaria (dry stenophora of Curcuma phaeocaulis Val., Curcuma kwangsiensis S. G. Lee et C. F. Liang, or Curcuma wenyujin Y. H. Chen et C.Ling) is a representative herb with clinical effects on liver diseases after being vinegar-processed. The crude Curcuma zedoaria and the processed Curcuma zedoaria (vinegar-boil) have been widely used as mixtures, but their equivalence has not been fully investigated. In this manuscript, quality markers of processed (vinegar-boil) Curcuma zedoaria were investigated by comparison of the compounds and hepatoprotective activities with the crude (three spices) ones. First, GC-MS-based untargeted metabolomics were applied to reveal the discriminatory components and discover potential markers. As a result, a total of six components were identified as potential markers. Then, the hepatoprotective activities were evaluated by dual cell damage models induced by a certain concentration of H2O2 or tertbutyl hydfroperoxide (t-BHP) (55 µM H2O2 or 40 µM t-BHP), which highlighted the potential of the processed Curcuma zedoaria on oxidative stress. Finally, epicurzerenone was identified as its quality marker on oxidative liver injury based on the above results and the cell-based biological assay. Overall, vinegar-processed Curcuma zedoaria was more suitable for the treatment of oxidative liver diseases, and epicurzerenone could be considered as its quality marker.