Cytotoxic diterpenoid dimer containing an intricately caged core from Euphorbia fischeriana.

Bislangduoids A and B, a novel class of dimeric diterpenoids based on ent-abietanes tethered by C-17-C-15' bridge, were identified as trace components from a traditional Chinese medicine Euphorbia fischeriana (Langdu). Bislangduoid A features a highly oxidized scaffold incorporating a cage-like pentacyclic core. Their structures were elucidated by extensive spectroscopic techniques, electronic circular dichroism, and NMR calculations. The biosynthetic pathway for the dimeric skeleton and the unique caged moiety via Michael and acetal-formation reactions was proposed. Bislangduoid A showed pronounced cytotoxicity against HepG2 cells through the mitochondria-dependent apoptosis pathway.

The nature compound dehydrocrenatidine exerts potent antihepatocellular carcinoma by destroying mitochondrial complexes in vitro and in vivo.

Cumulative evidence indicates that mitochondria dysfunction plays an important role in tumour treatment. Given the limited efficacy and toxicity of current mitochondria-targeted drugs, research into effective mitochondria-targeted anticancer agents remains an irresistible general trend. In this study, it was found that dehydrocrenatidine (DEC), a ß-carbolin alkaloid isolated from Picrasma quassiodes, displays a promising growth inhibitory effect in vitro and in vivo by inducing apoptosis of hepatocellular carcinoma (HCC) cells. Mechanistically, we provided that the possible target of DEC against HCC cells was determined by isobaric labels for relative and absolute quantification assay and validated them using further experiments. The results suggested that DEC can target and regulate the function of mitochondrial complexes I, III and IV, affecting oxidative phosphorylation and ultimately leading to mitochondrial dysfunction to exert its anti-HCC effects. In addition, the combination of DEC and sorafenib showed a synergistic effect and was also associated with mitochondrial dysfunction. Importantly, DEC did not show significant toxicity in mice. This study provided a new insight into underlying mechanisms in DEC-treated HCC cells, suggesting that DEC might be a mitochondrial targeting lead compound.

Identification of Escherichia coli β-glucuronidase inhibitors from Polygonum cuspidatum Siebold & Zucc

Abstract Gut bacterial β-glucuronidase (GUS) can reactivate xenobiotics that exert enterohepatic circulation- triggered gastrointestinal tract toxicity. GUS inhibitors can alleviate drug-induced enteropathy and improve treatment outcomes. We evaluated the inhibitory effect of Polygonum cuspidatum Siebold & Zucc. and its major constituents against Escherichia coli GUS (EcGUS), and characterized the inhibitory mechanism of each of the components. Trans-resveratrol 4'-O-β-D-glucopyranoside (HZ-1) and (-)-epicatechin gallate (HZ-2) isolated from P. cuspidatum were identified as the key components and potent inhibitors. These two components displayed strong to moderate inhibitory effects on EcGUS, with Ki values of 9.95 and 1.95 μM, respectively. Results from molecular docking indicated that HZ-1 and HZ-2 could interact with the key residues Asp163, Ser360, Ile 363, Glu413, Glu504, and Lys 568 of EcGUS via hydrogen bonding. Our findings demonstrate the inhibitory effect of P. cuspidatum and its two components on EcGUS, which supported the further evaluation and development of P. cuspidatum and its two active components as novel candidates for alleviating drug-induced damage in the mammalian gut.

UV-light-driven photooxidation of harmaline catalyzed by riboflavin: Product characterization and mechanisms.

ß-Carboline alkaloid harmaline (HA) is a candidate drug molecule that has been proven to have broad and significant biological activity. Herein, the effects of HA on the riboflavin (RF)-sensitized photooxidation under aerobic conditions were studied for the first time. The photooxidation reaction of HA catalyzed by RF is triggered by UV light at 365 nm and shows a time-dependent stepwise reaction process. Seven transformed products, including five undescribed compounds, oxoharmalines A-E (1-4 and 7), and two known compounds, N-(2-(6-Methoxy-2-oxoindolin-3-yl)ethyl)acetamide (5) and harmine (6), were isolated and identified from the reaction system, following as the gradual oxidation mechanisms. The rare polymerization and dehydrogenation processes in radical-mediated photocatalytic reactions were involved in the process. The transformed products 2-7 exhibited significant neuroprotective activity in a model of H2O2-introduced injury in SH-SY5Y cells, which suggested that the products of the interaction between HA and vitamins may be beneficial to health.

The identification of alkaloids from the stems of Picrasma quassioides via computer-assisted structure elucidation and quantum chemical calculations.

Four new alkaloids (1-4) and one known alkaloid were isolated from the stems of Picrasma quassioides. The structures of these isolated compounds were elucidated by spectroscopic analyses, a combination of computer-assisted structure elucidation software (ACD/Structure Elucidator) and gauge-including atomic orbital (GIAO) calculation of 1 D NMR data. All compounds were evaluated for their cytotoxic activities against hepatocellular carcinoma HepG2 and Hep3B cells. However, they did not show obvious inhibitory activities.[Figure: see text].

Guaiane-Type Sesquiterpenoids from the Roots of Daphne genkwa and Evaluation of Their Neuroprotective Effects.

Chromatographic purification of the roots of Daphne genkwa led to 11 new guaiane-type sesquiterpenoids (1-11), named genkwanoids A-K, and six known analogues (12-17). A comprehensive set of spectroscopic methods including IR, UV, HRESIMS, and 1D/2D NMR were used to elucidate the structures and relative configurations of 1-11. The absolute configurations were determined by the optical rotation calculations and the modified Mosher's method. The possible biosynthetic pathways for 1-11 are proposed. Furthermore, the neuroprotective effects of 1-17 on H2O2-induced damage in human neuroblastoma SH-SY5Y cells were screened using an MTT assay. Compounds 9, 10, and 16 exhibited moderate neuroprotective effects, with survival rates of 79.34%, 79.94%, and 75.64% after treatment with 12.5 µM, values that were comparable to the positive control, Trolox (78.65%). Furthermore, annexin V-FITC/PI staining of cells treated with 9, 10, and 16 showed that the neuroprotective effects of these compounds may arise from inhibiting cell apoptosis.

Effects of Enantiomerically Pure ß-Carboline Alkaloids from Picrasma quassioides on Human Hepatoma Cells.

Four pairs of ß-carboline enantiomers (1A: /1B: -4A: /4B: ), 2 ß-carboline derivatives (5:  - 6: ) with a single enantiomeric configuration, together with 2 known achiral congeners (7:  - 8: ) were isolated from the stems of Picrasma quassioides. Their structures were elucidated on the basis of extensive spectroscopic analyses and quantum mechanical calculations. Compound 5: possesses a 4,5-seco ß-carboline framework and represents the first example of this type of ß-carboline alkaloids from nature. A possible biosynthetic pathway is proposed to generate the racemate 4: and the enantiomerically pure compounds 5: and 6: . All isolates were screened for their cytotoxicity against hepatocellular carcinoma Hep3B and HepG2 cells, which revealed that enantiomeric compounds 4A: and 4B: had distinctive effects in HepG2 cells. Further investigation showed that 4B: could induce apoptosis in HepG2 cells.

Quassinoids from Picrasma quassioides and Their Neuroprotective Effects.

Quassinoids are a class of highly oxygenated degraded triterpenoids exclusively discovered from plants of the Simaroubaceae family. In this study, eight new (1-8) and 15 known quassinoids (9-23) were isolated from an extract of the stems of Picrasma quassioides. The structures were elucidated by spectroscopic analysis and electronic circular dichroism spectra combined with quantum chemical calculations. Compounds 4 and 5 represent the first examples of 18-nor-quassinoids from P. quassioides. All isolates were screened for their neuroprotective activities toward H2O2-induced cell damage in SH-SY5Y cells. Further study revealed that the potential protective activities of these compounds appeared to occur via the suppression of cell apoptosis and downregulation of caspase-3 activation.

New tirucallane triterpenoids from Picrasma quassioides with their potential antiproliferative activities on hepatoma cells.

Seven new tirucallane-type triterpenoids (1-7), kumuquassin A-G, along with 20 known analogues (8-27) were isolated from the stems of Picrasma quassioides. The structures and the absolute configurations of new compounds were elucidated by spectroscopic data, electronic circular dichroism (ECD) spectroscopic analyses and quantum ECD calculations. Notably, kumuquassin A (1) contains a rare Δ17, 20 double bond, kumuquassin B (2) is the first example of tirucallane triterpenoid possessing a 5/3 biheterocyclic ring system at the side chain. All the compounds were screened for the cytotoxicity against two human hepatoma cell lines, HepG2 and Hep3B, and several compounds exhibited promising activity. The most potential compound 3 was selected for cell cycle analysis, which showed that 3 could cause an accumulation of HepG2 cells at subG1 peak. Annexin V-FITC/PI staining further confirmed that compound 3 caused death of hepatoma cells through apoptosis induction.

Growth of pediatric recipients after renal transplantation from small pediatric deceased donors weighing less than 15 kg.

RTx is currently the best treatment for children with ESRD. This study retrospectively analyzed the outcomes of growth after RTx using the pediatric-to-pediatric allocation strategy and some factors that may affect it. From March 2012 to August 2016, 8 en bloc and 38 single pediatric RTxs were performed at our center using organs from small pediatric deceased donors (weight < 15 kg). Growth before and after RTx was analyzed according to the height-for-age z-score at RTx, the 3-year follow-up, and adulthood and compared between the procedures. The chi-square test and multiple linear regression analysis were used for statistical analyses. Overall, 79.2% of children were diagnosed with chronic nephritis before RTx; 14.6% of cases were due to congenital urinary tract malformation, and 6.3% of cases were due to unknown causes. All grafts and patients survived postoperatively. The mean estimated GFRs were 92.7 ± 28.6 mL/min/1.73 m2 , 100.9 ± 32.3 mL/min/1.73 m2 , and 110.1 ± 34.8 mL/min/1.73 m2 at 1, 2, and 3 years' postoperatively, respectively. The children's postoperative growth and development, particularly during the first year postoperatively, improved but were negatively correlated with age and the height-for-age z-score before RTx. The growth of children after RTx was moderate and accelerated during prepubescence. The rate of post-RTx growth during the first year postoperatively was unrelated to the recipient's sex or duration of dialysis (P > 0.05) but was negatively correlated with age at RTx (r = -0.349, P = 0.043). Future studies on the long-term outcomes are still needed.

Enantiomeric ß-carboline dimers from Picrasma quassioides and their anti-hepatoma potential.

Four pairs of enantiomeric ß-carboline alkaloids, (+/-)-kumudine A-D, along with their biosynthesis-related compound kumudine E, were obtained from the stems of Picrasma quassioides. Their structures, including the absolute configurations, were determined via extensive spectroscopic data combined with electronic circular dichroism (ECD) spectroscopic analyses and quantum mechanical ECD calculations. (+/-)-Kumudine A possessed a scaffold of ß-carboline-phenylpropanoid adduct, which were the first examples of this type of ß-carboline alkaloid from nature. The cytotoxicity assay against hepatocellular carcinoma Hep3B and HepG2 cells was evaluated by SRB assay, which showed that (-)-Kumudine B had stronger effect than its enantiomer (+)-Kumudine B in Hep3B cells. Moreover, further flow cytometry analysis also supported the enantioselectivity between (+)-Kumudine B and (-)-Kumudine B, suggesting that the compounds caused death of hepatoma cells through apoptosis induction.

Stereoisomeric guaiacylglycerol-ß-coniferyl aldehyde ether induces distinctive apoptosis by downregulation of MEK/ERK pathway in hepatocellular carcinoma cells.

Two 8-O-4'-type neolignan epimers erythro-guaiacylglycerol-ß-coniferyl aldehyde ether (1) and threo-guaiacylglycerol-ß-coniferyl aldehyde ether (2) were isolated from the stems of Picrasma quassioides. Further chiral separation gave two pairs of enantiomers 1a/1b and 2a/2b. The cytotoxicity assay against hepatocellular carcinoma Hep3B and HepG2 cells was evaluated by MTT assay. The results showed that 1b (IC50 = 45.56 µM) and 2b (IC50 = 39.02 µM) had more cytotoxic effect than its enantiomers 1a (IC50 = 82.66 µM) and 2a (IC50 = 67.97 µM) in Hep3B cells, respectively. Moreover, 1b and 2b could induce more apoptotic cells as well as higher reactive oxygen species (ROS) generation than 1a and 2a at 50 µM. In addition, a further study on the phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways was investigated. The results revealed that all compounds had no significant effect on PI3K/AKT pathway, however, 1b and 2b attenuated the relative levels of p-MEK and p-ERK when compared with 1a and 2a. Taken together, the absolute configurations of guaiacylglycerol-ß-coniferyl aldehyde ether had an impact on the inhibitory effect on Hep3B cells. The inactivation of MEK/ERK signaling pathway might contribute to apoptosis induction and ROS generation in 1b- and 2b-treated cells.

Bioactivity-guided isolation of ß-Carboline alkaloids with potential anti-hepatoma effect from Picrasma quassioides (D. Don) Benn.

ß-Carboline alkaloids in Picrasma quassioides (D. Don) Benn. have been proven to possess inhibitory activity against various cancer cells. However, their effect on hepatocellular carcinoma and structure-activity relationships (SAR) have not been systematically reported. In this work, bioactivity-directed fractionation of P. quassioides led to the separation of active fraction A2-2. A total of 39 ß-carbolines, including 4 new ones (1-4), were obtained from the active fraction. Moreover, all the isolated compounds were identified in the active fraction A2-2 by LC-MS. The cytotoxicity on HepG2 and Hep3B cells of all compounds was screened by MTT assay, and the SAR were established. The SAR were also supported by the apoptosis ratio of HepG2 cells using flow cytometry analysis after treatment with potential compounds 1, 2, 9, 10, 12, 29, 36 and 38. It suggested that these active compounds caused death of hepatoma cells through apoptosis induction. In addition, further study revealed that compounds 12, 29, 36 significantly activated caspase-3 in HepG2 cells.

Enantiomeric neolignans from Picrasma quassioides exhibit distinctive cytotoxicity on hepatic carcinoma cells through ROS generation and apoptosis induction.

Three pairs of enantiomeric neolignans 1a/1b-3a/3b were isolated from the stems of Picrasma quassioides, and separated successfully by chiral-phase HPLC. Their structures were established by comprehensive spectroscopic analyses as well as ECD spectroscopy. The in vitro cytotoxicity of the isolates was evaluated against human hepatocellular carcinoma HepG2 and Hep3B cells. Among them, 1 and its enantiomers 1a/1b, 3 and 3a/3b displayed similar cytotoxicity in pair-wise comparison against HepG2 and Hep3B cells, and the similar effects of 2 and 2a/2b were found in Hep3B cells. Interestingly, 2a and 2b had different cytotoxic activities on HepG2 cells with IC50 values of 35.6 µM and 104.4 µM, respectively. In addition, 2 exerted middle cytotoxicity against HepG2 cells with an IC50 value of 78.6 µM. The different cytotoxicity between enantiomers 2a and 2b attracted our interest. To investigate the underlying mechanisms responsible for the distinct cytotoxicity, we further assessed the effects of 2a and 2b on cell cycle distribution, cell apoptosis and reactive oxygen species (ROS) generation. The results indicated that 2a had more significant effect than 2b on apoptosis induction and ROS generation, but both had no obvious effect on cell cycle of HepG2 cells. It is concluded that the different configurations of 2a/2b determined the enantioselective cytotoxicity on HepG2 cells through apoptosis induction and ROS generation.

Mitochondria-targeted antioxidant peptide SS31 prevents hypoxia/reoxygenation-induced apoptosis by down-regulating p66Shc in renal tubular epithelial cells.

BACKGROUND/AIMS: Ischemia/reperfusion injury plays a crucial role in renal transplantation and represents a significant risk factor for acute kidney injury and delayed graft function. Mitochondria-targeted antioxidant peptide SS31 has been shown to attenuate ischemia/reperfusion injury by inhibiting oxidative stress. The present study was carried out to investigate whether the pretreatment of SS31 could reduce hypoxia/reoxygenation (H/R)-induced injury by inhibiting p66Shc. METHODS: The cultured rat renal proximal tubular cell line NRK52E cells were exposed to 24 h hypoxia (5% CO2, 1% O2, 94% N2) followed by 6 h reoxygenation (5% CO2, 21% O2, 74% N2). SS31 was added to the culture medium 4 h prior to the treatment. Then the cell viability, apoptosis, and oxidative stress levels were determined. In addition, western blot analysis was performed to determine the expression of p66Shc, p-p66Shc, cytochrome c, and caspase-3. RESULTS: H/R induced apoptotic cell death, accompanied with activation of total and p-p66Shc in NRK52E cells. Pretreatment with SS31 or overexpression of a dominantnegative Ser36 mutant p66Shc (p66Shc S36A) or p66Shc siRNA prevented cell death, whereas the protection effect of SS31 was completely blocked by overexpression of wild-type p66Shc. Furthermore, SS31 pretreatment reduced H/R-induced intracellular oxidative stress, cytochrome c translocation to the cytoplasm, and caspase-3 activation through inhibiting p66Shc. CONCLUSION: This study revealed that SS31 pretreatment serves a protective role against H/R-induced apoptosis of human renal tubular epithelial cells, and the mechanism is related to suppression of p66Shc.

In vitro effects of polyethylene glycol in University of Wisconsin preservation solution on human red blood cell aggregation and hemorheology.

Addition of hydroxyethyl starch (HES) to UW (University of Wisconsin) solution increases viscosity of the solution and red blood cell (RBC) aggregation. Recently, it was suggested that HES could be replaced by a new colloid, polyethylene glycol (PEG), in UW solution. The aim of this study was to see whether and how PEG affected RBC aggregation, and whether RBC aggregation parameters had any correlation with the molecular weight and concentration of PEG. After giving informed consent and signing consent documents, 12 healthy volunteers were enrolled in the study. Blood samples obtained from these volunteers were mixed with the test solutions with blood/solutions ratios of 5:1 and 1:1. Human RBC aggregation was investigated with an automatic hemorheological analyzer. Blood viscosity was measured with a cone-plate viscometer. Morphological characters of RBC aggregates were evaluated by light microscopy. It was found that viscosity was not affected by the Colloid-free UW solution. PEG20kDa (1 and 10 g/L) and PEG35kDa (1 g/L) had little effect on RBC aggregation, while PEG20kDa (30 g/L) and PEG35kDa (10 and 30 g/L) had a significant hyperaggregating effect on RBC. In conclusion, PEGs had a potential hyperaggregating effect on human RBC. This effect is correlated with PEG molecular weight and concentration. The use of large molecular weight and high concentration PEG in UW solution accounts for extended and accelerated aggregation of erythrocytes. The use of low concentration PEG35kDa (1 g/L) would be the optimal choice.